JP2001503631A - Glaucoma-associated proteins and corresponding nucleic acids and their use in therapy and diagnosis - Google Patents

Abstract

  (57) [Summary] Diagnosis of glaucoma based on detection of non-wild type GLC1A gene or protein in a tester, method of treating glaucoma, and assay for identifying an agent that modulates the activity of GLC1A and is therefore useful for treating or preventing glaucoma Is described.

Description

DETAILED DESCRIPTION OF THE INVENTION             Glaucoma-related proteins and corresponding nucleic acids Their use in treatment and diagnosis 1. Background art   Glaucoma is the second leading cause of total legal blindness in the United States, Are the leading cause of blindness in Descent Americans (Hiller, R. and HA Kahn, (1975) ) Am.J. Ophthalmol 80:62, and Kahn H. A. and H. B. Moorhead (1973) U S Public Health Service Publication NIH 73-427, 120). Primary open corner green Glaucoma (POAG) is the most common type of glaucoma, one of the population over 40 years of age. Or 2% are affected (JM Tielsch et al. (1990) Arch Ophthalmol. 108 : 286). Nearly 12,000 people are blinded by the disease each year in the United States (H.B. Moorhead (1973) US public Health Service Publication NIH 73-427 J., 1202-4; M. Tielsch et al. (1990) Arch Ophthalmol. 108: 286 and J.I. M . Tielsch, Transactions of the New Orleans Academy of Ophthalmology, Bal l, S. F. Franklin R. M. (Kugler, Amsterdam, 1993) , Pp. 61-68).   One way to identify genes involved in multifactorial diseases is to use similar phenotypes. To study a Mendelian disease with Juvenile open-angle glaucoma (JOA G) has an early onset age and a highly penetrant autosomal dominant inheritance pattern (A. T. Johnson et al. (1993) Ophthalmology 100: 524). In laboratory tests, young Patients with open-angle glaucoma and those with late-onset glaucoma Shows increased intraocular pressure and optic nerve cup formation in the presence of normal trabecular meshwork Are equivalent. Isolation of genes related to JOAG is useful for treating glaucoma and Necessary for development of diagnosis. 2. Summary of the Invention   In one aspect, the invention features an isolated GLC1A nucleic acid molecule. The disclosed molecules can be non-coding (eg, probe, antisense, or ribosome). Or a functional polypeptide (eg, human GLC1A). Determine at least one biological activity of the polypeptide, e.g., agonist or A polypeptide that specifically regulates by acting as either gonist) May be coded.   In a further aspect, the nucleic acid molecule is at least 70%, preferably 80%. %, More preferably 85%, and even more preferably at least 95%, Q ID Nos: the nucleic acid shown in 1 or 2, or SEQ ID Nos 1 is a GLC1A nucleic acid having the same sequence as the complementary strand of the nucleic acid shown in 1 or 2. Also In another embodiment, the nucleic acid molecule is at least 92%, more preferably At least 95% of the polypeptide and sequence shown in SEQ ID No: 3 have They encode similar polypeptides.   The present invention also relates to sequences represented as SEQ ID Nos: 1 or 2, EQ ID Nos: complementary strands of the sequence represented as 1 or 2, or Of at least six consecutive nucleotides of a naturally occurring mutant of A substantially purified oligonucleotide corresponding to the hybridizing nucleotide region Probes and primers containing nucleotides are provided. In a preferred embodiment In this case, the probe / primer has a detectable label attached to it. Contains.   For expression, the subject's GLC1A nucleic acid contains a transcriptional regulatory sequence, eg, At least one transcription promoter (eg, for constitutive or inducible expression). Or a transcriptional enhancer or suppressor sequence. The rows can be operatively linked to the GLC1A gene sequence. Such regulatory sequences Can be useful vectors for gene expression in cooperation with the GLC1A nucleic acid molecule. Noh. The present invention also relates to a prokaryote transfected with the expression vector. Alternatively, in vivo (for example, by using a eukaryotic host cell and the expression vector) GLC1A tamper in cell culture) and in vivo (eg, galvanic acid) The method for producing the glass will be described.   In another aspect, the invention provides an isolated GLC1A polypeptide, Or a substantially pure preparation (eg, purified plasma or produced recombinantly) GLC1A polypeptide). In one embodiment, the port The repeptide is equivalent to the GLC1A protein represented by SEQ ID No: 3. Or a similar polypeptide. Vertebrate and especially mammalian GLC1A Related members of the family are also within the scope of the invention. Preferably, G The LC1A protein is at least about 92%, and preferably at least about 9%. 5%, 96%, 97%, 98%, or 99% represented by SEQ ID No: 3 Has an amino acid sequence equivalent to the polypeptide to be prepared. In a preferred embodiment, The GLC1A polypeptide is represented by one of SEQ ID Nos: 1 or 2. Encoded by a nucleic acid that hybridizes to the nucleic acid sequence. Exhibitor's G LCLA proteins are also modified proteins that are resistant to post-translational modifications. Which include, for example, modification sites (tyrosine, threonine, serine, or Or glycosyl of the protein) With intracellular proteins that prevent cell transformation or are involved in signal transduction Mutations that inhibit the interaction.   The GLC1A polypeptide has the full length as represented by SEQ ID No: 3. Or one or more specific mochi Or any size, e.g. at least 5, 10, 25, 50, 100, 150, 175, 200, 225, 250, 275, 30 0, 325, 350, 375, 400, 425, 450, 475, 480, 48 Fragments corresponding to 5, 490, 495, or 500 amino acids in length Can be included.   Another aspect of the invention relates to chimeric molecules consisting of GLC1A proteins (eg fusions). Protein). For example, the GLC1A protein is a recombinant fusion protein. Can be provided as a protein, which is a second polypeptide moiety, for example, Has a (heterologous) amino acid sequence unrelated to the GLC1A polypeptide. A polypeptide (eg, the second polypeptide moiety is glutathione S Enzyme activity such as transferase, alkaline phosphatase, Is a topp tag).   Still another aspect of the present invention is to include a GLC1A polypeptide in an immunogen preparation. With respect to the immunogen comprising, the immunogen is an immunogen specific for a GLC1A polypeptide. Can elicit a response (eg, a humoral response, an antibody response, and / or a cell Sex reaction). In a preferred embodiment, the immunogen is an antigenic determinant, such as SEQ. D No. 3 contains unique determinants from the protein represented.   A further aspect of the present invention relates to a specific reaction with an epitope of the GLC1A protein. It features antibodies and antibody preparations that can be used. In a preferred embodiment The antibody is at least one of the epitopes set forth in SEQ ID No: 3. Specifically binds to   The present invention also includes the variant GLC1A gene described herein (preferred). Or further expressing the endogenous GLC1A gene. Non-human transgenic animal (eg, the subject GLC1A protein Animal in which expression of one or more of the proteins has been interrupted). Take The transgenic animals are mutated or mis-expressed GLC1A To study disorders of cells and tissues containing alleles of the It could serve as an animal model for use in cleaning. There Indicate that such transgenic animals are capable of expressing recombinant GLC1A polypeptide. It can be useful.   In still another aspect, the present invention relates to, for example, GLC1A proteins. For example, a virus, an extracellular ligand of the GLC1A protein, or the GLC Inhibitor of interaction between 1A protein and binding intracellular protein Or an assay for screening test compounds for the identification of potentiators. You. Representative methods include (i) a GLC1A polypeptide or a biologically active flag thereof. A GLC1A target molecule (such as a GLC1A ligand or GLC1A substrate). ), And the test compound, eg, the GLC1A protein Binding under conditions that allow the protein and the target molecule to interact; And (ii) forming a complex containing the GLC1A protein and the target polypeptide. , By direct quantification of the complex or by measuring the induction effect of GLC1A protein Or, in the case of a substrate, by measuring the conversion to product. Detection Includes the step of: Interaction between GLC1A and target molecule in the presence of test compound Reduction (as compared to that detected in the absence of the test compound). Statistically significant changes are associated with regulation (eg, GLC1A protein and its target molecule). Inhibition or enhancement of the interaction between).   Yet another aspect of the present invention relates to a method for modulating the biological activity of GLC1A ( For example, by protecting or disrupting the biological activity of GLC1A). General Whether it takes place in vivo, in vitro, or in situ This method should alter the biological activity of GLC1A compared to untreated cells. And treating with an effective amount of therapeutic GLC1A. Therefore, this method , Effects of GLC1A (eg, signal transmission from GLC1A protein or Activate or offset the ligand binding of the GLC1A protein, as described above. Peptides and peptidomimetics or other components identified in drug screening It is possible to use a therapeutic GLC1A, such as a child. Other therapeutic GLC1A Are antisense constructs for inhibiting expression of the GLC1A protein, Interaction and signal transduction upstream of type GLC1A protein Includes dominant negative mutants that inhibit competition by competition.   A further aspect of the invention arises from a GLC1A gene mutated in a subject. A method is provided for determining whether there is a risk of glaucoma or other disease. this The method comprises the steps of (i) encoding the GLC1A protein in the tester's tissue. Gene, for example, the gene represented by SEQ ID Nos: 1 or 2, or Mutation of their homologs; or (ii) less erroneous expression of the GLC1A gene. And detecting the presence or absence of a genetic injury characterized by one or the other. Like In a preferred embodiment, the detection of this genetic injury comprises one or more of the GLC1A genes. Or more nucleotide deletions; one or more nucleotides in the gene Addition of leotide, substitution of one or more nucleotides of the gene, Macroscopic chromosomal rearrangement of a gene; messenger RNA transcript Bell changes (eg, due to promoter mutations); The presence of a non-wild-type splicing pattern of the jar RNA transcript; And / or abnormal levels of soluble GLC1A protein. Be A confirmation of the presence of at least one of the   For example, damage to the gene can be caused by (i) the sense or antisense of the GLC1A gene. The sense sequence, or a naturally occurring mutant thereof, or the GLC1A Hybridizes to a 5 'or 3' flanking sequence naturally associated with the gene (Ii) providing a probe / primer comprising an oligonucleotide; Contacting the probe / primer with a suitable nucleic acid containing a sample; And (iii) hybridizing the probe / primer to the nucleic acid. And detecting the presence or absence of a genetic injury; for example, The detection comprises the nucleotide sequence of the GLC1A gene and optionally the flanking nucleic acid. Includes utilizing probes / primers to determine sequence. For example, Primers can be used in polymerase chain reaction (PCR) or ligation chain reaction (LCR). ) Can be used. In an alternative embodiment, the GLC1A protein Levels are determined by antibodies that can specifically react with GLC1A protein. Detected in the immunoassay used.   Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. Will be clear. 3. BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 shows the DNA sequence of the human GLC1A gene. The coding sequence is 5 ' And 3 'untranslated regions (UTRs) (SEQ. ID. No. 1) I have. Intron 1 is SEQ ID No. 9 and intron 2 is SE Each is provided as Q ID No. 10. Encoding protein and three DNA containing the two exon sequences is provided as SEQ ID No: 2. ing. The encoded protein is shown as SEQ ID No: 3. ing.   FIG. 2 shows the artificial dyeing of yeast containing the smallest tiled pathway contig. A color body (YACS) is shown. Black circles are included in each YAC marker Indicates the position of the marker (STSs) indicating that this is the case. Within the family Cessation of illness and shared haplotypes between families Are shown.   FIGS. 3A and 3B are found to accommodate sequence changes in glaucoma patients. Polymerase chain reaction (PCR) amplimers of the GLC1A gene (Amplimer). The wild type amplimer shown in FIG. Amplimer served as EQ ID No: 11 and also contains a mutation (CAC-TYR430His) is provided as SEQ ID No: 12. You. The wild type amplimer shown in FIG. 3B is SEQ ID No: 13 And the amplimer containing the mutation (GTC-GLY357 VAL and TAG-GLN361STOP) are SEQ ID No. 14 Has been served.   FIG. 4 shows GLY364 in a four generation family affected by open angle glaucoma. FIG. 1 outlines the segregation of VAL mutations. Black symbols in genealogy indicate open corners Shows individuals with recorded evidence of glaucoma. White symbols are not clinically affected Indicates a spouse. A photograph of the silver-stained SSCP gel is shown below the pedigree, Is the true symbol of the genealogy of the family tribute whose DNA is being analyzed in that lane. It is arranged to be located below. Families not clinically affected, for simplicity Members were not included.   FIG. 5 shows the fluorescent dye primer of the cloned PCR amplification product from the patient. Representative chromatograms obtained from the sequence, with unseen at codon 368. Expected to result in cessation of ripening (CLN368STOP), from C Shows the mutation to T. Prospective for the mutant and normal gene and The reverse sequence is shown: (A) normal, forward (SEQ ID No: 15) (B) mutant, prospective (SEQ ID No: 16); (C) normal, reverse Orientation (SEQ ID No: 17); (D) Mutant, reverse orientation (SEQ ID No: 17) No: 18). 4. Detailed description of the invention   4.1. Summary   As reported in this text, loci associated with JOAG were identified by genetic linkage analysis. And identified on chromosome 1q21-q31. Two large families of JOAG Glaucoma phenotype and recombination of highly polymorphic genetic markers In addition, the section containing the GLC1A gene is identified by the marker D1S366 on chromosome 1q. 5 and D1S3664 could be localized to the 3 cM region. Marker hap Further examination of rotypes indicates that each of the three glaucoma families The GLC1A gene was found to share the D allele of the marker. Must be in a narrower section, limited by 1S1619 and D1S3664 Suggested.   Several genes known to be in the GLC1A region of chromosome 1 It is considered a candidate for a gene that causes the disease. Three genes (LAMC1 (H. C. Watkins et al., (1993) Hum. Mol. Genet. 2: 1084), NPRI (DG. Lowe et al. (1990) Genomics 8: 304) and CNR2 (S. Munro et al. (1993) N. ature 365: 61) is a candidate by genetic linkage analysis using polymorphic markers within the gene. Excluded from the area. 5 additional YAC STS content mappings A novel candidate gene was determined to be within the recombination interval: Selectin E (MP Be (1989) Science 243: 1160) (GenBank accession number M24). 736); Selectin L (TF Tedder et al., (1989) J. Exp. Med. 170: 123) (G enBank accession number M25280); TXGP-1 (S. Miura et al., (1991) Mol. Ce. ll Biol. 11: 1313) (GenBank Accession No. MD90224); APT1L G1 (T. Takahashi et al., (1994) Int. Immunol. 6, 1567): and TIGR (Tr abecular meshwork Induced Glucocorticoid Response Protein) Corticoid-reactive protein) (J. R. Polansky et al. (1989) Prog. Cli n. Biol. Res 12: 113; (1995) J. Escribano et al. Biochem. 118: 921; International Patent Application Publication No. WO 96/14411) (GenBank Accession Number R954) 91, R95447, R95443, R47209). However, these genetics Two of the offspring (selectin E and selectin L) are shared in this approach It was found to be outside the haplotype interval. The remaining genes (AP T1LG1, TXGP-1, and TIGR) contain YACSTS content and release The most limited JOAG section by both Turned out to exist.   Two of these genes (APT1LG1 and TIGR) were Were screened for mutations in families with Primer should be From the column (T. Takahashi et al., (1994) Int. Immunol. 6, 1567; J. Escriban o et al. Biochem. 118: 921; International Patent Application Publication No. WO 96/144 11) (GenBank accession numbers R95491, R95447, R95443, R47209), single-stranded structural polymorphism analysis (BJ Bassam) Et al., (1991) Anal. Biochem. 196: 80) and direct sequencing of DNA Judged. Complete cDNA sequences of APT1LG1 and TIGR genes have been published However, the presence of intervening sequences is not Only 85-90% of the code sequence was made possible. This APT1LG Eight unrelated JOAG patients were screened by one assay and sequence alterations were detected. No differences were identified.   Affected individuals from a glaucoma family initially linked to four different 1q Screen affected four smaller families linked by data from To do so, TIGR gene analysis was used. In four out of eight families , An amino acid altering mutation was detected. Tyrosine at codon 437 The original family whose mutation to histidine (FIGS. 3A and 4) is linked to 1q (VC Sheffield et al. (1993) Nature enet . 4:47). Mutation of glycine to valine at codon 364 (FIG. 3B) But two families, including one family of unreported adult open-angle glaucoma with 15 affected individuals (FIG. 4). The codon at codon 368 (FIGS. 3B and 5) Nonsense mutations (to glutamine or termination) were detected in two families. The latter mutation is expected to result in cleavage of the gene product.   Then, in two PCR amplimers accommodating these three mutations The prevalence of the mutation was estimated by screening four different populations: Glaucoma patients with a family history of the disease; unselected primary in one clinic Open-angle glaucoma proband; (hereditary retinal disease from a family participating in a previous linkage study) General population (mocked by affected patient and spouse); and normal intraocular Unrelated volunteers over 40 who are under pressure and have no personal or family history of glaucoma A. Determine the sequence of the PCR product that has been determined by SSCP to contain a sequence mutation. Normal chromosomes in unaffected and unaffected individuals, and in affected individuals And the resulting sequence. Overall, a sudden change in missense or nonsense Differences are found in about 3-5% of unrelated glaucoma patients and in about 0.2% of controls. Was. Chi-square test showed that this difference was significant (p <0.001). table 1 indicates the GLC1A mutation and the polymorphism identified to date.   In summary, genetic linkage analysis and testing for shared haplotypes have shown that Obstacle section was able to be shortened. Genes in this section, the ciliary body (J . (1995) J. Escribano et al. Biochem 118: 921) and trabecular meshwork (J. R. Polansky) (1989) Prog. Clin. Biol. Res 12: 113, and International Patent Application Publication Number W O96 / 14411) Was. 13 individuals each carrying one of three different amino acid mutations Unrelated glaucoma patients (of the family whose glaucoma phenotype was originally linked to 1q Identification including the proband (VC Scheffield et al., (1993) Nature Genet. 4:47) Was done. Overall, mutations in this gene were previously associated with chromosome 1q. Communicating The cause of the attached glaucoma is notable evidence. In addition, Mutations were found in 15 affected members of a family affected by humans. In addition, an ongoing selection of unselected open-angle glaucoma patients A mutation was identified in 2.9%, indicating that this gene was associated with all open-angle glaucoma. It suggests that it plays a part. For two reasons: More than 3% of angle-retarded glaucoma is ultimately associated with mutations in this gene May be shown: 1) In this study, only some of the genes Screened; 2) The screening method used is not 100% sensitive Absent.   Genes whose mutations have been linked to the glaucoma phenotype can be And sequenced. 5 'and 3' terminal translation regions and two intron sequences This DNA sequence is shown in FIG. Importantly, this array International Patent Application Publication No. WO 96/14411) (GenBank Accession Number R9 5491, R95447, R95443, and R947209) It is substantially different from the GR gene sequence. In fact, as reported, The TIGR gene sequence does not encode a functional protein.   Summary of differences between the GLC1A and TIGR genes disclosed herein The results are shown in Table 2.  Identification of the gene for the disease will increase our understanding of the pathophysiology of glaucoma, which, in turn, Modulates the functional or mutant biological activity of a TIGR gene or protein Allow the development of analytical methods for the identification of molecules that act (eg, actuate or offset). Make it easier. Therapeutically effective amounts of these molecules are either glaucoma or glaucoma. To prevent or reduce the severity of the situation to potential examiners It is possible to administer. 4. 2 Definition   For the sake of convenience, the terms and words used in this description, the examples and the appended claims The meanings of the phrases are listed below.   As used herein, the term “agonist” refers to a GLC1A bioactivity, either directly or during Refers to an agent that directly enhances, supplements, or enhances (eg, a GLC1A therapeutic).   As used herein, the term "antibody" refers to GLC1A bioactivity either directly or Indirectly prevent, minimize or suppress agents (eg, GLC1A therapeutics) Point.   “Cells”, “host cells” or “recombinant host cells” are interchangeable here This is a term used in general. Such terms include not only the subject cell, Not to mention the progeny or potential progeny of such cells . Certain modifications may occur in subsequent generations due to mutations or environmental effects. Yes, such progeny may not actually be identical to maternal cells, but used here Are still included in the scope of such terms.   A "chimeric protein" or "fusion protein" is one of the subject polypeptides With the first amino acid sequence encoding Define regions that are heterogeneous and not substantially homogeneous (eg, polypeptide portions) And a fusion with the second amino acid sequence. Chimeric proteins are the first Present in organisms that also express proteins (although they may be in different proteins) B) it may represent a heterogeneous region, or a protein expressed by a different species of organism. There are also fusions such as “species” and “genes” of protein structure. Generally, fusion tamper The quality can be represented by the general formula: X-GLC1A-Y. Where GLC1A Represents a portion of the protein derived from one of the GLC1A proteins, X and Y independently include non-existent or naturally occurring mutants Amino acid sequence not related to one of the GLC1A sequences in the living organism.   As used herein, a "complementary sequence" is sufficient to hybridize to form a stable complex. A sequence having an incomplete complementarity.   As used herein, a “delivery complex” refers to a targeting means (eg, a gene, Higher affinity binding of protein, polypeptide or peptide to the target cell surface And / or molecules that enhance cellular uptake by target cells). Shall taste. Targeting means include sterols (eg, cholesterol), Pido (eg, cationic lipids, virosomes or liposomes), viruses ( Eg adenovirus, adeno-associated virus and retrovirus) or target Cell specific binding agents (eg, coordination recognized by target cell specific receptors) Children) are included. Preferred conjugates are sufficiently stable in vivo to target To prevent significant uncoupling prior to internalization. Only However, the complex is cleavable under compatible conditions within the cell and has a functional form Of the gene, protein, polypeptide or peptide.   As is well known, the genes for a particular polypeptide may be single or Exists as multiple copies. These replication genes can be the same, All encoding polypeptides having substantially the same activity, It may have certain modifications, including substitutions, additions or deletions of leotide. Therefore The term "DNA sequence encoding a gg polypeptide" refers to a particular single body. Refers to one or more genes. In addition, nucleotide sequences have Certain differences, called genes, may exist between each organism. This rivalry Genetic differences can also result in differences in the amino acid sequences of the encoded polypeptides. May not occur, but still have a protein with the same bioactivity. No.   As used herein, the term "gene" or "recombinant gene" refers to the structural sequence and ( (Optionally) an open encoding one of the polypeptides of the invention, including both intervening sequences A nucleic acid molecule having an open reading frame. "Recombinant gene" is a GLC1A polypeptide Refers to a nucleic acid encoding a chromosomal GLC1A gene or It may optionally contain intervening sequences derived from related chromosomal genes, Includes GLC1A coding structure array. Encodes a subject GLC1A polypeptide Typical recombinant genes are shown in the attached "Sequence Listing". Term “intervening” "Sequence" refers to the DNA sequence present in a given GLC1A gene, Are not translated and generally exist between the structural sequences.   "Homogeneity" or "identity" or "similarity" refers to the difference between two peptides or two A sequence similarity between two nucleic acid molecules. Homogeneity is measured within each sequence for comparison purposes. It can be determined by comparing the positions. The same base or amino acid at the position in the compared sequence When occupied by an acid, the molecule is homogeneous at that position. Array The degree of homogeneity between them is the It is a function of numbers. An "uncorrelated" or "heterogeneous" sequence is a GLC1A sequence of the invention. For one of the columns, preferably less than 25% identity, but less than 40% identity Share oneness.   As used herein, the term “interaction” is detected, for example, using a yeast two-complex assay. It is meant to include detectable interactions between molecules as possible. Interaction The term is also meant to include "binding" interactions between molecules. For example, mutual The effect may be protein-protein or protein-nucleic acid in nature.   "Isolated" as used herein with respect to nucleic acids such as DNA or RNA The term is derived from the group of DNA or RNA, each of which is present in the natural source of the macromolecule. Refers to separated molecules. For example, encoding one of the subject GLC1A polypeptides The isolated nucleic acid is directly and naturally linked to the GLC1A gene in genomic DNA. Adjacent nucleic acid sequences are reduced to only 10 kilobases (kb), more preferably Adjacent sequences that are naturally present are only 5 kb, more preferably Only 1 adjacent sequence exists. Contains 5 kb or less. Also used here isolated The term `` instrumentation '' refers to cellular matter, virus when produced by recombinant DNA technology. Or a culture medium, or, if chemically synthesized, a chemical precursor. Or a nucleic acid or peptide substantially free of other chemical agents. further, An "isolated nucleic acid" is not naturally occurring as a fragment, and is It is meant to include nucleic acid fragments that cannot be found in the form. Also "Isolated The term "isolated" also refers to polypeptides isolated from other types of cellular proteins. Including those that include both purified and recombinant polypeptides You.   As used herein, the term "adjustment" is used to refer to, for example, activation enhancement (i.e., Or stimulation), or low regulation by, for example, antagonism or GLC1A bioactivity. Refers to both reduction (ie, inhibition or suppression).   The "non-human animals" of the present invention include rodents, non-human primates, sheep, dogs, cattle Such as mammals, chickens, amphoteric webs, reptiles and the like. Of the genus Xenopus Supergenous amphoteric webs such as members and supergenetic chickens are also Can provide an important role in understanding and identifying agents that can affect formation Preferred non-human animals are rats and mice, most preferably mice including mice. Select from the system. The term "chimeric animal" refers herein to the presence of a recombinant gene. Or the recombinant gene was expressed in some but not all cells of the animal I refer to animals. The term "tissue-specific chimeric animal" refers to one of the recombinant GLC1A genes. One is present and / or expressed or divided in one tissue, but not in another Indicates no.   As used herein, the term “nucleic acid” refers to deoxyribonucleic acid (DNA), and In some cases, it refers to a polynucleotide such as ribonucleic acid (RNA). Also this term Is the equivalent of RNA or DNA made from nucleotide analogs as equivalents. Analogs, and single-stranded (sense or sense) as applicable to the described embodiment It should be understood that antisense) and duplex polynucleotides are also included.   As used herein, the term “facilitator” refers to a selection operably linked to the facilitator. Regulates the expression of the selected DNA sequence, and regulates the expression of this selected DNA sequence in cells. Means the DNA sequence that affects. This term refers to a “tissue-specific” promoter, The expression of the selected DNA sequence is restricted to a specific cell (eg, a cell of a specific tissue). And promoters that affect expression in E. coli. This term also applies to the selected D Regulates NA expression mainly in one kind of tissue, but also causes expression in other kinds of tissues Also includes so-called "leaky" facilitators. The term also includes non-tissue specific promoters. And are constitutively expressed or inducible (ie, the expression level is Facilitators).   The terms "protein", "polypeptide" and "peptide" are used to describe gene products. Are used interchangeably here.   The term “recombinant protein” is used to refer to the polypeptide of the present invention produced by recombinant DNA technology. Peptide, which in the art generally encodes a GLC1A polypeptide DNA to be inserted into a compatible expression vector, which is then used to transform host cells. Transform to produce a heterogeneous protein. Furthermore, the recombinant GLC1A gene The phrase "derived from" with respect to "recombinant protein" These proteins having the amino acid sequence of the natural GLC1A protein, Or replacement and deletion (including truncation) of the naturally occurring form of this protein These proteins having an amino acid sequence similar to that generated by a mutation containing Is also included.   As used herein, “small molecule” refers to a molecule of 5 kD or less, most preferably about 4 kD or less. A composition having a molecular weight. Small molecules are nucleic acids, peptides, polypeptides, peptide systems, Can be a carbohydrate, lipid or other carbon-containing or inorganic molecule You. Chemical or biological (eg, fungal, bacterial or algal extracts) An extensive library of compounds is available for screening with the assays of the present invention. .   As used herein, "specifically hybridize" or "specifically detect" The term binds, preferably at least about 6,12,2 of the GLC1A gene. 0, 30, 50, 100, 150, 200, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 13 00, 1350, 1400, 1450, 1460, 1470, 1480, 149 The ability of a nucleic acid molecule of the invention to hybridize to 0 consecutive nucleotides is referred to as GLC1A gene is CLC designated as one of SEQ ID No. 1 or 2. 1A sequence or a sequence complementary thereto or a naturally occurring mutant thereof This is a protein other than the binding GLC1A protein defined herein. Less than the cell nucleic acid encoding (eg, mRNA or genomic DNA) Both 10-fold hybridisation, preferably at least 50-fold hybridisation, even more preferably Indicates at least 100-fold hybridization.   "Transcriptional regulatory sequences" are used throughout this specification to describe initiation signals, enhancers and promoters. A generic term used to refer to a DNA sequence, such as a child, which DNA sequence Trigger or control the transcription of an operably linked protein coding sequence. Good In a preferred embodiment, transcription of one of the recombinant GLC1A genes is intended for expression. A promoter sequence (or other species) that controls the expression of the recombinant gene in the cell type Turn (A photoregulatory sequence). The recombinant gene is a GLC1A protein. Transcriptional regulatory sequences that are the same or different from sequences that control transcription in the naturally occurring form of It will also be appreciated that it is under column control.   The term "transfer" as used herein refers to a nucleic acid by nucleic acid-mediated gene transfer, for example, transfer. Means the introduction of the current vector into the recipient cell.   As used herein, "transformation" refers to the cellular uptake of exogenous damaged DNA or RNA. The process by which a cell's genotype changes as a result of its transformation and, for example, its transformation The isolated cells expressing a recombinant form of the mammalian GLC1A polypeptide; Or naturally occurring form of the GLC1A protein in the case of anti-from a transferred gene It refers to the process by which body expression divides.   As used herein, the term "transformation" refers to the transgenic animal into which it is introduced, or Nucleic acid sequences that are partially or wholly heterogeneous, ie, heterogeneous to cells (eg, For example, it encodes or encodes one of the mammalian GLC1A polypeptides. Antisense transcript) or the transforming gene into which it is introduced. Homologous to the endogenous gene of the product or cell, but the genome of the cell into which it is inserted Methods that change the position of the gene (e.g., Or its insertion results in a knockout). Means the designed or inserted nucleic acid sequence. For transformation, the selected nucleic acid One or more transcriptional regulatory sequences and intervening sequences may be necessary for optimal expression. Any arbitrary nucleic acid can be included.   A "transgenic animal" is any animal, preferably a non-human mammal, bird or both. One or more cells of a reticulate animal are well known in the art. Contains heterogeneous nucleic acids introduced by human intervention such as transformation techniques. micro Cell precursors by careful genetic manipulation, such as injection or infection by recombinant virus The nucleic acid is introduced directly or indirectly into the cell by the introduction into the cell. This gene The term manipulation does not include classical cross-breeding or in vitro fertilization, The introduction of DNA molecules. This molecule may be integrated into the chromosome or stained It may be a DNA that replicates outside the body. In the representative transgenic animals described here, this form Transformation gives cells a GLC1A protein, such as, for example, an agonistic or antagonistic form. One recombinant form of the protein is expressed. However, transgenic animals in which the recombinant GLC1A is silent are also expected, Are FLP or CRE recombinase dependent constructs described below, for example. You. “Transformed” animals also include one or more species of the GLC1A gene. Gene division is caused by human intervention, including both recombination and antisense technology The transgenic animals obtained are also included.   The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. . One type of preferred vector is episomal, i.e., capable of extrachromosomal replication. Nucleic acids. Preferred vectors are those that autonomously replicate the nucleic acid linked thereto and And / or capable of expression. The expression of a gene operably linked to it Vectors that can be directed are referred to herein as "expression vectors". Generally, recombinant DNA Expression vectors useful in the art are commonly referred to as circular double stranded DNA loops. In the form of “mid”, which is associated with the chromosome in its vector form. I haven't. As used herein, "plasmid" and "vector" are Since Smid is the most commonly used form of vector, it is used interchangeably. It is. However, the present invention performs equivalent functions and is well known in the art. It is intended to include other forms of expression vectors.   4. 3. Nucleic acid of the present invention   As described below, one aspect of the invention relates to a GLC1A polypeptide. An isolated nucleic acid having a coding nucleotide sequence, and / or such a nucleus Related to the acid equivalent. The term equivalent is a functionally equivalent GLC1A Activity of a polypeptide or binding GLC1A protein as described herein Including a nucleotide sequence encoding a functionally equivalent peptide having Understood. The equivalent nucleotide sequence contains only one, such as an allelic variant. Or sequences that differ due to substitution, addition or deletion of multiple nucleotides Therefore, due to the degeneracy of the genetic code, the GL shown in SEQ ID No. 1 or 2 Also included are sequences that differ from the nucleotide sequence of the C1A gene.   A preferred nucleic acid is a binding GLC1A nucleic acid. Particularly preferred GLC binding 1A nucleic acids are mammalian. Particularly preferred GLC1A nucleus, regardless of species The acid is a polypeptide that is at least 90% similar to the amino acid sequence of human GLC1A Is encoded. Preferred nucleic acids are, for example, one of SEQ ID No: 1 or 2. At least for the binding amino acid sequence of GLC1A, such as the sequence shown in GLC1A having an amino acid sequence of 90% homogeneity, more preferably 94% homogeneity Encode the polypeptide. Amino displayed in SEQ ID No. 1 or 2 At least about 95%, more preferably at least about 98-99, based on the acid sequence. Nucleic acids encoding polypeptides with a% similarity are also within the scope of the invention. Special In a particularly preferred embodiment, the nucleic acid of the invention comprises one of SEQ ID NO: 2. The amino acid GLC1A sequence shown in FIG. In one embodiment, The nucleic acid has at least one bioactivity of the subject GLC1A polypeptide. It is a cDNA that encodes a peptide possessed. Preferably, the nucleic acid is SEQ. The entire nucleotide sequence corresponding to the coding region of ID No. 1 or 2 or Includes some.   Yet another preferred nucleic acid of the invention is the amino acid residue of SEQ ID No: 2, For example, at least 2, 5, 10, 25, 50, 100, 150 or GL comprising a polypeptide sequence corresponding to all or part of 200 amino acid residues Encodes a C1A polypeptide. For example, probe / primer or anti Preferred for use as a sense molecule (ie, not encoding a nucleic acid molecule) Nucleic acid molecules are at least about 6, 12, 20, 30, 50, 100, 12 It can contain 5,150 or 200 base pairs, and the encoded nucleic acid molecule is about 200,2 50, 300, 350, 400, 410, 420, 430, 435 or 440 Can contain base pairs.   Another aspect of the invention relates to a nucleus represented by one of SEQ ID Nos: 1 or 2. Provided are nucleic acids that hybridize to acids. Compliant stringency conditions, for example, at about 45 ° C 6. DNA hybridization with 0x sodium chloride / sodium citrate (SSC) At 50 ° C. Cleaning of 0xSSC requires engineers in this field Is known, "Modern Protocols in Molecular Biology", John W. iley & Sons, N.M. Y. (1989), 6. 3. 1-6. 3. Look at 6 Can be For example, the salt concentration in the washing step is about 2. 0xSSC low From rigor to about 0. You can choose from up to 2 × SSC strictness. Addition Alternatively, the temperature of the washing step may be from low stringency conditions at room temperature, about 22 ° C to high at about 65 ° C. Can be increased to stringency conditions. Both temperature and salt concentration may be varied, or temperature or One of the salt concentrations may be kept constant and the other variable may be varied. In a preferred embodiment Thus, the GLC1A nucleic acids of the invention can be used under moderately stringent conditions, for example, about 2. 0xSSC And at about 40 ° C., to one of SEQ ID No: 1 or 2. Especially preferred In a preferred embodiment, the GLC1A nucleic acids of the invention are SEQ. Binds to one of ID Nos: 1, 3 or 4, but has SEQ ID Nos: 6, 8 , 9 or 11 does not bind.   Preferred nucleic acids are amino acid sequences of mammalian GLC1A, eg, SEQ ID   No: at least 75% homology to sequences as shown in one of 1 and 2 , More preferably 80% and even more preferably at least 85% homologous sequences Have. SEQ ID No: 1 or 2 At least 90%, more preferably 95%, and most preferably at least about 98%. Nucleic acids of -99% homogeneity are also naturally within the scope of the invention. Preferred In a preferred embodiment, the nucleic acid is a mammalian GLC1A gene, In a preferred embodiment, one code of SEQ ID No: 1 or 2 All or a part of the nucleotide sequence corresponding to the functionalized region.   Due to the degeneracy of the genetic code, one of SEQ ID Nos. Nucleic acids having a sequence that differs from the nucleotide sequence are also within the scope of the invention. like this A suitable nucleic acid is a functionally equivalent peptide (ie, the biological activity of a GLC1A polypeptide). Peptide), but due to the degeneracy of the genetic code, The sequence differs from the sequence shown. For example, the number of amino acids may be There is a designation. A codon specifying the same amino acid, or a synonym (eg, C AU and CAC each encode histidine) is a GLC1A polypeptide It can result in "silent" mutations that do not affect the amino acid sequence of the tide. However, DNA sequences which lead to variations in the amino acid sequence of the subject GLC1A polypeptides Polymorphisms are expected to exist among mammals. Someone with the technology in this field , Nucleic acids encoding polypeptides having mammalian GLC1A polypeptide activity For one or more nucleotides (eg, up to about 3-5% of the nucleotides) Where such variations exist between individuals of a given species due to natural allelic variations. You will understand that there are cases.   As exemplified by the examples below, the GLC1A protein-encoding nucleus Acids can be obtained from mRNA present in any of many eukaryotic cells. Also Nucleic acids encoding a mammalian GLC1A polypeptide of the present invention can be used in adult and fetal It is certainly possible to obtain from both the genomic DNA of the baby. For example, GLC1A Genes encoding proteins are described in the protocols described herein as well as in the art. The skilled artisan knows the cDNA or genome according to commonly known protocols You can clone from any of the libraries. Tissues suitable for isolation of subject nucleic acids Examples of and / or libraries are, inter alia, visual tissues. GLC1 The cDNA encoding the A protein can be used in binding cells, including fetal cells, It can be obtained by isolating total mRNA from cells such as natural cells or human cells. Double strand cDNA can then be prepared from the total mRNA, and can be prepared using any one of many well-known techniques. Followed by insertion into an appropriate plasmid or bacteriophage vector. I do. Also, the gene encoding the mammalian GLC1A protein is Established polymerase chain reaction based on nucleotide sequence information provided It can also be cloned using technology. The nucleic acid of the present invention may be DNA or RNA . Preferred nucleic acids are selected from the group consisting of SEQ ID No: 1 or 2. CDNA represented by the sequence shown.   4. 3. 1.   vector   The invention also provides a GL that is operably linked to at least one transcription regulatory sequence. An expression vector comprising a nucleic acid encoding a C1A polypeptide is provided. "Operational Linked to a nucleic acid sequence enables the expression of a nucleotide sequence into a regulatory sequence. It is meant to be connected so that Regulatory sequences are And control the expression of the subject mammalian GLC1A protein. Obedience Thus, the term “transcription regulatory sequence” refers to facilitators, enhancers and other expression control elements. Is included. Such regulatory sequences are described in Goeddel's “Gene Expression Technology”. A: Method 185 "in Enzymology, Academic Press, San D iego, CA (1990). In one embodiment, the expression vector Encodes a peptide having an operative activity of a subject GLC1A polypeptide. Or encodes a peptide that is an antagonistic form of the GLC1A protein Includes recombinant genes. Such expression vectors can be transferred to cells and thereby Polypeptides containing fusion proteins encoded by nucleic acids as described herein Can be used to make tides. Moreover, the gene constructs of the present invention may be used in gene therapy As part of the protocol, one of the active or one of the subject GLC1A proteins It can also be used to deliver nucleic acids encoding any of the antagonistic forms. Therefore Another aspect of the invention is to reconstitute the function of GLC1A-induced signaling in tissue. Or inhibits production of a GLC1A polypeptide of a particular cell type. It features an expression vector for transfer and expression in vivo and in vitro. For example, when a naturally occurring form of a protein is mis-expressed, or This would be desirable to deliver a form of the protein that alters differentiation. Ma The expression vector can also be applied to inhibit neoplastic transformation.   In addition to the viral transfer method as exemplified above, May also cause expression of a subject GLC1A polypeptide in animal tissues. it can. Many nonviral methods of gene transfer rely on macromolecule uptake and intracellular It depends on the canonical mechanism used by mammalian cells for transport. Preferred embodiment In one embodiment, the non-viral targeting method of the present invention comprises For uptake of the GLC1A polypeptide gene, we rely on the pathway by plasma membrane invagination. You. Typical targeting tools of this type include liposomal derivatization systems, poly-lysine Conjugates and artificial viral coats are included.   4. 3. 2.   Probes and primers   In addition, nucleotides determined from the cloning of the mammalian GLC1A gene The sequence may be a GLC1A analog in another cell type, such as from another tissue, And identifying and / or cloning GLC1A analogs from other mammals Enables the generation of probes and primers intended for use in . For example, the present invention provides probes / probes comprising substantially pure oligonucleotides. Also provided is a primer, which oligonucleotide has SEQ ID No: 1 and Or a sense or antisense sequence selected from the group consisting of At least about 12, preferably 25, more preferably 40 Hybridizes under harsh conditions to 50, or 75 contiguous nucleotides Includes regions of nucleotide sequence. For example, as shown in SEQ ID Nos. Primers based on the indicated nucleic acids clone GLC1A analogs in a PCR reaction Can be used to A preferred primer of the present invention is SEQ ID No: 4 And 5 and SEQ ID Nos. 5 and 6.   Similarly, a probe based on the subject GLC1A sequence will It can be used to detect transcripts or genomic sequences encoding proteins. Good In a preferred embodiment, the probe is further linked to and detectable. Labeling groups, such as radioisotopes, fluorescent compounds, It is selected from enzymes and enzyme cofactors.   As will be discussed in more detail below, such a probe is a GLC1A protein. As part of a diagnostic test kit to identify cells or tissues that are mis-expressing Can be used to detect the level of GLC1A-encoding nucleic acid in cell samples from patients. Measurement of GLC1A mRNA level, To determine if the murine GLC1A gene has been mutated or deleted . Briefly, the nucleotide probe is linked to the presence of the GLC1A-encoding transcript. (Or absent) histological screen of intact tissues and tissue samples It can be generated from the subject GLC1A gene, which facilitates ligation. Similar to the diagnostic use of anti-GLC1A antibodies, GLC1A messages or genomic Applications of probes directed to the murine GLC1A sequence include, for example, neoplastic or highly plastic Conflicts manifested in sexual diseases (eg, unnecessary cell growth) or abnormal differentiation of tissues It can be used for both predictive and therapeutic evaluation of genetic mutations. Described here When used in conjunction with an immunoassay that performs Involvement of certain abnormalities linked to the expression (or lack thereof) of the LC1A protein It can help facilitate the determination of molecular criteria for a developing disease that will occur. For example, a mutation in polypeptide synthesis can be a mutation in the coding sequence. Can be distinguished.   4. 3. 3. Antisense, ribozyme and triplex technology   One aspect of the invention relates to the use of isolated nucleic acids in "antisense" therapy. I do. As used herein, “antisense” therapy refers to one of the subject GLC1A proteins. Cellular mRNA and / or Genomic DNA Encoding Species or Plural Cells Specifically hybridize (e.g., bind) under statistical conditions, e.g., transcription and / or Or an oligo that inhibits the expression of the protein by inhibiting translation. It refers to the administration or in situ generation of nucleotide molecules or derivatives thereof. This result If they are due to normal base pair complementation or, for example, DNA duplex Via specific interaction in the main groove of the double helix in the case of binding to May be. In general, “antisense” therapy is an area of technology commonly used in the field. Includes any therapy that relies on specific binding to an oligonucleotide sequence .   The antisense constructs of the present invention, for example, when transcribed into cells, R that is complementary to at least a unique portion of the cellular mRNA encoding the protein It can be delivered as an expression plasmid that produces NA. Or this anti The sense construct is an in vitro generated oligonucleotide probe, Is the mRNA and / or genomic sequence of the GLC1A gene when introduced into cells. Hybridization with the rows causes inhibition of expression. Oligonuk like this Reotide probes are exonucleases and / or endonucleases. Repairs that are antagonistic to endogenous nucleases such as Decorated oligonucleotides are preferred. Use as antisense oligonucleotide Exemplary nucleic acid molecules include phosphoramidates, phosphothioates and methylated DNA. It is an analog of lephosphonate. (Also see U.S.A. S Patent 5,176,996; 5,2 64,564; and 5,256,775). In addition, General techniques for constructing oligomers useful for transduction therapy are described, for example, in Vander K. (1988) Biotechniques 6: 958-976. And Stein et al. (1988) Cancer Res. 48: 2659 -2668. For antisense DNA, translation initiation site For example, between the -10 and +10 regions of the subject GLC1A nucleotide sequence. Derived oligodeoxyribonucleotides are preferred.   Antisense approaches include oligonucleotides that are complementary to GLC1A mRNA ( It involves the design of either DNA or RNA. Antisense oligonucleotides are Binds to GLC1A mRNA transcript and inhibits translation. Absolute complementarity is preferred Is not required. The sequence "complementary" to a portion of the RNA referred to herein is its RN A that has sufficient complementarity to hybridize with A and forms a stable duplex Means double-stranded, in the case of a double-stranded antisense nucleic acid, the single strand of the duplex DNA is Can be tested or triplex formation can be assayed. Hybridization performance is complementary It will depend on both the degree of sex and the length of the antisense nucleic acid. Generally, hybrid formation As nucleic acids become longer, base mismatches with RNA increase, but this is not Contain (and sometimes form a triplex) and still form it . Those skilled in the art will be able to fuse complexed complexes using standard procedures. By measuring the points, an acceptable degree of mismatch can be determined.   Oligonucleotides complementary to the 5 'end of this message, such as AUG start Up to 5 'untranslated sequences, including codons, are most effective at inhibiting translation I do. However, 3 'untranslated sequences of mRNAs also inhibit translation of mRNAs. Have recently been shown to be effective (Wagner, R.M. 19 94 Nature 372: 333). Therefore, 5 'of the GLC1A gene or Is an oligonucleotide complementary to any of the 3 'untranslated, non-coding regions Can be used in antisense techniques to inhibit the translation of endogenous GLC1A mRNA Become. Oligonucleotides complementary to the 5 'untranslated region of the mRNA are AUG-opened. Must contain the complement of the start codon. Anti-sense complementary to mRNA coding region S-oligonucleotides are less effective as translation inhibitors, but in accordance with the invention Can be used. Hybrid forms in the 5 ', 3' or coding region of GLC1A mRNA If designed, the antisense nucleic acid should have at least six nucleotides in length. Should preferably be 6 to about 50 oligonucleotides in length. You. In some embodiments, the oligonucleotide comprises at least 10 nucleotides. At least 17 nucleotides, at least 25 nucleotides or fewer At least 50 nucleotides.   Regardless of the choice of target sequence, an in vitro study is first performed to To quantify the ability to inhibit nucleotide gene expression, this antisense Preferably, the performance of the lignonucleotide is expressed quantitatively. In these studies, oligos A control that distinguishes between antisense gene inhibition of nucleotides and nonspecific biological effects It is preferable to use it. In these studies, the target RNA or protein It is also preferred to compare the level with an internal reference RNA or protein. in addition The results obtained using this antisense oligonucleotide were compared with the control oligonucleotide. Plan to compare with results obtained using leotide. Control oligonucleotide The oligonucleotide is approximately the same length as the oligonucleotide being tested. The nucleotide sequence of the nucleotide is specific for the target sequence relative to the antisense sequence. It is preferred that there is no more than the difference necessary to prevent seed formation.   The oligonucleotide may be DNA, RNA, a chimeric mixture, derivative, or Modified versions thereof may be single-stranded or double-stranded. Oligonucleotide , Base moiety, sugar moiety or phosphate skeleton to stabilize molecules and hybrids Performance can be improved. Oligonucleotides are peptides (eg, host cells in vivo). (For targeting vesicular receptors), agents that facilitate transport across cell membranes (Le tsinger et al., 1989, Proc. Natl. Acad. Sci. U. S . A. 86: 6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84: 648-652; PCT Publication No. WO8 8/09810, 1988. Published on December 15) or blood-brain barrier (for example, P CT Publication No. WO 89/10134, 1988. See announcement on April 25 ) Hybridization-Triggered Cleavage Agents (eg, Krol et al., 1988, BioTechni) ques6: 958-976) or intercalating agents (eg, Zon, 1 988, Pharm. Res. 5: 539-549), etc. Species added groups can be included. Oligonucleotides are separate molecules for this purpose , Eg, peptides, hybridization-crosslinking agents, transport G -Agents, hybrid formation-bound to trigger cleavable agents, etc.   The antisense oligonucleotide comprises at least one modified base moiety, This includes, but is not limited to, 5-fluorouracil, 5-bromouracil, Lolouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetone Tylcytosine, 5- (carboxyhydroxymethyl) uracil, 5-carboxy Methylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyl Lacil, dihydrouracil, β-D-galactosyl eosin, inosine, N6 -Isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2 -Dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcysteine Tosin, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methyl L-aminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β- D-mannosyl eosin, 5'-methoxycarboxymethyluracil, 5-me Toxiuracil, 2-methylthio-N6-isopentenyladenine, uracil- 5-oxyacetic acid (v), wybutoxocin, pseudouracil, queosin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thio Uracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, Uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3- (3- Amino-3-N-2-carboxypropyl) uracil, (acp3) w and 2 , 6-diaminopurine.   Antisense oligonucleotides include, but are not limited to, arabinose, Select from group containing fluoroarabinose, xylulose and hexose At least one modified sugar moiety.   In yet another embodiment, the antisense oligonucleotide comprises a host Holothioate, phosphorodithioate, phosphoramidothioate, phosphoramido Dirt, Phosphoradiamidate, Methylphosphonate, Alkylphosphotries And a group selected from the group comprising formacetal or analogs thereof. Contains at least one modified phosphate backbone.   In yet another embodiment, the antisense oligonucleotide is an anolyte. It is a mer oligonucleotide. The anomeric oligonucleotide is complementary RN A forms a specific double-stranded hybrid with A, where unlike normal units, the chains Running in parallel (Gautier et al., 1987, Nucl. Acids Re s. 15: 6625-6641). The oligonucleotide is 2'-O-methyl Ribonucleotides (Inoue et al., 1987, Nucl. Acids Res . 15: 6131-6148) or chimeric RNA-DNA analogs (Ino 1987, FEBS Lett. 215: 327-330).   The oligonucleotide of the present invention is an automated DNA synthesizer (Biosear-ch). , Available from the Applied Biosystem, etc.). And can be synthesized by standard methods well known in the art. For example, phosphorothioers Oligonucleotides can be prepared by the method of Stein et al. (1988, Nucl. Aci ds Res. 16: 3209). Trioligonucleotides are controlled porous glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U. S. A. 85: 744 8-7451).   Antisense nucleotides complementary to the GLC1A coding region sequence can also be used. However, those complementary to the transcribed untranslated region are most preferred.   It is necessary to deliver this antisense molecule to cells expressing GLC1A in vivo. There is. Several methods have been developed to deliver antisense DNA or RNA to cells. ing. For example, an antisense molecule can be injected directly into a tissue site. Or Modified antisense molecules designed to target the desired cells A peptide or antibody that specifically binds to the expressed receptor or antigen. Sense) can be administered systematically.   However, sufficient intracellular concentrations of antisense to suppress the translation of endogenous mRNAs It is often difficult to achieve. Therefore, the preferred approach is to use antisense Oligonucleotide is under the control of potent polIII or polII promoter , Utilizing recombinant DNA constructs. Such a configuration for transferring target cells into a patient The single-stranded RN that forms a complementary base pair with the endogenous GLC1A transcript A sufficient amount of As will be transcribed, thereby translating the GLC1A mRNA. To prevent. For example, take up vectors by cells and direct antisense RNA transcription You As described above, a vector can be introduced into this organism. Such vectors can be used as long as they are transcribed to produce the desired antisense RNA. Either somatic retention is possible or becomes chromosomally integrated. This Such vectors are constructed using standard recombinant DNA technology techniques in the field. I can do it. Vectors are plasmids used for replication and expression in mammalian cells. Virus, virus or other species known in the art. Sign antisense RNA The expression of the activating sequence is a function of this fraction acting in mammalian, preferably human, cells. It is performed by promoters well known in the field. Such facilitators remain inducible. Or structural. Such promoters include, but are not limited to, SV40, early facilitator region (Brnoist and Chambon, 1981) Nature 290: 304-310), the 3 'long end of Rous sarcoma virus. Promoters contained in repeats (Yamamoto et al., 1980, Cell 22: 7 87-797), herpes thymidine kinase promoting factor (Wagner et al., 1 981, Proc. Natl. Acad. Sci. U. S. A. 78: 1441 -1445), the regulatory sequence of the metal thionein gene (Brinster et al., 198). 2, Nature 296: 39-42) and the like. Plasmid, cosmid , YACs or viral vectors can be used to prepare recombinant DNA constructs. These can be used to produce tissue sites such as the choroid network or the hypothalamus Can be introduced directly. Alternatively, a virus vector that selectively infects the desired tissue Can also be used (for example, a herpesvirus vector can be used in the brain) In this case, administration is completed by another route (eg, systematically).   Ribozyme molecules designed to catalytically cleave GLC1A mRNA transcripts Can also be used to prevent translation of GLC1A mRNA and expression of GLC1A. (For example, PCT International Publication WO90 / 1136 published on October 4, 1990) 4: Sarver et al., 1990, Science 247: 1222-1225. checking). Ribozymes that cleave mRNA at sites specific to the recognition sequence Can be used to destroy GLC1A mRNAs, but hammer-like (hamm The use of the ribozyme of the present invention is preferred. Hammerhead ribozyme is the target m At the position designated by the region on the side forming complementary base pairs with the RNA, m Cleave the RNA. Target mRNA has a two base sequence: 5'-UG-3 ' That is the only requirement. The composition and yield of hammerhead ribosomes is not The well-known, and more completely, Natl of Haseloff and Gerlach ure, 1988, 334: 585-591. Person GLC1Ac There are hundreds of potential hammerhead-like ribozyme cleavage sites in the nucleotide sequence of DNA. There is also. Preferably, the cleavage recognition site is located near the 5 'end of GLC1A mRNA. To improve efficiency and minimize intracellular deposition of non-functional mRNA transcripts. Ribozymes are engineered to achieve   The ribozymes of the present invention include Tetrahymena thermophila (Known as IVS or L-19IVS RNA) More widely described by Thomas Cech and co-workers (Zang et al., 1984, Sc 224: 574-578; Zang and Cech, 1986, S. science, 231: 470-475; Zang et al., 1986, Nature. 324: 429-433; International Patent Application No. published by the University Patent Association. WO8 8/04300; Been and Cech, 1986, Cell, 47: 207. -216) endoribonuclease (hereinafter referred to as “Cech-Thailand”). Privozyme) is also included. Cech-type ribozymes are capable of opening target RNA. Has eight base-pair active sites that hybridize to the target RNA sequence after cleavage has occurred . The present invention targets eight base pair active site sequences present in GLC1A. These tech-type ribozymes.   As in the antisense approach, ribozymes are modified oligonucleotides (Eg, for improved stabilization, targeting, etc.) Deliver to GLC1A-expressing cells in the hypothalamus and / or choroid plexus There is a need. Preferred methods of delivery include strong constitutive pol III or po Use of DNA constructs that "encode" ribozymes under the control of II promoters Involved and transfected cells destroyed the endogenous GLC1A message and translated Yield sufficient ribosomes to inhibit ribosomes. Riboza unlike antisense molecules Because im is catalytic, lower efficiency is required for intracellular concentrations. Again Targeted homologous recombination (eg, Smithes etc, 1985, Nature 317: 230-234; Thomas & Capec. chi, 1987, Cell 51: 503-512; Thompson et al., 1 989, Cell 5: 313-321. Each is in the references here Using the GLC1A gene or its promoter. Activation or "knock-out" can reduce this. For example, sudden change Heterologous, endogenous GLC1A gene (coding region or regulatory region of GLC1A gene) Non-functional GLC1A flanked by DNA of homogeneity to either of the Is a completely unrelated DNA sequence) with a selectable marker and / or a negative selectable marker. Use in presence or absence of Kerr to transfer cells expressing GLC1A in vivo Can be Insertion of the DNA construct via targeted homologous recombination resulted in Inactivation of the GLC1A gene occurs. Such a method is called ES (embryonic Stem) Producing animal progeny with inactive GLC1A using modifications to cells It is particularly suitable in the field of agriculture (for example, Thomas & Capec). chi 1987 and Thompson 1989, supra). But for example Hell for delivery to brain tissue such as the hypothalamus and / or choroidal retina Recombinant DNA using a compatible viral vector, such as a pessvirus vector If the construct is administered or targeted directly to the required site in the body, this approach will Can be adapted for use.   Alternatively, a deoxyribonucleotide sequence complementary to the regulatory region of the GLC1A gene Columns (ie, GLC1A facilitator and / or enhancer) as targets Forming a triple helix structure that prevents transcription of the GLC1A gene in target cells Can reduce endogenous GLC1A gene expression (generally, Helene . C. 1991, Anticancer Drug Des. , 6 (6): 5 69-84; Helene. C. Et al., 1992, Ann. N. Y. Acad. S ci. 660: 27-36; and Maher, L .; J. , 1992, Bio assays 14 (12): 807-15).   Similarly, the anti-sense constructs of the present invention can be used to construct one common GLC1A protein. By antagonizing biological activity, for example, both in vivo and in vitro tissue cultures Can be used for tissue manipulation such as tissue differentiation.   Moreover, anti-sense techniques (eg, injection of anti-sense molecules or transcripts thereof) Transfer of plasmids that are anti-sense with respect to GLC1A mRNA or gene sequence Using GLC1A in developmental events, as well as in adult tissues. The normal cell function of GLC1A can be studied. Such techniques can be used for cell culture. However, it can also be used to create transformed animals as described in detail below.   Ribozymes are enzymatic RNA molecules that can catalyze the specific cleavage of RNA . The mechanism of ribozyme action involves the arrangement of the ribozyme molecule on the complementary target RNA. It involves row-specific hybridization followed by endonucleolytic cleavage. Ribozyme content The composition of the offspring must differ to include one or more sequences complementary to the target gene mRNA. And must contain the well-known catalytic sequence responsible for mRNA cleavage. this For sequences, U.S.A. S. Patent, No. 5 , 093, 246. Thus, within the scope of the present invention, GL Specific for endonucleolytic cleavage of RNA sequence encoding C1A protein A hammerhead-like motif ribozyme molecule that catalyzes and efficiently catalyzes Is started.   A specific ribozyme cleavage site within any potential RNA target has the sequence: GU A, Molecules of interest for ribozyme cleavage sites including GUU and GUC By scanning, it is first identified. Once identified, include the cleavage site. Short RNA of between 15 and 20 ribonucleotides corresponding to the region of the target gene Sequences can be evaluated for predicted structural features, such as secondary structure, which This oligonucleotide sequence could be inappropriate. Of the candidate sequence Compatibility can also be determined by using ribonuclease protection techniques to avoid interference with complementary oligonucleotides. It can be assessed by testing its accessibility to speciation.   The nucleic acid molecule used for triple helix formation to inhibit transcription is preferably a single-stranded And is composed of deoxyribonucleotides. These oligonucleotides The base composition of otide has a triple helix formation via Hoogestin base pairing rules. Must be promoted and triple helix formation is present on one strand of the duplex Generally requires a fairly large stretch of either purines or pyrimidines . The nucleotide sequence may be pyrimidine-based and the resulting triple La This can result in TAT and CGC triplets crossing the three linked strands of the strand. And The molecules of the pyrimidine karchi are placed in a parallel orientation with this strand. The single-stranded purines of the plex provide base complementarity to the region of the rich. Addition Instead, for example, a purine-rich nucleic acid molecule containing expansion and contraction of G residues can be selected. These molecules form a DNA duplex and a triple helix with rich GC pairs. Where the majority of the purine residues are Located on the single-strand, resulting in three strands in the triplex. Produces a matching CGC triplet.   Alternatively, potential sequences that can be targeted for triple helix formation are the so-called "switches". Can be increased by creating back "nucleic acid molecules. Switchback molecules Base pairs with one strand of them or the duplex first and then the other Either purines or pyrimidines to be on one strand of the duplex Alternating 5'-3 ', which removes the requirement for a fairly large stretch of It is synthesized in a 3'-5 'manner.   The anti-sense RNA and DNA N ribozymes and triple helix molecules of the invention comprise D Preparation of NA and RNA by any method known in the art. Can be manufactured. This includes, for example, solid phase phosphoramidite chemical synthesis Well known oligodeoxyribonucleotides and oligoribonucleotides And the technology for chemically synthesizing Or the RNA molecule is an antisense RNA molecule May be generated by in vitro and in vivo transcription of a DNA sequence encoding This DNA sequences such as T7 or SP6 Incorporate into a wide range of vectors incorporating compatible RNA polymerase promoters be able to. Or, constitutively or inducibly, depending on the promoter used. Antisense cDNA constructs that synthesize antisense RNA can be stably introduced into cell lines. You.   Moreover, various well-known modifications to nucleic acid molecules have been shown to improve intracellular stability and semi-cellularity. It could be introduced as a way to increase the lifespan. Possible modifications include, but are not limited to, 5 'of the molecule on the side sequence of the ribonucleotide or deoxyribonucleotide And / or addition to the 3 'end, or phosphorothioate or oligodeoxy. 2'O-methyl not a phosphodiesterase bond in the silribonucleotide backbone Including the use of Four. Four. Polypeptide in the present invention   The present invention makes available isolated GLC1A polypeptides available. Isolated GLC1 A polypeptide is isolated and substantially free of other cellular proteins. May be normally associated with those not present, especially the GLC1A polypeptide. No signaling or transcription factors are substantially absent. "other Cellular protein is virtually absent ”(here“ mixed protein ” Or "substantially pure or purified preparation". Is a GLC1A polypeptide mixture containing about 20% (dry weight) or less of protein. GLC1A polypeptide preparation where preparation and protein contamination of about 5% or less is preferred Defined as a product. For the first time, use cloned genes as described here Then, as a purified preparation, the functional form of the target polypeptide is adjusted. "Purify" This means that when referring to peptide, DNA or RNA sequences, they are like other proteins. The specified molecule exists where the physical macromolecule does not substantially exist Say that The term “purified” is used herein to refer to the same type of biological In the presence of macromolecules (water, buffers and other small molecules, especially those with molecular weights below 5000) At least 80% by dry weight is desirable, preferably 95-99% For at least 99. Use 8% as the most desirable. To say “smart” The term has the same numerical definition here as "purified" described immediately above. Use it with it. "Isolated" and "purified" refer to the state as it is (For example, in acrylamide gel) Does not contain substances. However, pure substances and solutions (for example, no contaminating proteins, Alternatively, chromatographic reagents such as denaturants and polymeric substances, for example, Lilamide and agarose) are not limited to this. The purified GLC1A preparation is Lack of contaminating proteins from the same animal in which the GLC1A is normally produced desirable. For example, human GLC1A protein can be engineered in non-human cells. That is, it is obtained by recombinant expression.   One or two specific motifs or domains, or files of any size Small-sized proteins or fragments are, for example, at least 5,10 , 25, 50, 75, 100, 125, and 150 amino acids are within the scope of the invention.   For example, the isolated GLC1A polypeptide is SEQ ID No. G represented in 3 Contains all or part of the amino acid sequence corresponding to the LCIA polypeptide . For the isolation of the peptidyl portion of the GLC1A protein, such peptides Screen for recombinantly produced peptides from the corresponding nucleic acid fragment encoding It is obtained by performing In addition, the fragments are from the traditional Merrifield solid phase. F-mock or t-bock chemistry . For example, the GLC1A polypeptides in the present invention do not overlap each other May be divided arbitrarily by the desired length and overlap if desired Could be broken into pieces like Generated fragments (recombinantly or chemically Confirm the peptidyl fragment. Peptidyl fragments are wild-type (ie, "Genuine") can function as an agonist or antagonist of GLC1A protein.   Another feature of the present invention relates to a recombinant form of the GLC1A protein. You. In addition to the original GLC1A protein, the recombinant polypeptide used in the present invention The peptide must have at least 92% of the amino acid sequence represented by SEQ ID Nos: 3 For example, 94% most preferably has 95% homology. SEQ ID No. Three The polypeptide is at least a sequence selected from the group consisting of It is within the scope of the invention to have 98-99% homology. GLC in the present invention Preferably, the 1A protein is a GLC1A protein . GLC1A protein is SEQ ID No. Including one of the functional arrays in 3 Especially desirable. The GLC1A protein has the biological activity of GLC1A Especially desirable.   The present invention further relates to a recombinant form of the subject GLC1A polypeptide. You. The subject GLC1A polypeptide is encoded by a gene derived from a mammal, Q ID No. Amino acid sequences that are evolutionarily related to the GLC1A protein shown in 3. Have a column. Such a recombinant GLC1A polypeptide is provided with the attached sequence listing Bioactivity of wild-type ("real") GLC1A protein in Can act as either an agonist or antagonist for one of them . “Evolutionarily related” means that the amino acid sequence of GLC1A protein Polypeptides with naturally occurring amino acid sequences, and combinatorial mutations By reference to both variants of the GLC1A polypeptide are indicated. Book Such evolutionarily related GLC1A polypeptides desired in the invention are GL The amino acid sequence published as SEQ ID No: 3 has C1A biological activity and It has at least 92%, preferably 94%, most preferably 98-99% homology. GLC1A protein is SEQ ID No. Contains three amino acid functional sequences Is particularly desirable.   Generally, it has the activity (having "biological activity") of the GLC1A protein referred to here. Polypeptides are those of SEQ ID No. Of the GLC1A protein shown in 3 A polypeptide containing an amino acid sequence corresponding to all or part of the amino acid sequence All or all of the biological / biochemical activities of the GLC1A protein in its natural state A polypeptide that mimics or antagonizes a portion. Biochemical Activity is associated with gene expression, pituitary development, and umbilical yolk artery expression in the abdomen It is desirable to be involved in the outbreak.   Other biological activities of the subject GLC1A protein are described here. Or will be announced in some specialty. According to the present invention, If the peptide is a specific agonist or antagonist of natural GLC1A protein, Has biological activity.   The invention further relates to a method of making the subject GLC1A polypeptide. For example A nucleic acid vector expressing a nucleotide sequence encoding the polypeptide of interest The host cell into which the protein has been introduced is cultured under appropriate conditions to express the peptide. Fine The vesicles are collected and lysed to isolate the protein. Cell culture involves host cells, culture media, And other by-products. The culture medium suitable for cell culture is technically good Are known. Recombinant GLC1A polypeptides can be prepared by methods well known in the art. To isolate from cultures, host cells, or both. For protein purification, On-exchange chromatography, Kel filtration chromatography, ultrafiltration, Electrophoresis and immunoaffinity purification using antibodies specific for such peptides Made There is. Recombinant GLC1A polypeptide has a domain to facilitate purification. Fusion proteins such as GST fusion proteins and poly (His) fusion proteins Is desirable.   More generally, under certain circumstances, one of the subject GLC1A polypeptides may have Providing molog is considered convenient. Organism of natural protein To promote or inhibit only a subset of the activities, the subject GLC1A poly Peptides are limited to either GLC1A agonistic (mimic) or GLC1A antagonistic Function. Therefore, processing with a homolog that has only limited functions Can elicit more specific biological effects. It is treated with agonists and antagonists. Have fewer side effects than The action of agonists and antagonists is similar to natural GLC1A It is because of all the biological activities of the protein.   Each homologue of the target GLC1A protein has a discontinuous point mutation or truncation. It can be created by mutations such as For example, due to mutation Thus, a homologue having the same biological activity as the original GLC1A polypeptide Creating a homolog as a mere subset of the original GLC1A polypeptide Can be. Another approach is to create antagonistic proteins and use GLC1A protein Natural forms, such as competitive binding upstream or downstream of biochemical pathways There is also a method of inhibiting the function of a protein. In addition, creating action-type proteins, There is also a method of maintaining an activated state. Therefore, provided by the present invention Human GLC1A protein and its homolog are regulated in gene expression. Can work both positively and negatively.   The recombinant GLC1A polypeptide of the present invention is a homolog of the real GLC1A protein. Also includes logs. It can modify, for example, ubiquitin binding or Proteolysis by mutations that alter the target of other associated enzymes Mutations in the protein that make it resistant to gender cleavage.   GLC1A polypeptides can be chemically modified to create GLC1A derivatives . Modifications include glycosyl groups, lipids, phosphates, acetyl groups, etc. By forming a covalent or cohesive bond with a chemical group. Sharing of GLC1A protein The conjugation derivative is a functional group of N-terminal or C-terminal amino acid side chains Conversion It is obtained by crosslinking a chemical group.   Improves therapeutic and preventive effects and improves stability (shelf life and resistance to tampering) To secure or post-translational modifications (changing the phosphorylation pattern of proteins) To this end, the structure of the GLC1A polypeptide is modified. Pepti so modified May have at least one of the activities of the native protein, or When designed to be a potent antagonist, the GLC1A described herein in detail It will be functionally equivalent to the polypeptide. Such modifications of the peptide For example, this can be achieved by amino acid substitution, deletion or addition.   For example, leucine to isoleucine or valine, aspartic acid to glutamine Acid, threonine to serine, or one amino acid Substitution of amino acids does not significantly affect the biological activity of the resulting molecule It is natural to expect. Conservative replacement is the side chain It is performed in a family of amino acids that are related to Encoded These amino acids can be generally divided into four families. (1) Acidity = Aspartic acid, glutamic acid; (2) basicity = lysine, arginine, histidine (3) non-polar = alanine, valine, leucine, isoleucine, proline, Enylalanine, methionine, tryptophan and (4) uncharged = glycine , Asparagine, glutamine, cysteine, serine, threonine, tyrosine. Similar In such a manner, amino acids are divided into repertoires as follows. (1) Acidic = aspartic acid, glutamic acid; (2) basic = lysine, arginine, arsenic Stidine; (3) aliphatic = glycine, alanine, valine, leucine, isoleucine , Serine, threonine, and serine and threonine are further aliphatic hydroxyl groups. (4) aromatic = phenylalanine, tyrosine, trypto Fan; (5) amide = asparagine, glutamine; and (6) containing sulfur = Cysteine, methionine (for example, Biochemistry 2nd edition, WH Freeman & Co 19 81 L. See Stryer). GLC1 with functional changes in the amino acid sequence of the peptide A homolog (meaning functional means that the polypeptide mimics wild-type function or antagonizes it) Whether to create a wild type tag in the cell. By examining whether the mutant peptide shows a similar reaction to the protein, Or by investigating whether to competitively block such reactions I understand. Polypeptides with more than one substitution can be examined in a similar manner. it can.   The present invention further provides truncation of mutant GLC1A proteins. We also plan to create combinatorial mutations as well as mutations. It is gene expression The possibility of a mutant sequence (for example, a homolog) that functions to regulate Especially useful for: Screening such combinatorial mutation libraries The purpose is, for example, to have both action and antagonism, in other words, to include both. To create a new GLC1A homolog with a new activity.   Moreover, this combination approach of selectively inhibiting gene expression results in GLC 1A homologs can be created. For example, mutations cause other signals GLC1A homologs that bind to pathway proteins and interfere with signal transduction it can. For example, a homolog is a dominant negative mutation. In addition, GL Manipulating a C1A domain makes it more suitable for using fusion proteins Can be obtained.   In one embodiment of the invention, the GLC1A mutation is caused by a combinatorial mutation at the nucleic acid level. Create a different variegated library. It is encoded in a variegated gene library Have been. For example, a mixture of synthetic oligonucleotides is genetically Denatured with the potential GLC1A sequence bound to the sequence Large fusion proteins containing the GLC1A sequence so that they can be expressed as (For phage display).   A library of such potential GLC1A sequence homologs is There are many ways to create from nucleotide sequences. Chemical synthesis of degenerate gene sequences It can be dynamically generated by a DNA synthesizer, and the synthesized gene can be converted to an appropriate expression vector. Connecting. The purpose of such a series of degenerate genes is the potential for their potential in one mixture. All sequences encoding a preferred set of GLC1A sequences are included. That is. The synthesis of degenerate oligonucleotides is well known in the literature. ( See literature, Narang, SA (1983) Tetrahedron 39: 3; Itakura et al. (1981) Reco mbinant DNA, Proc 3rd Cleveland Sympos. Macromloecules, ed. AG Walton, A mste rdam: Elsevier ppg. 273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53 : 323; Itakura et al. (1984) Science 198: 1056; Ike et al. (1983) Nucleic Acid Res. 11: 477). Such technology directly advances other proteins (See literature, Scott et al (1990) Science 249: 386-390; Robe rts et al. (1992) PNAS 89: 2429-2433; Devlin et al. (1990) Science 249: 4 04-406; Cwirla et al. (1990) PNAS 87-6378-6382; as well as U. S. Patents N os. 5,223,409, 5,198,346, and 5,096,815).   In addition, variegated populations of GLC1A fragments are created and screened for biological activity. Of functional sequence fragments for the GLC1A clone to select for functional fragments Bally is provided. Various methods of creating such libraries include chemical synthesis It is well known in the literature, including: As one embodiment, the function distribution is performed as follows. Create a library of column fragments. (i) One nicking occurs per molecule Treating the double-stranded PCR fragment of the GLC1A functional sequence with nuclease under such conditions; i) denaturation of double-stranded DNA; (iii) sense-antisense pair from other nicking products Of DNA to make double-stranded DNA containing DNA; (iv) Restoration by S1 nuclease treatment (V) removing the single-stranded portion from the pair; Incorporate in. In this method, N-terminal or C-terminal or intermediate sites An expression library of fragments of various sizes can be created.   Some gene products from combinatorial libraries by point mutation or truncation Or cDNA libraries for gene products with specific characteristics For this reason, a wide variety of techniques are known in the literature. Such a technique is known as a GLC1A homolog. Rapid screening of gene libraries created by combinatorial mutation of logs Generally applicable. Ideal for screening large gene libraries A widely used method is to place a gene library in a replicable expression vector. Cloning and introducing the vector library into appropriate cells. And expressing the combination gene under certain conditions. The conditions and Encodes a gene whose product can be detected by detecting a suitable activity The vector is relatively easy to isolate. Examples described below Each of the tests that yields a number of modified G Follow the advanced throughput analysis required to screen LC1A sequences .   Combinatorial mutations can create large sized mutant proteins. So Has a size of the order of 10.sup.26. Combination libraries of this size Technically challenging despite throughput screening assays It may be something. New methods have recently been developed to overcome this problem. Supplement It is a sufficient ensemble mutation (REM). It's in the random library Avoid most non-functional proteins and simply increase the frequency of functional proteins. And It reduces the complexity required to perform array space sampling. REM If appropriate selection or screening methods are used, library A type of algorithm that increases the frequency of mutation. (Arkin and Yourvan , 1992, PNAS USA 89: 7811-7815; Yourvan et al. , 1992, Parallel Problem Sol vjng from Nature, 2. , In Maenner and Manderick, eds. , Elsevir Publishing  Co. , Amsterdam, pp. 401-410; Delgrave et al. , 1993, Protein Engineering 6 (3): 327-331).   The invention also provides for the reduction of the GLC1A protein. It is a mammal in the present invention. Prevents binding of GLC1A polypeptides both upstream and downstream of the component To create mimetics, ie peptide or non-peptide agents, that can It is. Mutation techniques such as those described above provide GLC for protein-protein interactions. It is also useful for mapping determinants of 1A protein. For example, the target GL The C1A polypeptide functions upstream (in some cases it is an active activator, Repressor), or GLC When binding to a protein or nucleic acid that functions downstream of the 1A polypeptide, Includes both negative control and positive control. To illustrate, upstream and downstream of GLC1A Determining important residues of the subject GLC1A polypeptide required for molecular recognition of the components A GLC1A-derived peptide that competitively inhibits binding to a real GLC1A protein Used to create dometics. For example, binding to other extracellular proteins To map the amino acid residues of each of the subject GLC1A proteins involved in GLs that promote interaction by using scanning mutations in Peptidomimetic compounds that mimic C1A protein residues can be created You. Such mimetics also interfere with the normal function of the GLC1A protein Could be used to For example, hydrolysis with such residues None of the peptide analogs are benzodiazeprine, a It can be created by using Zepin. (See literature, Freidinger et al . in Peptides: Chemistry and Biology, G. R. Marshall ed. , ESCOM Publisher: L eiden, Netherlands, 1988), azepine (e. g. , See Huffman et al. in Peptides: Chemistry and Biology, G. R. Marshall ed. , ESCOM Publisher: Leiden, Nether lands, 1988), substituted gammal lactam rings (Garvey et al. in Peptides: C hemistry and Biology, G. R. Marshall ed. , ESCOM Publisher: Leiden, Netherl ands, 1988), keto-methylene pseudopeptides (Ewenson et al. (1986) J Med C hem 29: 295; and Ewenson et al. in Peptides: Structure and Function (Proceed ings of the 9th American Peptide Symposium) Pierce Chemical Co. Rockland , IL, 1985), b-turn dipeptide cores (Nagai et al. (1985) Tetrahedron Lett 26: 647; and Sato et al. (1986) J Chem Soc Perkin Trans 1: 1231), and b-amin oalcohols (Gordon et al. (1985) Biochem Biophys Res Commun 126: 419; and Dan n et al. (1986) Biochem Biophys Res Commun 134: 71). Four. Four. 1． Cells expressing recombinant GLC1A polypeptide   The present invention provides a method for introducing a nucleic acid to express a recombinant form of a subject GLC1A polypeptide. The associated host cells are also involved. Host cells can be prokaryotic or nucleated cells . GLC encoding full or full size protein Using the nucleotide sequence obtained by cloning the 1A protein, A recombinant GLC1A polypeptide was made via a nucleated cell process. Expression A nucleotide sequence is connected to a gene construct such as a Yeast, birds, insects or mammals) or prokaryotic cells (bacteria) The method of switching among them is the standard for creating other well-known proteins. It is a way. Well-known proteins include, for example, map kinase, pg. 53, W TI, PTP phosphatase, SRC, etc. Similar method or improvement In accordance with the subject invention, the recombinant G LC Create a 1A polypeptide.   Recombinant GLC1A genes can be found in prokaryotic or nucleated cells, or both. Encoding the GLC1A protein or a part thereof in a vector suitable for expression of By connecting different nucleic acids. Recombinant GLC1A polypep Expression vectors for making tides contain plasmids and other solids. You. For example, vectors suitable for expressing a GLC1A polypeptide include the following: Such as: pBR322-derived plasmid, pEMBL-derived plasmid, pEX Derived plasmids, pBTac-derived plasmids, and prokaryotic cells such as E. coli. PUC-derived plasmid for expression.   There are many vectors for expressing recombinant proteins in yeast. For example , YEP24, YIP5, YEP51, YEP52, pYES2 and YRP17 were obtained by converting the gene construct into cerevisiae (see, eg, Broach et al., Ex, hereby incorporated by reference). perimental Manipulation of Gene Expression Academic Press 1983 p.83 Cloning and expression vectors that are useful for introduction into Academic Press) is there. These vectors replicate in E. coli in the presence of pBR322, and Replication of the determinants of the plasmids. Replicate in cerevisiae. Further Ann Use a drug resistance marker such as picillin. Examples include the GLC1A polypeptide The peptide is a functional sequence of one of the GLC1A genes shown in SEQ ID Nos: 1 and 2. Using an expression vector obtained by subcloning create.   Popular mammalian expression vectors promote the growth of the vector in bacteria. One or more nucleated cells that contain prokaryotic cell sequences for expression Contains a cell transcription unit. PcDNAI / amp, pcDNA / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-ne o and pHyg are mammalian expression vectors suitable for transfecting nucleated cells This is an example. Some of them promote replication in both prokaryotic and nucleated cells, Modified with a bacterial plasmid sequence such as pBR322 to facilitate resistance selection There are also things. Others include cattle for transient expression of proteins in nucleated cells. Papilloma virus (BPV-1) or Epstein-Barr virus (pH EBo, pREP origin and p205) may be used. Transformation of host organisms and plasmids The various methods used for the adjustment of are detailed in the literature. For both prokaryotic and nucleated cells Other suitable expression systems and common recombination techniques are described in Molecular Cloning. Laboratory Manual ”(2nd edition, Shamrock, Fritz and Maniatis, Cold Surling Harbor. See Lab Publishing) Chapters 16 and 17.   In some cases, recombinant GLC by utilizing a baculovirus expression system It may be desirable to express a 1A polypeptide. Such a vacu Examples of virus expression systems include pVL-derived vectors (eg, pVL1392, pV139). L1393 and pVL941), pAcUW-derived vectors (e.g., pAcUW1), and pBlueBac- There are derived vectors (eg, pBlueBaeIII containing beta-gal).   If it is desired to express only a portion of the GLC1A protein, for example, Want truncation mutants lacking the N-terminus, such as truncation mutants lacking peptides If the starting codon (A TG) must be added. N-terminal methionine is methionine aminopeptide It is well known in the literature that enzymatic cleavage can be achieved by using titase (MAP). Have been. MAP is used for E. coli (Ben-Bassat et al. (1987) J.C. Bacteriol. 169: 751-757) , And Salmonella cloned, and the in vitro activity is (Miller, et. A1. (1987) PNA S84: 2718-1722). So, if you wish, To express a polypeptide derived from GLC1A in a host making MAP Thus, in vivo (E. coli or CM89, or S. aureus). cerevisiae), purified MAP In vitro (by the method of Miller et al.) Allows the use of N-terminal methionine Can be removed.   In another embodiment, transgenics used to create recombinant proteins are used. The animals are described in detail below. Four. Four. 2． Fusion proteins and immunogens   This is another embodiment. The functional sequence of a polypeptide Incorporated as part of a fusion gene that contains the encoding nucleotide sequence I will. If you want an immunogenic fragment of the GLC1A protein, The system is useful. For example, the rotavirus VP6 capsid protein is GL Used as an immunocarrier protein for portions of the C1A polypeptide. So It may be a monomer or a virus particle. Produce antibody of target GLC1A protein Incorporate the nucleic acid sequence corresponding to the part to be ligated into the fusion gene construct . The construct contains the functional sequence of the late vaccinia virus structural protein. A fusion protein containing a GLC1A epitope as a virus particle portion. To produce the expressed recombinant virus. Hepatitis B surface antigen fusion protein The used immunofusion protein is also known. Similarly, recombinant hepatitis B virus particles Use it. Similarly, the GLC1A protein portion and the poliovirus cap Chimeric constructs encoding fusion proteins, including sid proteins, Enhances the antigenicity of the polypeptide. (See literature, EP Publication No: 0259149; and  Evans et al. (1989) Nature 339: 385; Huang et al. (1988) J. Virol. 62: 3855; and Schlienger et al. (1992) J. Virol. 66: 2).   Multi-antigen peptide system for peptide-based immunization creates an immunogen Used to do. Organization of peptides on oligomer-branched lysine cores The desired portion of the GLC1A polypeptide can be obtained directly by chemical synthesis. ( See literature, Posnett et al. (1988) JBC 263: 1719 and Nardelli et al. (1991) J . Immunol. 148: 914). The antigenic determinants of GLC1A protein are also found in bacterial cells. It can appear and be presented.   In addition to utilizing fusion proteins to increase immunogenicity, fusion proteins Is widely recognized to promote protein expression. Therefore, in the present invention, GL The fusion protein is used for expression of the C1A polypeptide. For example, GLC1A tan Protein is produced as glutathione-S-transferase (GST fusion) protein . Such GST fusion proteins have, for example, a glutathione derivative substrate Thus, a purified product of the GLC1A polypeptide can be easily obtained. ( See literature, Current Protocols in Molecular Biology, eds. Ausubel et al. ( N. Y. : John Wiley & Sons, 1991)).   This is another embodiment. The fusion gene encoding the purified leader sequence For example, poly- (His) entero at the N-terminus of the desired portion of the recombinant protein Ki Affinity chromatography using nickel resin like the sequence of the enzyme cleavage site The fusion protein expressed can be easily purified by the chromatography. Purification The leader sequence is subsequently removed by enkerokinase treatment and the purified protein is removed. Can be obtained. (See literature, Hochuli et al. (1987) J.C. Chromatography  411: 177; and Janknecht et al. PNAS 88: 8972).   Techniques for making fusion genes are described in specialized journals. Various polypeptide arrangements Joining the various DNA fragments encoding the sequence follows conventional methods. Do Use blunt or Strager ends for splicing and Terminate the ends with restriction enzymes to fill the binding ends and avoid improper conjugation. Alkaline phosphatase treatment and enzymatic splicing . In another embodiment, fusion genetics can be performed using conventional methods, including automatic DNA synthesizers. Combines the children. Alternatively, gene fragments are amplified by PCR using anchor primers. Width. In doing so, a complementary overhang between two consecutive gene fragments And then annealing to create one chimeric gene sequence be able to. (See literature, Current Protocols in Molecular Biology, eds. Au subel et al. John Wiley & Sons: 1992). Four. Four. 3． antibody   Another feature of the invention is that antibodies, particularly those that react with GLC1A protein, Body. For example, an immunogen derived from the GLC1A protein based on a cDNA sequence Anti-protein / anti-peptide antiserum and Clonal antibodies can be created. (See literature, Antibodies: A Laboratory Man ual ed. by Harlow and Lane (Cold Spring Harbor Press: 1988)). Mouse and ham A mammal, such as a star or rabbit, is immunized with the immunogenic peptide. (GL CIA polypeptide or an anti-protein capable of producing an antibody reaction as described above. Original fragments or fusion proteins). Carrier bonding and other tamper Methods for imparting antigenicity to proteins and peptides are well known in the literature. Anti-GLC1A protein The immunogenic moiety is administered with an adjuvant. Immunity progresses in serum or plasma Check by monitoring the antibody titer. To evaluate normal antibody levels A standard ELISA or other immunoassay using the immunogen used as the antigen Do. Antibodies of interest are antigenic determinants of mammalian GLC1A protein, , An antigenic determinant of the protein represented by SEQ ID No: 2, or extremely Close homologs (94% or more homology is preferable if at least 92% homology can be achieved) It is desirable that the antigenic determinant of (1) has immunospecificity.   Following animal immunization with an antigenic portion of a GLC1A polypeptide, anti-GLC1A An antiserum can be obtained. If desired, polyclonal anti-GLC from the serum The 1A antibody can be isolated. To make a monoclonal antibody, Collect antibody-producing cells (lymphocytes) from the animal, and follow standard somatic cell fusion methods. The hybridoma cells are obtained by fusing with immortalized cells such as myeloma cells. That Such techniques are well known in the literature. For example, the hybridoma method (literally, originally veloped by Kohler and Milstein, (1975) Nature, 256: 495-497), human B cell Hybridoma method (Literature, Kozbar et al. , (1983) Immunology Today, 4:72), and And EBV-transformed hybridoma method for obtaining human monoclonal antibodies (literature, Cole et al. Alan R., (1985) Monoclonal Antibodies and Cancer Therapy. Lis s, Inc. pp. 77-96). Specifically reacts with the GLC1A polypeptide of the invention The hybridoma cells producing the antibodies are screened immunochemically. Monoclonal antibodies can be isolated from cultures of such hybridomas.   As used herein, the term antibody is a member of the mammalian GLC1A polypeptide of interest. If they specifically react with each other, the fragments are also included. Antibodies are conventional Can be fragmented. Same as above, before fragmentation Screening antibody fragments by the method. For example, F (ab)_TwoFragment pepsin antibody Obtained by treating That F (ab)_TwoReduces disulfide bonds in fragments This results in an F (ab) fragment. The antibody of the present invention has at least the CDR region of the antibody. GLC1A also has a bispecific and chimeric molecule with affinity It is.   An antibody that specifically binds to a GLC1A epitope is the subject GLC1A polypeptide. Tissue samples to determine their expression levels and patterns. Also used for staining. Anti-GLC1A antibodies are used to test G in tissues as part of a clinical test. To detect and evaluate LC1A protein levels, use immunoprecipitation or immunoblotting. Diagnostic methods may be used. For example, such a measurement is It is useful for predicting the onset and progress of the disease. Similarly, individual individuals monitor GLC1A protein levels. Being able to monitor the treatment of patients with such diseases You will also know the effect. GLC1A polypeptide levels can be measured in cerebrospinal fluid or amniotic fluid. Can be measured using cells in body fluids such as described above and tissue samples obtained by biopsy. Anti Diagnosis using the GLC1A antibody includes, for example, early diagnosis of degenerative diseases, particularly at birth. Immunological tests for diagnostic purposes are also included. Diagnosis with anti-GLC1A polypeptide antibody Diagnosis also includes immunoassays for the early diagnosis and typing of neoplastic and hyperplastic diseases It is.   Another application of the anti-GLC1A antibody in the present invention includes gtll, gt18-2 3.Immunization of cDNA libraries constructed in expression vectors such as ZAP and ORF8 There is staring. Proper reading frame and orientation This type of messenger library with inserted functional sequences Make the impact. For example, gt11 has an amino terminus of beta-galactosidase. A fusion protein consisting of a foreign polypeptide at the carboxy terminus create. The antigenic epitope of GLC1A protein is that it is specific for GLC1A. Detect antibodies with other A-protein orthologs or paralogs of the same species be able to. For example, a nitrogenase transferred from a plate on which phages are growing React the membrane with anti-GLC1A antibody. Positive files found by this test The phage is then isolated from the growing plate. Thus, the GLC1A homolog Detected a different isoform (including splicing mutation) from human For cloning from other animals. 4.5. How to treat the disease   In the present invention, the pathological conditions targeted for GLC1A treatment are various. May. Various types of glaucoma are one of the obvious examples.   “GLCIA Therapy” Becomes Active or Antagonistic to Wild-Type GLC1A To work, any suitable adjustments, such as those described above, to the polypeptide , Gene therapy constructs, antisense molecules, peptidomimetics, small Includes molecules, non-nucleic acids, non-peptides, or drugs identified in drug assays herein In.   In some cases, the gene is upregulated in some diseases and Is regulated in an inactive state, so when treating with the substances and methods described here Activates or suppresses GLC1A biological activity in some situations It is desirable. Some genes are under-expressed in certain diseases. GLC1A gene product Activity is impaired for some reason and progresses towards the development of disease symptoms. That's it Regulation of GLC1A gene expression or reduction of GLC1A protein activity Can cause or exacerbate the disease itself.   May be used to ameliorate disease symptoms including GLC1A gene misexpression Some approaches include, for example, antisense, ribozyme, There are main-chain molecules. Linking upstream or downstream elements in a signal cascade In this case, compounds that compete with the GLC1A protein are Antagonism, which exerts a therapeutic effect. Examples of suitable compounds are detailed above. The antagonists and homologs described above. Another example is the biological activity of GLC1A protein. It is probably desirable to enhance sex, for example, Use GLC1A agonists and mimetics and perform gene replacement therapy This is possible.   Confirm that GLC1A gene expression and protein activity are enhanced or suppressed. The compound is effective in treating patients to treat or improve glaucoma-related symptoms Doses. 4.5.1. Effective dose   The toxicity and therapeutic effects of such compounds can be measured using standard drugs using cell culture and laboratory animals. I studied it in a scientific way. In examining, LD₅₀(50% fatality rate of the population) and ED₅₀(Group At a dose that produces a 50% therapeutic effect. The dose ratio between toxic and therapeutic effects is the therapeutic index And LD₅₀/ ED₅₀Expressed as a ratio. Compounds with a large therapeutic index are preferred. on the other hand, When using compounds with toxic side effects, care must be taken with the administration method. Absent. Affected tissue to minimize potential damage to intact cells It is the administration method that determines the site. Thereby reducing side effects You.   Using data from cell culture and animal studies to determine the range of calculate. The dose of such compounds should be₅₀ Or a circulating concentration range including Dosage should be in this range depending on the route and mode of administration. Fluctuate within. Therapeutic effect on any compound used in the method of the invention Fruit doses are first calculated by cell culture assays. IC defined by cell culture₅₀(Symptoms Circulating plasma concentrations, including the concentration of the test compound that suppresses half of the maximum) The dose is determined in an animal model as described. Based on such information, the dose in humans Make more accurate decisions. Levels in plasma, e.g. high performance liquid chromatography Measure with Five. 2． Formulation and use   In accordance with the present invention, a pharmaceutical composition for use may have one or more physiologically acceptable It is formulated in a usual manner using a carrier or excipient obtained. Therefore the compound Physiologically acceptable salts and solvents may vary depending on the method of administration, e.g., injection, inhalation or Is another inhalation (by mouth or nose) or oral, sublingual, parenteral or direct Intestines, determined against.   For such treatments, the oligomers of the invention may be administered systemically, dermally Alternatively, it is formulated for various administration loads such as local administration. Technology and formulations For generalization, see Remington's Pharmaceutical Sciences, Meade Publishing Co. , Easton, PA. For systemic administration, injection is preferred. that is Injection, intramuscular injection, intravenous injection, intraperitoneal injection and subcutaneous injection. Book for injection The oligomer of the invention is made into a liquid dosage form. It's like Hanks solution or Ringer's solution Preferred physiologically compatible buffers are preferred. Further, the oligomer was once in a solid dosage form It can be dissolved or suspended immediately before use. Lyophilized products are also included.   For oral administration, the drug composition is passed through a pharmaceutically acceptable excipient. For example, tablets or capsules are made in the usual way. The excipient is a binder (eg, Corn, starch, polyvinylpyrrolidone, or Is hydroxypropyl methylcellulose); filters (eg lactose, fine Microcrystalline cellulose or calcium hydrogen phosphate); lubricating oils (eg, steer Magnesium phosphate, talc powder or silica); disintegrants (eg potatoes) ・ Starch or sodium starch glycolate or wetting agent Sodium uryl sulfate). Tablets are coated in a manner well known in the literature . Liquid preparations for oral administration may take the form of liquids, syrups or suspensions. Or, provided as a dry product and dissolved in water or another suitable solvent immediately before use There is also. Such liquid preparations are routinely prepared using pharmaceutically acceptable excipients. Adjust by law. Additives include, for example, suspending agents (eg, sorbitol syrup , Cellulose derivatives or hydrogenated edible fats); emulsifiers (eg lecithin) Or acacia); water-insoluble solvents (eg, almond oil, oil S Ter ethyl alcohol or fractionated vegetable oils) and preservatives (eg, Methyl or propyl-P-hydroxybenzoate or sorbic acid) You. Formulations add buffer salts, flavor, color and sweetness as appropriate.   For formulations for oral administration, the active form should be in a form suitable for controlled release. Take.   For sublingual administration, it takes the form of tablets or candy according to the usual formulation method.   For administration by inhalation, the use of the compounds according to the invention takes place under pressure. In the form of a spray in a container or a nebulizer with a suitable propellant take. Propellants include, for example, dichlorodifluoromethane, Tan, dichlorotetrafluoroethane, carbon dioxide or other suitable gas It is. In the case of a pressurized sprayer, the dosage unit may be with a valve to inject the indicated amount. And can be determined. Capsules such as gelatin for use in inhalers Powders and cartridges contain a powdered mixture of the compound and an appropriate powdered material such as lactose or starch. Make by mixing the base material.   Compounds for parenteral administration by injection, for example, by physician injection or infusion Adjust things. Injectable preparations can be prepared in ampoules or multi-dose containers Take the form for each dosage unit. Its composition includes suspension, liquid, and oil Milky or dissolved in water. Such formulation agents are included. Other active ingredients include sterile pyro It is a suitable solvent, such as water without gen, in powder form before use.   The compounds are also adjusted for rectal administration, such as a suppository or retention enema. Common suppository bases are cocoa butter and other glycerides.   In addition to the formulations described previously, the compounds may also be prepared for a depot preparation. That's it Long-acting preparations such as those implanted (eg, subcutaneously or intramuscularly) or injected intramuscularly Is administered. So, for example, the compound may be a suitable polymer or hydrophobic substance (such as Emulsions in acceptable oils) or ion exchange resins, or slightly It is prepared as a solubilized derivative, for example, as a solubilized salt.   Systemic administration includes transmucosal or transdermal routes. Transmucosal or transdermal administration For proper penetration to infiltrate through barriers like skin and mucous membranes Adjust the agent. Such penetrants are known in the literature, for example, transmucosal In such cases, bile salts or fuchsic acid derivatives are used. In addition, surfactants promote penetration I do. Transmucosal administration can be through nasal sprays or by suppositories. local For topical administration, the oligomers in the present invention may be ointments, as is known in the literature, It takes the form of an ointment, gel or cream.   In clinical practice, a method for administering a gene to a patient for GLC1A gene therapy is Many are known. It is detailed in the literature. For example, gene therapy It is administered systemically, for example by intravenous injection. And the specific tamper of the target cell Transduction depends on the specificity of the transfection. Transfer The specificity of the action depends on the expression of the gene transfer vector, cell type and tissue type. I'm involved. They support the expression of receptor genes controlled by transcriptional regulatory sequences. Are arranged. In other embodiments, the initial delivery of the recombinant gene is transfer to the animal. It is quite limited, being totally localized. For example, gene delivery vehicles With a catheter (US Patent 5,328,470) or stereotactic injection (Chen et al. 1994 PNAS9 1: 3054-3057). Glue that constitutes SEQ ID NO: 1 or 2 Any of the sequences shown in the list may be used, but the GLC1A gene or its homologue In a manner described, for example, Dev et al. , (1994 Cancer Treat Rev 2 0: 105-115) by electroporation using a gene therapy construct Introduce. Virus-containing genes with specific effects on local treatment of eye diseases The therapeutic vector is published International Patent Application No. WO 95/34580 to U. It is described in Eriksson et al.   Gene therapy construct formulations are essentially gene delivery systems in diluent System or with a base that slowly releases the gene delivery vehicle Take the wrapped form. In addition, complete gene delivery and system Some are produced. For example, retroviral vectors A type of preparation consisting of one or more cells that produce a child delivery system.   The composition may, if desired, be packaged in one or more unit doses containing the active ingredient. Presented at the dispensing device. Packaging is gold, like plastic wrap, for example. Made of genus or plastic foil. Packaging or dispensing equipment Instructions. Diagnosis and prognosis   The following mutations have been identified in glaucoma patients: (1) GLN368TOP, (2) GLY364V AL, (3) TYR437HIS, (4) ILE477ASN, (5) a 6 bp insersion in codon 396, (6) VAL 495ILE, (7) ALA455VAL, (8) THR377MET, (9) GLN19HIS, (10) ARG82CYS plus Such mutations are known among healthy subjects. (1) tYR347TYR, (2) THR325THR, (3) V AL439VAL, (4) ARG398LYS (5) Pro13Pro.   The prevalence of the nucleotide sequence reported here is the prevalence of glaucoma in the general population. Together, glaucoma in approximately 100,000 patients in the United States has a GLC1A gene It suggests that the mutation is the cause. Therefore, the GLC1A gene is It is the best known molecule in extinction. For comparison, in Japan It has been estimated that only 2,000 people have a mutation in the rhodopsin gene. You. The gene is considered to be the causative gene of retinitis pigmentosa. A specific glaucoma source The discovery of a causative gene means that children have a high risk of the disease before losing vision. It allows for treatments that measure and preserve vision.   In addition to the GLC1A nucleic acid molecules and polypeptides described above, In a prognostic test, the present invention provides at least one of the nucleic acids set forth in SEQ ID Nos: 1 or 2. Or the amino acid sequence shown in SEQ ID No: 3 And a polypeptide containing a part thereof.   The method provides a method for determining whether a subject is at risk for glaucoma are doing. The method comprises the steps of detecting genetic damage having at least one of the following characteristics: It is desirable to have the property of detecting the presence or absence of Good. (i) It affects the integrity of the gene encoding GLC1A protein. (Ii) Misexpression of the GLC1A gene. To explain, such a genetic loss Scratches can be detected by confirming at least one of the following: (i) GLC1A remains Deletion of one or more nucleotides in the gene, (ii) one in the GLC1A gene (Iii) the addition of one or more nucleotides in the GLC1A gene (Iv) rearrangement of the chromosome containing the GLC1A gene, (v) GLC Alteration of messenger RNA transcription level of 1A gene, (vi) Methylation of genomic DNA Abnormal modifications of the GLC1A gene, such as patterns, (vii) Presence of non-wild type in splicing of messenger RNA transcription, (viii) GLC1 Non-wild type level of A protein, (ix) loss of allele of GLC1A gene, (x) Inappropriate post-translational modification of GLC1A protein. As described below, the present invention provides a G A number of methods are provided for detecting damage to the LC1A gene. The important thing is G Causes of abnormal proliferation or proliferation of cells depending on LC1A or the possibility of differentiation To provide the ability to identify various molecules.   As a specific example, the sense or antisense sequence of the GLC1A gene Oligonucleotides containing (purified) nucleotide sequence regions that can be rediscovered Provided are nucleic acid components made of a tide probe. For example, something like SEQ ID: 1 and 2. Or a naturally occurring mutation, or the subject GL 5 'or 3' flank sequence or intron sequence naturally associated with C1A Or a spontaneous mutation of it. Make cell nucleic acids easy to hybridize Purify and dissolve. Exposing the probe to the sample nucleic acids. Sample nucleic acid and probe Hybridization with the probe. Such technology is used for mRNA transcription level. As well as deletions or replacements at the genomic level or at the mRNA level. Used to detect injured damage.   As mentioned above, one of the features of the present invention is that cells isolated from a patient are used. To determine if one or more of the sample cells has a mutation in GLC1A. It is related to such diagnostic tests. In this method, the subject is Risk of disease characterized by overgrowth or differentiation of vesicles Provide a way to determine whether Of the gene encoding GLC1A Genetic damage characterized by changes that affect integrity Who have the characteristic of being able to detect whether or not there is Law is preferred. Illustratively, such genetic damage is at least one of the following: Detected by checking one. (i) one or more in the GLC1A gene (Ii) one or more nucleotides in the GLC1A gene (Iii) replacement of one or more nucleotides in the GLC1A gene (Iv) non-wild in splicing of messenger RNA transcript of GLC1A gene The existence of a type. As described below, the present invention detects damage to the GLC1A gene. It offers many ways. Importantly, GLC1A-dependent abnormal proliferation of cells Provides the ability to identify a variety of molecules that are likely to multiply and / or differentiate It is doing.   To detect damage, use the polymerase chain reaction (PCR) (US Patent Nos. 4,683,195 and 4,683 , 302)), for example, anchor PCR or RACE PCR or ligation strand Reaction (LRC) (see literature, Landegran et al. (1988) Science 241: 1077-1080; and Nak azawa et al. (1994) PNAS 91: 360-364), the latter being particularly abrupt in the GLC1A gene. Useful for detecting mutations (see literature, Abravaya et al (1995) Nuc Acid Re s 23: 675-682). It is only an illustrative example. The method includes the following steps: You. (i) collection of cells from a patient, (ii) nucleic acid (genomic nucleic acid, m (Iii) isolation of the GLC1A gene (if any) Highly specific for the GLC1A gene under conditions such that Bringing one or more primers into close contact with the sample nucleic acid, (Iv) Detection of the presence or absence of amplification products, or detection of amplification product size and control Comparison with the rule sample. How to detect the mutations described here and PCR or LCR for primary amplification in combination with any method used It seems to be desirable to use.   Other amplification methods include self-sustaining sequence replication (Literature, Guatelli, J. et al. C. et al. , 1990 , Proc. Natl. Acad. Sci. USA 87: 1874-1878), transcription amplification system (literature, Kwoh , D. Y. et al. , 1989, Proc. Natl. Acad. Sci. USA 86: 1173-1177), Q belt ・ Replicase (Literature, Lizardi, P. M. et al. , 1988, Bio / Technology 6: 1197 ) Or other nucleic acid amplification methods, amplified molecules by methods well known in the literature It is like detecting. These detection schemes have very few nucleic acid molecules Useful for detecting such molecules when present only in numbers.   It is an example of a sample test. Restriction of GLC1A gene mutation in sample cells It can be seen as a change in the cleavage pattern of the enzyme. For example, samples and controls DNA is isolated, amplified, digested with one or more restriction endonucleases, and Check the fragment length by electrophoresis. Furthermore, sequence-specific ribozymes (US Patent No. . 5,498,531) causes specific sudden abrupt You can know the presence of the mutation.   This is still another embodiment. Using various sequencing reactions known in the literature, The sequence of the GLC1A gene is determined by The sequences of the 1A gene (control) are compared. Magzam and Kilbert (literature, Proc. Natl Acad Sci USA (1977) 74: 560) or Sanger (literature, Sanger et. al (1977) Proc. Nat. Acad. Sci 74: 5463). Has become. It is also conceivable to use automated sequencing for specimen testing. (Literature Biotechniques (1995) 19: 448), which also includes a mass spectrometer. (Literature See PCT publication WO 94/16101; Cohen et al. (1996) Adv Chromatogr 36: 1 27-162; and Griffin et al. (1993) Appl Biochem Biotechnol 38: 147-159) In some instances, although obvious in the literature, one or two Three nucleobases are required. For example, if you find only one nucleic acid, you can achieve it. You.   This is another embodiment. Cleavage reagent (nuclease, hydroxylamine or Protection (along with smith tetroxide and piperidine) Used to detect matched bases or RNA / DNA heteropairs (Literature, Myers, e t al. (1985) Science 230: 1242). Generally, mutations obtained from tissue samples are RNA or DNA capable of functioning and RNA or DNA containing the wild-type GLC1A sequence The heterozygous pair can be formed by the hybridization, and the mismatch “cleavage starts. Treat the reagent with a mismatched base pair between the sample and the control. For single-stranded regions. For example, an RNA / DNA pair is treated with RN ace, / DNA is treated with S1 nutase to enzymatically digest the mismatched region. Other practices It is an example. Piperidine to digest mismatched regions in both DNA / DNA and RNA / DNA pairs Together with hydroxylamine or osmium tetroxide. mismatch Material obtained after digestion of the region was denatured to determine the mutation site Determine size with polyacrylamide gel. (See literature, Cotton et al (1988 ) Proc. Natl Acad Sci USA 85: 4397; Saleeba et al (1992) Methods Enzymod. 217: 286-295). Control DNA and RNA should be marked for detection .   This is another embodiment. In the mismatch cleavage reaction, G obtained from the sample cells was used. A system for detecting or mapping point mutations in LC1A cDNA And proteins that recognize mismatched base pairs in double-stranded DNA (so-called “DNA mismatches”). Use more than one enzyme (eg, the E. coli mutY enzyme is a G / A mismatch) Thymidine DNA glycosylate obtained from HeLa cells To cut the T. (Literature, Hsu et al. (1994) Carcinogenesis 15: 1657-1662). You see According to a specific example such as a book, GLC1A, for example based on wild-type GLC1A The probe and the cDNA of the test cell or other DNA are hybridized. salt The base pair is treated with a DNA mismatch repair enzyme. The cleavage product was detected by electrophoresis. For example, US Patent No. 5,459,039.   This is another embodiment. A change in the mobility of electrophoresis causes the GLC1A gene to protrude. Confirm the mutation. For example, single-stranded polymorphisms (SSCPs) Nucleic acid differences are detected by electrophoretic mobility. (Literature, Orita et al. (1989) Proc Nat l. Acad. Sci USA 86: 2766, see also Cotton (1993) Mutat Res 285: 125-144; a nd Hayashi (1992) Genet Anal Tech Appl 9: 73-79). Samples and controls The single-stranded fragment of GLC1A nucleic acid is denatured and reconstituted. Secondary structure of single-stranded nucleic acid Depends on the sequence. If you check the mobility by electrophoresis, you can see the change of one base Can be turned on. The DNA fragment is labeled or labeled with a labeled probe Is detected. Using RNA (rather than DNA) increases the sensitivity of this test. Its secondary This is because the structure is more sensitive to changes in the arrangement. The method of interest is based on electrophoretic mobility. It is desirable to use heteropair analysis to separate double-stranded heteropair molecules New (Literature, Keen et al. (1991) Trends Genet 7: 5).   In another embodiment, a gradient that denatures the movement of the mutant and wild type fragments is used. Another method is to use a polyacrylamide gel (DGGE). (Literature, Myers et  al (1985) Nature 314: 495) When using DGGE as a test method, DNA is completely altered. Add a GC clamp of approximately 40 base pairs of GC-enriched DNA by PCR to avoid inactivation. Furthermore, Heat the denaturing reagent gradient to identify differences in mobility between control and sample DNA. (See Lisen, Rosenbaum and Reissner (1987) Biophys Chem 265: 127) 53).   Examples of other methods for detecting point mutations include, but are not limited to, selection. Selective oligonucleotide hybridization, selective amplification, or selection This is a typical primer extension. For example, a known mutation Place in the middle of the primer and hybridize only when a perfect match is obtained. Hybridize under the condition that it can be synthesized (Literature, Saiki et al. (1986) Natur e 324: 163); Saikietal (1989) Proc. Natl Acad. Sci USA 86: 6230). Like that Allele-specific oligonucleotide hybridization methods are When the oligonucleotide hybridizes with the target DNA amplified by PCR, 1 To test for two mutations or to hybridize oligonucleotides Hybridizes with labeled DNA by adhering to a membrane for It is used to check for various mutations.   Alternatively, allele-specific amplification that relies on selective PCR amplification The width method may be used with instant inventions. Primers for specific amplification Oligonucleotides used as primers must either have a mutation in the middle of the molecule (amplification). Is dependent on differential hybridization), (literature, G ibbs et al (1989) Nucleic Acids Res. 17: 2437-2448) or one ply Insert the 3 'end of the mer to prevent mismatches under appropriate conditions and extend the polymerase To (Literature, Prossner (1993) Tibtech 11: 238. Furthermore, cut-based detection It is desirable to insert a new restriction enzyme site in the mutated region to , Gasparini et al (1992) Mol. Cell Probes 6: 1). For some amplifications, Taq G) Ligase may be used (Literature, Barany (1991) Proc. Natl. Acad. Sci USA 88: 189). In such a case, the 3 'end of the 5' Since splicing can be performed, the presence or absence of amplification can be determined by identifying the presence or absence of amplification. Mutations can be detected.   For mutations for early termination in protein translation, see Application testing (PTT) is an effective diagnostic approach (Literature, Roe st, et. al. , (1993) Hum. Mol. Genet. 2: 1719-21; van der Luijt, et. al. , (1 994) Genomics 20: 1-4). For PTT, RNA is first isolated from tissue. Reverse it Transcribe and amplify by PCR. Reverse transcription PCR products are Template for embedded PCR amplification with primers containing nucleated cell translation initiation sequences Use it as After amplifying the target region, the unique mochi incorporated into the primer Allows for in vitro continuous transcription and translation of PCR products. Translation product Truncated by sodium silyl sulfate polyacrylamide gel electrophoresis The appearance of the polypeptide is a mutation that causes premature termination of translation. That is, there is. Applications of this method include target area If derived from a son, DNA (as the corresponding RNA) can be used as a template for PCR. There is also.   In another embodiment of the invention, an oligonucleotide comprising a region of nucleotide sequence A nucleic acid component containing a reotide probe is provided. The area is GLC1A Whether the target GLC can hybridize with the gene sense or antisense sequence Mutation to intron sequence or 5 'or 3' flank sequence linked to 1A gene What can be caused or its mutation can occur naturally It is. Cell nucleic acids are dissolved and purified for easy hybridization. Exposing the probe to the sample nucleic acids. Hybridization of probe to sample nucleic acid Detect the zation. Such methods include: Used to detect damage at the genomic or mRNA level, such as replacement. like that Oh Rigonucleotide probes can be used for neoplastic or hyperplastic diseases (eg, abnormalities). (Cell proliferation) for diagnostic or predictive evaluation of gene mutations Used.   The method described herein may be used, for example, for at least one nucleic acid probe described herein. Performed by using pre-packed diagnostic kits containing lobes or antibodies. It is. Patients who have or have a family history of the disease or illness involving the GLCA gene It may be convenient to use it for clinical use to diagnose.   Any cells or tissues where GLC1A is expressed, preferably monocytes, endothelium Cells and smooth muscles are preferred, but can be used for the diagnosis described below. For example, The body fluid of the specimen (eg, blood) is obtained in a known manner (eg, venipuncture). You. In other methods, nucleic acid testing can be performed on dry samples (skin or hair). You. Fetal nucleic acid samples are available from International Patent Application No. WO 91/7660 Obtained from mother's blood as described in to Bianchi. Other than that, amniotic fluid cells Villi are used for prenatal diagnosis.   Diagnostic methods include direct tissue sections (fixed) using patient tissue obtained from biopsy or resection. Or freezing). It does not require nucleic acid purification. That's it For such methods, nucleic acid reagents are used as probes or primers (see literature, Nuovo, G. J. , 1992, PCR in situ hybridization: protocols and applications , Raven Press, NY).   In addition to focusing on one nucleic acid sequence, such detection schemes Feel is also evaluated. The fingerprint profile can be Use differential display, Northern analysis and RT-PCR.   Antibodies to wild-type and mutant GLC1A proteins are as discussed above. It is also used for diagnosis and prediction. Such diagnostics include the expression of the GLC1A protein. Abnormal levels or structurally, GLC1A proteins in tissues, cells and subcells This is to detect abnormalities in the localization of the work. The structural difference is, for example, normal GLC1 Differences in size, charge and antigenicity of mutant GLC1A protein compared to A protein It is. Proteins from cells and tissues to be analyzed are not limited to Western blots Can be easily detected and isolated by methods well known in the literature. Wester N • For a detailed description of blots, see Sambrook 1989, Chapter 18. here The method of protein detection and isolation used in You. (Literature, Harlow, E. and Lane, D. , 1988, "Antibodies: A Laboratory Manua l ", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) It is referred to here as a whole.   This includes, for example, fluorescently labeled antibodies (see below) and light microscopy or flow cytometry. It can be performed by an immunofluorescence method that combines tomometry and fluorometry. In the present invention Antibodies (or fragments thereof) can also be used for in situ GLC1A protein It is also used histologically in immunofluorescence and immunoelectron microscopy for detection of bleeding. In situ Detection involves taking a tissue sample from the patient and reacting with the labeled antibody of the present invention. antibody( Or fragment) by placing the labeled antibody (or fragment) on top of the biological sample It is preferred to react. Through such a method, the GLC1A tan You can know not only the presence of Park but also its distribution. Using the present invention, Various histological methods (eg, staining methods) can be used to achieve in situ detection. Method) can be improved.   Solid supports and carriers are often used as supports for binding antigens and antibodies. Well-known supports and carriers include glass, polystyrene, polypropylene, Kisstran, nylon, amidase, natural and modified cellulose, polyacrylic Amido, gabbro and magnetite. The carrier is soluble to some extent Insoluble for light purposes. The support substance is a molecule that couples It has a tertiary structure that can bind to the body. Thus, the support structure is Such as spherical, or cylindrical, such as the inner surface of a test tube, or the outer surface of a rod It is something like In another case, the surface can be as flat as paper or test paper is there. Preferred supports are polystyrene beads. Suitable for binding antibodies and antigens The carriers and their experimental methods are well described in the literature.   One method of labeling a GLC1A protein-specific antibody is through an enzyme. You. It is used for enzyme immunoassay (EIA) (Literature, Voller, "The Enzyme Linked Imm unosorbent Assay (ELISA) ", Diagnostic Horizons 2: 1-7, 1978, Microbiologic al Associates Quarterly Publication, Walkersville, MD; Voller, et al. , J. Clin. Pathol. 31: 507-520 (1978); Butler, Meth. Enzymol. 73-482-523 (1981 ); Maggio, (ed. ) Enzyme Immunoassay, CRC Press, Boca Raton, FL, 1980; awa, et al. , (Eds. ) Enzyme Immunoassay, Kgaku Shoin, Tokyo, 1981). Antibody The bound enzyme reacts with the appropriate substrate. The substrate is preferably a chromogenic one . Such methods include, for example, spectrophotometry or fluorescence, or visual inspection. Can be detected by any means. Enzymes that label antibodies are not limited to this Not listed below. Malate dehydrogenase, staphylococcal nuclease, delta -5-steroid isomerase, yeast alcohol dehydrogenase, alpha glyce Lophosphate dehydrogenase, tyrosine / phosphate / isomerase, horseradish / peroki Sidase, alkaline phosphatase, asparaginase, glucose oxy Sidase, beta-galactosidase, ribonuclease, urease, catala , Glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholine Esterase. Detection is performed using a chromogenic substrate for the enzyme. Inspection On exit, the enzyme reaction of the substrate is compared to a similarly treated standard. Check the amount visually.   Detection is also performed by other immunoassays. For example, radioactive antibodies or antibody fragments By labeling with a substance, you can use a radioimmunoassay (RIA) to Wild type and mutant peptides (see literature, We intraub, B. , Principles of Raddioimmunoassays, Seventh Training Course o n Radioligand Assay Techniques. The Endocrine Society, March, 1986, whic h is incorporated by reference here). Radioactive isotope is Gamma Coun It can be measured with a counter, scintillation counter, or autoradiography.   Antibodies can also be labeled with a fluorescent material. Fluorescently labeled antibody When exposed to long light, fluorescence can be detected. Most useful for labeling compounds Commonly used fluorescent isothiocyanates, rhodamine, phycoerythri , Phycocyanin, allophycocyanin, ortho phthalaldehyde and fluoro It is Resamine.   Antibodies label fluorescent metals such as 152Eu and other lanthanides Can also be detected. These metals are diethylenetriaminepentovinegar acid Adhesion to antibodies with chelating agents such as (DTPA) and ethylenediaminetetraacetic acid (EDTA) Wear.   Antibodies can also be detected by labeling a chemiluminescent substance. Chemiluminescence mark The presence of the antibody marked with is indicated by detecting the luminescence generated during the chemical reaction. Chemical Typical light substances are luminol, isoluminol, Lidinium esters, imidazoles, acridinium salts and oxalates Tell.   Similarly, in the present invention, a bioluminescent substance is used for labeling an antibody. Bioluminescence is Chemistry found in biological systems where catalytic proteins increase the efficiency of chemiluminescence Light emission. The presence of a bioluminescent protein can be determined by detecting luminescence. Labe Important bioluminescent materials used in luciferins are luciferin, luciferase and alcohol. It is a kuolin.   Monitor changes in the GLC1A gene and its products throughout treatment and therapy It will be appreciated that any of the above methods can be used to accomplish this. Four. 7． Drug screening assays   Drug based on identifying specific GLC1A mutations in glaucoma Many standard assays are used to screen agents. Drugs for glaucoma It slows or prevents progress. From the molecular level verification of glaucoma, these Are expected to be better than current treatments.   For example, if the exact phenotype associated with these mutations can be determined, Functionally important regions of the protein can be identified. That way, these specific Mutations can occur in other forms, such as overexpression in cell lines or creation of transgenic animals. Can be used for experiments. Ideally, a mutation that repeats glaucoma at various times of life Is to find out. And the mutation is the expression system and trans The purpose is to show behavior similar to that of a transgenic animal.   GLC1A causes glaucoma through action in the trabecular meshwork and ciliary body However, an important part of the pathogenesis of GLC1A-related glaucoma is an indirect mechanism. Eye or whole body to make sure that it is not caused by It is important to examine the expression of this gene. Therefore, using standard methods, Confirm the tissue distribution and time course of gene expression (from both protein and DNA) Is important.   In addition, proteins that interact with the GLC1A gene product are different types of green cells. GLC1A gene product and protein A protein that interacts with a gene corresponding to a protein can be identified.   The mechanism by which the GLC1A gene causes disease is dependent on other important genetics. It has been shown to cause glaucoma in humans because it may alter the expression of offspring products Whether the mutation alters protein trafficking in animal models and tissue culture Research is also being conducted to find out. For example, at the ER level, The overall protein pattern in the cell caused by the removal of This can happen, as trafficking is affected.   In addition to the pathogenesis studies described above, PCR-based screening assays have been improved. To fully test the functional sequence of GLC1A in glaucoma patients Identify the exact location and sequence of the intron within the GLC1A gene. Also It is also important to identify sequences involved in the regulation of the GLC1A gene . For example, some glaucomatous mutations in the GLC1A gene include There are things in the upstream sequence that change the current to negative or positive. In addition, a similar tone The gene receiving the node may be important for glaucoma (eg, GL Gene sharing the C1A gene and the upstream regulatory region).   In order to identify a new class of drugs useful for the treatment of glaucoma, It is helpful to understand more. For example, pre- It has been suggested that exposure to GLC1A induces the GLC1A gene. Therefore, this Drugs that can block steroid effects should be tested for their ability to prevent or reduce the progression of glaucoma. Used for inspection.   As described further below, in vitro suitable for screening compounds Perform the test. The simplest example of this approach is for the GLC1A gene product. Using antibody, we developed ELIZA method to examine induction of GLC1A gene product, Screening large amounts of drugs that effectively block steroid induction in the product The To perform this assay, perform the assay in a 96-well microtiter plate. You. Thus, screening thousands of potentially effective drugs An automated method is used.   In addition, knowledge of the structure and function of the GLC1A gene implies other glaucoma. It has been suggested that the gene is involved. Such clues are homology, Evolution, evaluation of structural motifs in genes, and diseases that cause polygenes Genetic studies using analytical methods to identify genes reveal.   The first linkage study described here identified 22 glaucoma genes. Three of his carriers did not develop severe glaucoma. This information is GLC1 It suggests the presence of other genes that alleviate the effect of the A mutation. That's it Different ways to find glaucoma in different contexts To express the gene. To do this, create transgenic animals The gene causing cataract is introduced into a genetically different strain of mice. If the system If the phenotypes are different, the animals are backcrossed to identify palliative genes.   Some of the assays described above are detailed below. Four. 7. 1． Non-cell based assays   Drug screening pros for screening compound libraries and natural extracts Grams are often higher to maximize the number of compounds in a given time Assays with throughput are desirable. Such as with purified or partially purified protein Assays performed in non-cellular systems may be preferred as “primary” screens. And many. It is quick and relatively immune to changes in the target molecule by the test compound. It is easy to detect. Furthermore, the toxicity and bioavailability of test compounds to cells Typically, in vitro systems, i.e. binding affinity with upstream or downstream elements Unique focus on the effect of the drug on the target molecule, e.g. The test did not tell. Therefore, in the screening assay of the present invention, Things, whether they are positively or negatively regulated, are GLC1A polypeptides. Is upstream of the protein (including its activator and repressor) Or a protein or nucleic acid that functions downstream. Compounds and upstream or downstream elements of Add the GLC1A polypeptide into the mixture. GLC1A in upstream or downstream element Detecting and quantifying the polypeptide complex means that GLC1A and GLC1A A method to find compounds that inhibit complex formation between binding members. Compound Efficacy is dose-dependent from data obtained using various concentrations of test compound Evaluate by making a sex curve. In addition, the control test sets the standard for comparison. Done for In the control assay, the isolated and purified GLC1A polypeptide was used. The peptide is added to the composition containing the GLC1A binding element and its complex formation is tested Quantification in the absence of substances.   Complexation of a GLC1A polypeptide with a GLC1A binding element can be accomplished in a variety of ways. Can be detected. Changes in the structure of the complex, such as proteins labeled for detection, That is, GLC1A polypeptides labeled with radioactive, fluorescent, or enzymatic It can be detected by immunoassay or chromatocra using pide.   Typically, GLC1A or binding tandem is used, as well as familiarity with automated assays. Immobilizes Parka to promote separation of uncomplexed from complex Is desirable. GLC1A binding to upstream or downstream elements in the presence of candidate drug And in a suitable container containing the reactants in the absence. For example, it is my Clotiter plates, test tubes and microcentrifuge tubes. Take an example. There is a fusion protein with a domain added so that the protein binds to the matrix. You. For example, glutathione S transferase / GLC1A (GST / GLC1 A) The fusion protein is glutathione sepharose beads (Sigma Chemical) , St. Louis MO) and glutathione-treated microtiter pluto Can be So combine with cell lysis. For example, 35S label, test Compounds are required to be slightly harsher than physiological conditions. The mixture was prepared and incubated. Do not bind after incubation Wash the beads to remove any unwanted labels, immobilize the matrix, and Directly (eg, put the beads in scintillant) or dissociate the complex After that, measure the supernatant (whisper). Another method was separated from SDS-PAGE Separated from gel by matrix and standard electrophoresis as described in attached examples The complex is separated from the level of GLC1A binding protein in the separated bead fraction.   Other methods for immobilizing proteins on matrices can also be used in target assays. For example, GLC1A or its binding protein is composed of biotin and streptavidin. The solid phase can be immobilized by using an union bond. For example, a biotinylated GLC1A molecule has been described in the literature. Methods described (literature, biotinylation kit, Pierce Chemicals, Rockford, IL) ) With biotin-NHS (N-hydroxysuccinimide) Secure the wells of a 96-well plate (Pierce Chemical) coated with treptavidin Can be phased. Another method involves reacting with GLC1A, but upstream or downstream. This is fixed to the well of the plate using an antibody that does not interfere with the binding to the element. By binding to the body, GLC1A can be captured in the well. GL as described above The C1A binding protein and the test compound were placed in the wells of a plate on which GLC1A was immobilized. Incubate and quantify the amount of complex captured in the well. GST fixed In addition to phased complexes, methods for detecting such complexes include the GLC1A binding member Or reacts with GLC1A protein to compete with the binding element. Includes immunodetection of the conjugate using the body. It is unique or not This is similar to the enzyme-linked assay in which the enzyme activity associated with the binding element is assayed. In the latter example Is that the enzyme is chemically bound or as a fusion protein GLC1A-BP Provided. To illustrate, GLC1A-BP is chemically associated with horseradish peroxidase. Or are genetically fused. Of the polypeptide captured by the complex The amount is 3,3 'diaminobenzadine tetrahydroxy chloride which is a chromogenic substrate Alternatively, it can be evaluated with 4-chloro-1-naphthol. Similarly, polypeptides and groups A fusion protein comprising lutathione S transferase is provided. Complex formation is Quantify by detecting GST activity using 1-chloro-2,4-dinitrobenzene ( Literature, Habig et al (1974) J Biol Chem 249: 7130).   During an immunoassay to quantify one of the proteins captured by the complex, anti-G Antibodies to proteins such as the LC1A antibody are used. Another way The protein to be detected in the complex is an "epitope-marked" fusion protein It is possible. This is to facilitate the production of antibodies in addition to the GLC1A sequence. A second polypeptide has been added (eg, from a commercial route). for example For example, as described above, GST fusion protein can be quantified using an antibody against GST. Can be Another useful epitope marker is the myc epitope (see See Ellison et al. (1991) J Biol Chem 266: 21150-21157). It is pFLAG Systems (International Biotechnologies, Inc. ) Similarly, 10 residues of c-myc In. Alternatively, there is the pEZZ protein of the A system (Pharamacia, NJ). Four. 7. 2-cell based assay   In addition to the non-cell based assays described above, the mutations provided by the present invention Different and functional sources of GLC1A nucleic acids and proteins regulate the potency and antagonism of small molecules It can be used for cell-based assays. For example, target cell-mediated Assay for GLC1A response in recombinant G in the presence or absence of the test agent of interest. Overexpress LC1A protein. GLC1A dependent, as in non-cell based assays Identify agents that statistically significantly alter sexual responses (whether suppressive or synergistic) can do. In an illustrative example, GLC1A expression and activity is regulated intracellularly. Of the target drug in target readout (eg, tissue differentiation, proliferation, tumorigenesis) Measure the fruit. For example, to enhance the GLC1A-dependent signal cascade Alternatively, the expression of a suppressively regulated gene can be assayed. In the preferred example Regulates, such as, for example, the 5 'flank promoter and enhancer region Genetic products are easier if the region is labeled with a detectable label (eg, luciferase) Can be detected.   Common mammalian cells such as HeLa cells and Kos cells (COS-7, ATCC♯CRL-1651) In addition to cell lines, cells and cell lines derived from eye tissue (trabecular meshwork or ciliary epithelium) Used. Further, the transgenic animals discussed herein may comprise one or more glaucoma cells. Used to create cell lines containing the mold. It can be a cell culture model for this disease . Although the present invention also utilizes primary cultured cells derived from glaucoma transgenic animals, Creation of long-term cultured cell lines is desirable. For example, from transgenic animals See the literature (Small et al. , 1985, Mol. Cell Biol . 5: 642-648).   Using these cells, cell proliferation, migration, phagocytosis, adhesion and biosynthesis (and For example, the effects of test compounds from various viewpoints, such as extracellular matrix components) Examined. Then, without being limited to changes in morphology, growth, migration and adhesion, The phenotype of the cells associated with the disorder was examined.   GLC1A protein binds to DNA by itself or in a complex with other proteins And the ability to modify gene transcription In addition, transcription-based assays have been used, for example, in the GLC1A responsive regulatory region. Link detectable marker genes.   Monitor compound effects in cells with only basic drug screening It also applies to clinical trials. In such clinical trials, certain drug treatments The expression of the gene panel is used as a "read-out" of the effect.   Another feature of the invention is that the subject GLC1A polypeptide binds to GLC1A. Or interacting intracellular proteins (GLC1A binding proteins, Is a "two-hybrid" assay for isolating functional sequences of GLC1A-bp) (See literature, U. S. Patent No. 5,283,317; Zervos et al. (1993) Cell 72: 223-23 2; Madura et al. (1993) J Biol Chem 268: 12046-12054; Bartel et al. (1993) Bi otechniques 14: 920-924; Iwabuchi et al. (1993) Oncogene 8: 1693-1696; and Br ent WO94 / 10300).   Briefly, a two-hybrid assay is a method that uses two separate fusion proteins. Rebuilding a functional transcriptional activator protein in vivo . In particular, the method utilizes a chimeric gene expressing a hybrid protein . To illustrate, the first hybrid gene is a functional arrangement of the GLC1A polypeptide. Function of the DNA binding domain of a transcriptional activator fused in-frame to a sequence Contains an array. The second hybrid protein is from a cDNA library Encodes a transcriptional activation domain fused in frame with a sample gene are doing. If the bait interacts with the sample hybrid, for example, If they could form a GLC1A-dependent complex, they would have two transcriptional activators. It will be very close to the main. This distance drives transcription of the reporter gene. Enough to wake up. Reporter gene is transcription that responds to transcription activator Linked to a regulatory site, reporter gene expression is detectable, GLC It can be used to measure the interaction of 1A with sample proteins. 4. 8 transgenic animals 4. 8. 1. Animal-based system   Another aspect of the invention comprises a transgen of the invention and preferably comprises ( But optionally) an exogenous GLC1A protein in one or more cells of the animal A transgenic animal consisting of cells (of this animal) that express GL The C1A transgene can encode a wild-type protein, or Both gonists and antagonists and their homologs containing antisense structures Can be coded. In a preferred embodiment, the expression of the transgene is For example, cells and tissues that utilize cis-acting sequences to regulate expression in a desired pattern Or restricted to a special subgroup of growth stages. In the present invention, the GLC1A protein Mosaic expression may be essential for many forms of phylogenetic analysis And even for example in small patches of tissue in otherwise normal embryos A means to assess the impact of the lack of GLC1A, which could significantly alter growth Can be given. To this end, tissue-specific regulatory and condition regulatory sequences Can be used to regulate transgen expression in some spatial pattern. Wear. Furthermore, the temporal pattern of expression can be determined, for example, by conditional recombination systems or prokaryotic translocation. It can also be provided by a copy adjustment array.   Regulate transgene expression in vivo by site-specific gene amplification Genetic techniques are known to those skilled in the art. For example, catalysis for gene recombination of the target sequence Utilizes a genetic system that allows for regulated expression of recombinases that provide It is possible. As used herein, the phrase "target sequence" is used by recombinase Refers to a genetically modified nucleotide sequence. The target sequence is a recombinase Flanked by alternative sequences and generally expresses recombinase activity Excised or inverted in cells. Recombinase catalyzed The recombination event is that recombination of the target sequence activates the expression of one of the subject GLC1A proteins. Or it can be designed to provide suppression. For example, an antagonist Recombinant GLC1A gene encoding homologue or antisense transcript Excision of the target sequence that interferes with the expression of the offspring Can be designed as follows. Interfering with the expression of this protein can be Or different mechanisms, such as spatial separation of the GLC1A gene from the internal stop codon? Can be generated. In addition, the coding sequence of the gene Flanked by sequences and initially 3 'to 5' with respect to the promoter element Produce a transgene that will be transfected into the cells in the orientation. Can be. In such cases, the 5 'end of the coding sequence may be transcribed from the promoter. Target sequence by placing it in an orientation relative to the promoter element that drives activation Will cause the gene of interest to be reoriented.   All of the transgenic animals of the present invention contain the cells of the present invention in a plurality of their cells. It contains a transdiene, which transgene is responsible for cell growth, death and / or Alter the phenotype of a "host cell" with respect to or regulating differentiation. Listed here The transgenes of the present invention may be utilized utilizing one or more transgene structures. General description is generally exogenous because it is possible to produce nick organisms Will be directed to the production of transgenic organisms by matching genetic material . This general description is provided by one of ordinary skill in the art to the individual traffic utilizing the methods and materials described below. It can be applied to the introduction of transgenic sequences into organisms.   In one exemplary embodiment, the cre / loxP recombinase system of bacteriophage P1 is (Lakso et al. (1992) PNAS 89: 6232-6236; Orban et al. (1992) PNAS 89: 6861- 6865) or Saccharomyces cerevisiae FLP recombinase system (O 'Gorman et al. (1991) Science 251: 1351-1355; PCT publication WO 92/15694) Create an in vivo site-specific recombination system using any of can do. Cre recombinase is an intervening target sequence located between loxP sequences Catalyze site-specific recombination of rows. The loxP sequence is Cre 34 base-to-nucleotide cycling sequences linked to the recombinase. Necessary for combinase-mediated genetic modification. Cre recombinase When present, the orientation of the loxP sequence is such that the intervening target sequence is excised or reversed. Is determined (Abremski et al. (1984) J.C. Biol. Chem. 259: 1509-1 514), when the loxP sequence is directly oriented as a repeat, Mediate, and the loxP sequence is oriented as an inverted repeat. Catalyzes the reversal of the target sequence.   Therefore, the recombination of the target sequence depends on the expression of Cre recombinase. You. The expression of the recombinase is regulated by a promoter element, such as tissue -Specific, growth stages-specific, induced or suppressed by externally applied agents Factors to be adjusted. Recombinase expression is promoter When mediated by an agent, this coordinated repression is only present in cells in the target sequence. Genetic recombination will occur. Therefore, activated expression of recombinant GLC1A protein Can be adjusted by regulating recombinase expression.   The use of cre / loxP to regulate the expression of recombinant GLC1A protein was Contains transgenes encoding both the combinase and the target protein Requires the creation of transgenic animals. Cre recombinase and recombination Animals containing both the GLC1A gene of the rodent have "double" transgenic movements. It can be provided by creating a product. A common method of providing such animals is transgene The two genes each contain, for example, the GLC1A gene and the recombinase gene. To breed one transgenic animal.   Contains GLC1A transgene in a recombinase-mediated expressible manner One advantage derived from the initial creation of transgenic animals that Transgenic animals with target protein of either May be degradable in the expression in In such cases, the subject Transgene breeds and maintains sources that are latent in all tissues can do. Each of these sources may be associated with, for example, one or more tissues and And / or mating with an animal expressing the recombinase in the desired temporal pattern. Can be Thus, for example, an antagonistic GLC1A transgene Creation of a source that is latent will allow testing of offspring from the source. Where GLC1A-mediated induction in certain tissues or at certain developmental stages is mixed. The perturbation will, for example, result in a lethal phenotype.   Prokaryotic proteins that are also expressed to promote expression of the GLC1A transgene Providing similar conditional transgenes using prokaryotic promoters that require white matter can do. Exemplary promoters and corresponding trans-acting prokaryotic proteins The quality is shown in U.S. Pat. No. 4,833,080.   In addition, conditional transgene expression can be induced by methods such as gene therapy. Where trans-acting proteins, such as recombinase or Genes encoded in prokaryotic proteins are sent to tissues and are, for example, cell type specific Is expressed by a standard method. By this method, the GLC1AA transgene is Can remain latent in adults until "activated" by introduction of lance-acting factor Was.   In an exemplary embodiment, a "transgenic non-human animal" of the invention is a transgenic non-human animal. Into the germline of a non-human animal. At various stages of growth Transgens can be introduced using embryonic target cells in a cell. Embryo target cells Different methods are used depending on the growth stage. Used to implement the invention Animal specific strains generally have good health, good embryo yield, and good pre- Selected for nuclear visibility and good reproduction match. In addition, haplotypes It is an important factor. For example, when trying to create a transgenic mouse For example, strains such as C57BL / 6 or FVB strains are often used. (Jackson Laboratory, Bar Harbor, ME). A preferred strain is H-2^b, H-2^dAlso Is H-2^qWith haplotypes, eg C57BL / 6 or DBA / 1 ,. The strains used to carry out the invention are themselves transgenic. Nicks and / or knockouts (e.g., That is, a movement having one or more genes that are partially or completely suppressed. May be obtained from a product).   In one embodiment, the transgene construct is introduced into a single stage embryo. Contact Coalescence is the best target for microinjection. In mice, male pronuclei have 1-2 pl of D Approximately 20 micrometers in diameter to allow reproducible injection of NA solutions Reach. The use of conjugates as targets for gene transfer is almost always The main advantage is that the isolated DNA will be guided into the host gene before the first division. With dots (Brinster et al. (1985) PNAS 82: 4438-4442). As a result, the transformer All cells of the transgenic animal will have the transgene introduced. Raw This generally occurs because 50% of the cultured cells will have the transgene. It will also be reflected in the effective transmission of the transgene to the offspring of the source.   Usually, fertilized embryos are cultured in a suitable medium until the appearance of pronuclei. Almost this At time, the transgene containing nucleotide sequence is either female or Introduced into the male pronucleus. For some species, such as mice, the male pronucleus is preferred. New Before the exogenous genetic material is processed by the oocyte nucleus or zygotic female pronucleus Most preferably, it is added to the male DNA complement of the zygote. For male DNA complement The influencing oocyte nucleus or female pronucleus release molecule is probably the protamines of female DNA Is replaced by histones, thereby facilitating the combination of female and male DNA complement This is believed to form a diploid conjugate.   Therefore, exogenous genetic material can be transferred to the male complement of DNA or other complement of DNA. Is added before it is affected by the female pronucleus. For example, exogenous inheritance The offspring material enters the early male pronuclei when the male and female pronuclei are well separated and Is also added as soon as possible from the production of the male pronucleus so that it is also located near the cell membrane. You. The formation of exogenous genetic material in the nucleus of the sperm causes it to undergo decondensation Added after the Then adding sperm containing exogenous genetic material to the egg Or transfer the decondensed sperm to the egg to which the transgene construct has been added. Can be added as soon as possible.   Introduction of the transgenic nucleotide sequence into the embryo is a procedure known in the art. Steps, for example, can be performed by microinjection, electroporation, or lipofection . Embryo changes in vivo after introduction of the transgenic nucleotide sequence into the liver May be cultured for a certain amount of time, or may be re-transplanted into a surrogate host, Or both. In vitro culture to maturity is within the scope of the present invention. One A common method is to culture embryos in vitro, either by seed or for about 1-7 days. And then re-transplant them into a surrogate host.   For the purposes of the present invention, a conjugate is a diploid cell that can grow into an essentially complete organism. It is the formation of a vesicle. In general, a zygote comprises two members from one or more gametes. From eggs containing nuclei, produced naturally or artificially by fusion of haploid nuclei Will be. Therefore, gamete nuclei are inherently compatible, ie, undergo differentiation. And anything that will produce a viable conjugate that can grow into a working organism, No. Generally, euploid conjugates are preferred. A non-euploid zygote is obtained The number of chromosomes in relation to the positive multiple of the organism from which gametes are oriented Must not be changed more than once.   In addition to similar biological considerations, physical considerations should also be in the nucleus of the zygote or The amount (eg, volume) of exogenous genetic material that can be added to the genetic material forming the head Distribute. If genetic material is not removed, the amount of exogenous genetic material that can be added is Limited by the amount that would be absorbed without being destroyed. Generally, inserted The volume of exogenous genetic material used will not exceed 10 picoliters. Additives The physical effect must not be so great as to physically destroy the viability of the zygote. DN The biological limitations of the number and type of A sequences depend on the specific zygote and exogenous genetic material. Will vary with function and include exogenous genetic material of the resulting zygote Genetic material initiates and maintains the differentiation and growth of zygotes into working organisms Must be biologically possible to Will be.   Copy number of transgen construct added to zygote is exogenous inherited It will depend on the total amount of progeny material and will be the amount that can cause gene conversion. Reason In theory, only one copy is needed, but generally one copy is active. To ensure that there is a transgenic construct, for example 1,000-20, As many as 000 copies are used. According to the present invention, an exogenous DNA sequence One functional core of each inserted exogenous DNA sequence to promote phenotypic expression of Having a peak is often an advantage.   Techniques that allow for the addition of exogenous genetic material into nuclear genetic material are Unless it is disruptive to the vesicle, nuclear envelope or other existing cells or genetic structure Can be used. Exogenous genetic material is preferably present in minor amounts in nuclear genetic material Inserted by injection. Microinjectors of cells and cell structures are known and Used in technical fields.   Reimplantation is performed using standard methods. Generally, the surrogate host is anesthetized Is applied and the embryo is inserted into the fallopian tube. The number of embryos transferred to a particular host depends on the species Although it will vary more, in general the species will be comparable to the number of offspring that naturally give birth There will be.   Transgenic progeny of the surrogate host can be transfected by an appropriate method. And / or may be screened for expression. Screening is Saza At least one of the transgenes by immunoblot or Northern blot analysis. Often done with a probe to replenish the part. Coded by Transgen Transform Western blot analysis using antibodies against the Used as an alternative or additional method to screen for the presence of Can be used. Typically, DNA is produced from tail tissue and transfected. Analyzed by Southern blot analysis or PCR for sugen. Or Tissues or cells believed to express transgenes at the highest level Analyzed by Southern blot analysis or PCR for transgenics, Tissue or cell types may be used for this analysis.   Alternative or additional methods for assessing the presence of transgens Without limitation, suitable biochemical assays, such as enzymes and / or immunology Assay, histological staining for specific markers or enzyme activities, flow cytometry Including analysis. Blood analysis detects the presence of transgene products in blood For the level of various types of blood cells and other blood components It may be useful to assess the effects of Gen.   Progeny of the transgenic animal breed the transgenic animal with a suitable partner And / or eggs obtained from transgenic animals Is obtained by in vitro fertilization of sperm. When trying to cross with the other party The partner may be transgenic and / or knockout. Or it may not, and if it is transgenic, Or different transgenes or both. Or phase The hand may be a parent line. If in vitro fertilization is used, fertilized embryos May be transplanted into a surrogate host or cultured in vitro, or both. You may. Using any method, the offspring may be as described above or other suitable methods Can be used to assess for the presence of the transgene.   Transgenic animals produced according to the present invention may contain exogenous genetic material. U. As noted above, the exogenous genetic material may in some embodiments be GLC1A protein (A Gonist or antagonist), and antisense transcript, or GL It would be a DNA sequence that would produce a C1A mutant. Furthermore, such In embodiments, the sequence enables expression of the transgene product in a specific type of cell. Transcription inhibitors, such as promoters.   Using retroviral infection to introduce transgenes into non-human animals it can. Growing non-human embryos can be cultured in vivo to the blastocyst stage . During this period, blastomeres may be targets for retroviral infection (Jaenich , R .; (1976) PNAS 73: 1260-1264). Effective infection of blastomeres eliminates Zona Persida (Manipulating the Mouse Embryo, Hogan eds. (Cold S pring Harbor Laboratory Press, Cold Spring Harbor, 1986). Transgen The viral vector system used to introduce the It is a replication-defective retrovirus with a chromosome (Jahner et al. (1985) PNAS 82: 6 927-6931; Van der Putten et al. (1985) PNAS 82: 6148-6152). Virus blasting Easy and efficient transfection by culturing on a monolayer of production cells (Van der Putten, supra; Stwart et al. (1987) EMBO J .; 6: 383-388). Alternatively, the infection can be performed at a later stage. Virus or virus-production details Vesicles can be injected into a blast cocell (Jahner et al. (1982) Nature 298: 623-628). Transduction is performed within a subgroup of cells that produced the transgenic non-human animal. Most sources will be mosaic for transgenes . In addition, sources are generally located at different locations in the genome that will segregate into progeny May contain various retroviral insertions of the transgene. In addition, uterine retrovirus infection of embryos in the second trimester can transgene It is also possible to introduce them into columns (Jahner et al. (1982) supra).   The third type of target cells for transgen transfer is embryonic stem cells (ES). You. ES cells were obtained from pre-transplant embryos cultured in vitro and fused with embryos. (Evans et al. (1981) Nature 292: 154-156; Bradley et al. (1984) Nature 3 09: 255-258; Gossler et al. (1986) PNAS 83: 9065-9069; and Robertson et al. . (1986) Nature 322: 445-448). Transgen is a DNA transfectant in ES cells. Efficient by Sfection or by retrovirus-mediated transduction Can be introduced. Such transformed ES cells are subsequently transformed into non-human Can be combined with a blastocyst from an object. ES cells then colonize the embryo And contribute to the germline of the resulting chimeric animal. For an overview, see Jaenisch, R. . (1988) Scinence 240: 1468-1474.   In one embodiment, the method uses cognate recombination to modify the genome of the animal. Changes can be made in embryonic stem cells cultured using certain gene targeting You. By targeting the GLC1A gene in ES cells, Can be added into the animal germline to generate chimeras. Gene targeting process Containing a fragment homologous to the target GLC1A locus in tissue culture cells and Includes intended sequence modifications to the genomic sequence (eg, insertions, deletions, point mutations). This is accomplished by adding a DNA target-setting construct. The treated cells are then To screen for appropriate targets and identify those that are properly targeted To isolate.   Gene targeting in embryonic stem cells is actually one or more GLC1A genes. Targeting transgen constructs designed to undergo homologous recombination with nome sequences As a means for disrupting GLC1A gene function through use of the body, It is the scheme illustrated. Recombination of targeting constructs with factors of the GLC1A gene In addition, a positive selectable marker is inserted (or replaced) into the coding sequence of the gene And so on. The inserted sequence functionalizes the GLC1A gene Breaks down, but also gives positive selection properties. An example GLC1A target setting construct is shown below. Are described in more detail.   Generally, embryonic stem cells (ES) used to produce knockout animals The cell) will be of the same species as the knockout animal to be developed. It Thus, for example, mouse embryonic stem cells are commonly used for the development of knockout mice. Will be.   Embryonic stem cells are described, for example, in Doetschman et al. (1985) J.C. Embryol. Exp. MoGLC1Ah ol. 87: 27-45) using methods known to those skilled in the art, such as those described by Manufactured and maintained. Although any lineage of ES cells can be used, Strains that are typically growing to produce germline transmission of knockout constructs Selected for the ability of cells to be integrated and become part of the embryonic germline You. Therefore, any ES cells that are believed to have this ability will Suitable for use. One mouse strain typically used for the generation of ES cells is 129 J strain. Another ES cell line is the mouse cell line D3 (American Type Culture). re Collection, catalog no. CKL 1934). Yet another preferred ES cell line is W W6 cell line (Ioffe et al. (1995) PNAS 92: 7357-7361). These cells For example, Robertson in: Teratocarcinomas and Embryonic Stem Cells: A Practical  Approach, E. J. Robertson, ed. IRLPress, Washington, D. C. [1987]); byBrad ley et al. (1986) Current Topics in Devel. Biol. 20: 357-371); and by Hogan  et al. (Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY [1986]) For knockout construct insertion using methods known to those skilled in the art, such as Cultured and made.   Insertion of the knockout construct into ES cells can be performed, for example, by electroporation, microinjection, and Using various methods known in the art, including calcium phosphate treatment Can be. The preferred method of insertion is electroporation.   Each knockout construct to be inserted into a cell must initially be in a linear form No. Therefore, the knockout construct is contained in a vector (described below). If inserted into the DNA, linearization cuts the DNA only within the vector sequence and Appropriate restriction end selected to not cleave within the knockout construct sequence It is performed by digestion with nuclease.   For insertion, the knockout construct is inserted into a chosen insertion method known to those skilled in the art. Add to ES cells under conditions appropriate for the method. Introducing more than one construct into ES cells In this case, each knockout construct should be introduced at the same time, i.e. at once. Can be.   If an ES cell is to be electroporated, the ES cell and knockout configuration Body DNA is subjected to electrical pulses using an electroporator and according to the manufacturer's instructions. I do. After electroporation, ES cells are typically recovered under appropriate culture conditions. Fine The vesicles are then screened for the presence of the knockout construct.   Screening can be performed using various methods. Marker gene is anti A biomaterial resistance gene, for example, ES cells have a lethal concentration The cells may be cultured in the presence of a raw material. These surviving ES cells are probably knocked Incorporates an out construct. The marker gene is not an antibiotic resistance gene In some cases, Southern blots of ES cell genomic DNA were Probing with a DNA sequence designed to form a bridge Can be. Alternatively, PCR can be used. Finally, the marker gene Genetics encoded by an enzyme whose activity can be detected (eg, b-galactosidase) If so, the enzyme substrate is added to the cells under appropriate conditions and the enzyme activity is assayed. Can be analyzed. One skilled in the art will recognize other useful markers and those in particular cells. Will identify the means of detection. Such markers are within the teachings of the present invention. It is considered to be included.   Knockout constructs may be integrated at several locations in the ES cell genome, and And integrates into different positions in each ES cell genome due to the occurrence of irregular insertion events Is also good. The desired insertion position is the DNA sequence to be knocked out, eg, GL This is an interpolation position for the C1A coding sequence, transcription control sequence, and the like. Typically, About 1-5% of the ES cells that absorb the knockout construct will actually Will be incorporated into the lockout structure. Suitable insert for knockout construct In order to identify those ES cells with Can be extracted using a conventional method. The DNA was then analyzed on a Southern blot. Designed to hybridize to genomic DNA digested with constant restriction enzymes Testing can be performed using one or more probes. Instead, In addition, genomic DNA can be used to propagate DNA fragments of a particular size and sequence. Can be propagated by PCR using a specially designed probe (That is, only cells that contain the knockout construct in the appropriate location are of the appropriate size. Will produce a DNA fragment).   After appropriate ES cells containing the knockout construct in the appropriate location have been identified , These cells can be inserted into the embryo. Insertions can be made by various means known to those skilled in the art. While this may be done by a method, the preferred method is by microinjection. For microinjection Me For this, approximately 10-30 cells are collected in a micropipette and knocked out. Proper growth allowing integration of heterogeneous ES cells containing adult in developing embryos Inject into staged embryos. For example, as described in the appended examples, ES cells can be microinjected into blastocysts.   Appropriate developmental stages for embryos used for insertion of ES cells are very species dependent. Yes, but for mice, it's about 3. 5 days. Embryo irrigates pregnant female uterus Obtained by flowing. Suitable methods for doing this are known to those skilled in the art. And, for example, Bradley et al. (Above).   Embryos in their developmental stage are suitable for use, but preferred embryos are male. With a mouse Preferred embryos are those that differ from the coat color encoded by the ES cell gene. It also has a gene encoding a color. In this way, the mosaic coat color (Indicating that ES cells were introduced into the developing embryo) It can be easily screened for the presence of an out construct. Therefore, For example, if the ES cell line has a gene for white hair, Will have a gene for black or brown hair.   After introducing ES cells into the embryo, the liver is transferred to the uterus of the pseudopregnant stepmother for gestation. It may be transplanted to. Either stepmother may be used, but the stepmother typically has female Selected for their ability to give birth and recover well and for their ability to care for their children. Selected. Such stepmothers are typically crossed with vasectomized males of the same species. Created. The stage of the stepmother during pseudopregnancy is important for transplant success, and It is species dependent. For mice, this stage is about 2-3 days of pseudopregnancy It is.   The coat color selection method (described above and in the accompanying examples) is used The offspring born from the stepmother should first be screened for the mosaic coat color. Keep leaning. Additionally or alternatively, DNA from progeny tail tissue is Knockout configuration using Southern blots and / or PCR as described above Screen for body presence. They are in their germline If you believe you have a knockout construct, homozygous knockout animals Are crossed with each other to form a mosaic. Of this cross Product mice and known homozygous mice and wild-type mice Homozygotes were identified by Southern blotting of equal amounts of genomic DNA from Is determined.   Other means of identifying and characterizing knockout progeny are also available. For example Genes that were knocked out using Northern blots, marker genes, or Or probe the mRNA for the presence or absence of the transcripts encoding both. Can be inspected. In addition, Western blots can be used to Blots to generate antibodies or marker genes for specific GLC1A proteins If this gene is expressed using an antibody against The expression of the knocked-out GLC1A gene in various tissues of the offspring The current level can be evaluated. Finally, the in situ distribution of various cells from the progeny Analysis (eg, fixing cells and labeling with antibodies) and / or FACS ( Fluorescence-activated cell sorting) analysis was performed using the appropriate antibody to The presence or absence of offspring products can be found.   Still other ways to produce knockout or disrupted transgenic animals Generally known. For example, Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. , 1986). Les Combinase-dependent knockout can be used, for example, for cognate recombination to the inserted target sequence. Tissue-specific and / or temporary suppression of GLC1A-gene inactivation It can also be generated so that it can be suppressed by a recombinase sequence (described below).   More than one knockout construct and / or more than one transgen Animals containing the expression constructs are produced in any of several ways. Preferred manufacturing The method comprises a series of mammals each containing one of the desired transgenic phenotypes. It is to raise animals. Such animals are a series of matings, reverse matings and selections And ultimately all desired knockout constructs and / or Generate a single animal containing the expression construct, where this animal is not Common to wild-type except for the presence of knot-out constructs and / or transgenes It is of the genetic lineage (genetically identical).   The invention is further described by the following examples, which are controlled in any way. It is not intended to limit. All cited references (cited throughout this application) (Including published paper references, issued patents, and published patent applications) This is the content of the present invention. The practice of the present invention will not Nouchi cell biology, cell culture, molecular biology, transgenic biology, microorganisms General techniques of immunology, recombinant DNA, and immunology will be used. these The technology is detailed in the following document: For example, Molecular Cloning A Laboratory M anual, 2nd Ed. , Ed. By Sambrook, Fritsch and Maniatis (Cold Spring Harbo r Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed. Oligonucleotide Synthesis (M. J. Gait ed. Mullis et al. U. S. Patent No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J . Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. H iggins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss , Inc, 1987); Immobilized Cells And Enzymes (IRL Press, 1986); Perbal , A Practical Guide To Molecular Cloning (1984); the treatise, Methods I n Enzymology (Academic Press, Inc. , N. Y. ); Gene Transfer Vectors For Mamm alian Cells (J. H. Miller and M. Pm. Calos eds. , 1987, Cold Spring Harbor Lab oratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds. ), Imm unochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds . , Academic Press, London, 1987); Handbook Of Experimental Immunology, V olumes I-IV (D. M. Weir and C. C. Blackwell, eds. , 1986); Manipulating the  Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor , N. Y. , 1986).   The present invention is further described by the following examples, which are limited in either way. It is not what we are trying to determine. All cited references (cited throughout this application) Published paper references, issued patents, published patent applications and ongoing applications (Including patent applications therein) are incorporated herein by reference. 5. 1 Genetic linkage of familial open-angle glaucoma to chromosome 1q21-q31 Materials and methods Pedigree   Fifth consecutive generation infected with young-onset open-angle glaucoma without iris corneal corneal abnormalities Identified. This family is a woman who moved from Germany to the Midwest in the late 1800s. Consists of descendants. Disease symptoms in infected family members up to age 30, normal Arterial atrial angle, high intraocular pressure, absence of systemic or other eye abnormalities, and infection Included the need for surgery to control glaucoma in affected individuals. 35 people Refractive vision, slit-lamp at 50% risk of glaucoma for all family members Intravital microscopy, applanation tomography, angle spectroscopy, stereo disk photography and hand A complete eye examination was performed, including a Free, Goldman or Octopus perimeter. Two other infected patients were confirmed by reviewing the records of other ophthalmologists. Suffering Recorded any pressure greater than 30 mmHg and evidence of ocular nerve or visual field loss. Or when they have an intraocular pressure greater than 22 mmHg and apparently infected Having a child was considered infected for linkage. Infected family structure Members have early stages of diagnosis, trabecular meshwork that appears normally, very high intraocular pressure (often 50 mmHg or more), and relatively pressure-resistant ocular nerves. DNA classification   All surviving infected family members with blood samples and infected patients with children Obtained from six spouses. Place 10 ml of blood in each EDTA-containing glass tube for each patient I got it. DNA was made from blood using a non-organic extraction process (Grimberg, J. et al. Nucl. Acids Res 17, 8390 (1989)). Minutes beyond the entire autosomal genome Short tandem repeat polymorphisms (STRPs) that are propagated can be found in the literature or in J. L. By Weber Selected from those provided. Most are [dC-dA]-[dG-dT] dinucleotide repeats Was part. Orthonucleotide primers flanking each STRP are standard Phosphoramidite Chemistry (Applied Biosystems model 391 DNA synthesizer) Synthesized. The growth of each STRP was reduced using 50 ng of each patient's DNA. Contains the following 8. Performed in 351 PCR: 1. 25 liters of 10X Buffer (100 mM Tris-HCl pH 8. 8,500 mM KCl, 15 mM gCl_Two, 0.01% weight / volume gelatin), 300M dCTP, dGTP, respectively and dTTP, 37M dATP, 50 pmol of each primer, 0.25 liter of³⁵S-dATP (Amersham,> 1000 Ci mmol^-1), And 0.25U Taq polymerase (Perkin-Elmer / Cetus). Transfer the sample to DNA Thermosa The following conditions for 35 cycles in Ecler (Perkin-Elmer / Setus) Cultured under: 94C for 30 seconds, 55C for 30 seconds, and 30C 72C for seconds. After growth, 5 liters of stop solution (95% formamide, 10 mM NaOH, 0.05% bromophenol blue, 0.05% xylene Anol) was added to each sample. After denaturation at 95C for 3 minutes, 5 1 liter of each sample is immediately pre-warmed on a polyacrylamide gel (6 % Polyacrylamide, 7M urea) and charged for 3-4 hours Electrophoresed. The gel is then placed on Whatman 3 mm paper and slab gel dried Dried in the machine. Kodak Kisomat AR film is applied to dried gel Autoradiographs were made by exposing for 6 hours. Linkage analysis   Genotype data from autoradiographs on a Macintosh computer Put in. Hiber-card based programs (Nichols, BE et al., AmJ  Hum Genet 51 A369 (1992)) to store and retrieve marker data. And computer program LINKAGE (version 5.1) (Lathrop, GM and La Louel, JM359, 794-801 (1992)) and transferred to a DOS-compatible machine for analysis. Allele The frequency was assumed equal for each marker. MLINK rules for paired analysis Was used. All possible disease orders and two markers (D1S191 And D1S194) were performed under the ILINK program. Meaning of chain Justice is a standard (Z_max> 3.0). result Clinical discovery   All of the 37 family members tested were of known infected parents or siblings. Therefore, the disease risk was 50%. 19 of these patients were initially released There was elevated intraocular pressure and visual field loss consistent with a diagnosis of angular glaucoma. 3 more Patients had moderately elevated intraocular pressure and apparently infected children . Linkage analysis   Linkage exceeds 90 before detection with marker mapping long arm of chromosome 1 Short tandem repeat polymorphisms were classified within families. All available family members and -Point maximum equivalence calculation using chromosome 1 markers of 33 and 33 And significant linkage (Table 2). D1S212 is perfect for all members of the family Information and paired linkage analysis yielded a rod score of 6.5 (= 0) . Multiple point linkage analysis was not added to the peak rod score. Therefore, the location of glaucoma is D An area of about 20 centiMorgan (cM) with dimensions between 1S191 and D1S194 It was decided to be located. Both of these markers are labeled with multiple recombinants (each , 2 and 3) were shown in infected individuals in the family. D1S191-Glaucoma-D The degree of 1S194 is almost 1,000 times more than the other two possible degrees. Was. Table 2 Paired chain data Recombinant fraction 0.05 0.19 5.2 Genetic fine mapping of the juvenile primary open-angle glaucoma locus and glaucoma lesions Identification and properties of genes   Once the primary linkage has been identified, positional cloning can be used to identify disease genes. Next step is to narrow the candidate loci to the smallest possible gene region . The first study described in Example 5.1 showed that the primary open-angle glaucoma gene was chromosome 1q Approximately 20 flanked by markers D1S194 and D1S191 above It is shown that it is within the cM region. Another marker and tribe is obtained and this The loci are probed into the 2.5 cM region using two of these families. The third tribe The spacing should be even smaller.   1q glaucoma using polymorphic DNA markers and genetic maps, in addition to family members Scrutinize loci. Using STRPs, the genotype of each family member is determined. Was. Propagation of each STRP was performed using the following recipe:   1) Dilute genomic DNA (about 1 g / liter) 1/50, ie 2     0 liters of “raw” DNA and 980 ddH_Two0.   2) Use 2.5 liters of "diluted" DNA as template for PCR     You.   3) Prepare a PCR reaction mixture as follows:       1.25 liter of 10X buffer (Stratagene)       0.12 liter of each primer (50 pmol of each primer)       0.5 liter of dNTPs (5 mM C, T, and G, and 0.6 mM         25 mM A "cold")       3.5 liters of ddH_TwoO       0.25 liter³⁵S-dATP       0.1 liter of Taq polymerase     Oil (1 drop)   4) PCR was performed under the optimal conditions for the specific primer (generally 9430 seconds, 5     530 and 7230 seconds) and run for 35 cycles.   5) 5 liters of stop solution (95% formamide, 10 mM NaOH, 0.0     5% bromophenol blue, 0.05% xylene cyanol) to each tube     You.   6) The sample was denatured at 95C for 3 minutes and immediately pre-warmed     hand     On a polyacrylamide gel.   7) Dry the gel on Whatman paper and autoradiograph     The system is exposed for 1-2 minutes.   If possible, different STRPs on the gel were loaded multiple times. One gel hit Up to six markers were successfully filled. Further PCR amplification (up to 3 markers) Was confirmed. Based on recombination observed in the tested families, juvenile glaucoma Gene is between markers AFM238 and AT3 (8 centimorgan interval) I can believe. Haplotype analysis between families shows this interval to be D1S210 and AT3. Between 2 cm Morgan intervals.   Because the gene spacing is so narrow, the use of physical mapping Can be. Whole human genomic yeast for identifying YACs mapping to the region Nearest furan for screening artificial chromosome (YAC) libraries King marker. The CEPH and CEPH mega-YAC libraries are used for this purpose. Can be used for (Center d'Etude du Polymorphisme Humain (CE PH) Available from Paris, France). In the CEPH Mega-YAC library 44% of the clones have an average size of 560 kb and another 21% have an average size of 800 kb. Have an average size, and 35% have an average size of 120 kb. This library -Only the 50-200 PCR reaction identified a unique YAC containing STS. Gridded fines such as need to be performed using specific sequence labeling (STS) It can be used in a titration plate system. YAC content identified by CEPH And start constructing the contig beyond the 1q candidate region (see FIG. 3). Configure a YAC Container that uses YAC ends to identify additional YACs Can be achieved. Immobilized PCR (Riley, J. et al (1990) Nucleic Acid Res 18: 2887-2890) can be used to rescue the YAC ends and Can then be sequenced and this sequence is used to sequence tagging sites (S TS) can be grown. Reload the YAC library using STS Screening can yield overlapping adjacent YACs.   Are some YACs, especially those from the mega YAC library, chimeric? Or have been shown to contain deletions or transpositions, Check the accuracy of each YAC container by creating a field map It is. Furthermore, single dyeing by in situ fluorescence hybridization (FISH) YAC map for chromosomes or pairing to the same chromosome using monosomatic somatic cells The two chimeric YAC end maps (NICMs Panel 2) ACs are minimized. In addition, the single-YAC At least two overlapping independent Y's to ensure coverage of a particular area without relying on Obtaining AC can minimize the YAC chimera problem.   Once a YAC contig that expands the candidate region is isolated, this reagent can be used to It is possible to create another genetic marker that enables a finer genetic map Wear. In addition, YACs can be used, for example, in region-specific lambda and cosmid Higher resolution physical mapping reagents can be produced. Weather Lambda and cosmid talone can be used for cogene isolation. D Xenon multiplication (Buckler, A.J. (1991) Proc Natl Acad Sci USA 88: 4005-4009) Propagation of "exon trapping" (Duyk, G.M. (1990) Proc Natl Acad  Sci USA 87: 8995-8999) to identify exons from genes within the region can do. Exon Trapped from Candidate Region as Probe To screen cDNA libraries to isolate cDNAs be able to. If necessary, c in the YAC fragment subcloned into the cosmid Screening of DNA libraries, zoom blot analysis, for example, biotin- Direct selection of cDNA using treptavidin-tagged cosmid clones (Morgan, J.G. et al (1992) Nucleic Acid Res 20 (19): 5173-5179) Time cloning strategy and HTF island analysis (Bied, A.P. (1987) TrendsGe net 3: 342-247) to identify genes in genomic DNA using other methods. Can be. Possible genes include GC-immobilized denaturing gradient gel electrophoresis (S heffield, V.C. et al (1989) Genomics 16: 325-332), single-strand gel Polymorphism (SSCP) analysis (Orita, M. et al (1989) Proc Natl Acad Sci USA 86: 2766-2770) and mutations caused by direct DNA sequence formation. Would. 5.3 Primers for use in identifying patients with predisposition for glaucoma versus   190 base pairs from human genomic DNA with mutations causing glaucoma Two primers that can be used with the polymerase chain reaction to grow rows The pair (primers 1 and 2 in Table 3) has been identified.   Trabecular Meshwork Induced Glycocorticoid (T IGR) gene (International publication number 96/14411 for Nguyen et al.) Were used to screen 410 glaucoma patients and 81 normal persons. 4 types Amino acid alteration sequence changes were detected in a total of 12 glaucoma patients (2.9%). No change in the amino acid sequence was found in normal subjects.   The specific mutations observed are shown below the normal sequence in FIG. These programs The incidence of mutations in fragments of DNA propagated by primer pairs There are about 100,000 use of these primers in the United States alone along with detection methods Suggests that it can be used to identify predisposition to glaucoma in patients .

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 39/395 A61K 48/00 48/00 A61P 27/06 A61P 27/06 C12N 1/15 C12N 1/15 1/19 1/19 1/21 1/21 5/00 A 5/10 A61K 37/02 (31) Priority claim number 08 / 822,999 (32) Priority date March 21, 1997 (1997. 3.21) (33) Priority country United States (US) (81) Designated country EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC NL, PT, SE), AU, CA, JP (72) Inventor Sheffield, Val 52240 Coralville NE Oak Park Place 13 (72) Inventor Orward, Wallace et al. Le M United States Iowa 52245 A Iowa City Ridgeway Drive 2015

Claims (1)

[Claims] 1. a) SEQ. ID. Nos. A nucleic acid represented by either 1 or 2;   Is SEQ. ID. Nos. A complementary strand of the nucleic acid represented by either 1 or 2;   And     b) a nucleic acid capable of hybridizing with the nucleic acid of a) under appropriate stringency   An isolated nucleic acid molecule comprising a member selected from the group consisting of: 2. 2. The isolated nucleic acid molecule of claim 1, wherein said suitable stringency is   A molecule characterized by mild stringency. 3. 2. The isolated nucleic acid molecule of claim 1, wherein said suitable stringency is   A molecule characterized by a high degree of stringency. 4. 3. The isolated nucleic acid molecule of claim 1, further comprising a label.   And a molecule characterized by: 5. 2. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid is functional.   A molecule characterized by encoding a GLC1A biological activity. 6. 6. The isolated nucleic acid molecule of claim 5, wherein said nucleic acid is glaucoma.   It encodes a protein that can treat or prevent   Molecule. 7. A vector comprising the nucleic acid molecule according to claim 1. 8. The vector according to claim 7, which is capable of replicating in a cell. 9. A host cell comprising the vector according to claim 7. Ten. A transgenic animal comprising the vector of claim 7. 11． A method for treating glaucoma in a human tester, comprising the generation of the GLC1A gene.   A therapeutically effective amount of a compound that modulates the activity of a current or GLC1A gene product,   Administering to the test subject. 12． 12. The method of claim 11, wherein said compound, protein, or peptide is used.   Selected from the group consisting of tides, peptidomimetics, small molecules, or nucleic acids   Features method. 13. 13. The method according to claim 12, wherein said nucleic acid is a gene, an antisense gene.   Ribozymes, and triplex nucleic acids.   how to. 14. 12. The method of claim 11, wherein said compound is a normal GLC1A residue.   A method characterized by being an agonist of a gene or gene product. 15． 15. The method of claim 14, wherein said compound is a therapeutic gene.   A method comprising: 16． 15. The method of claim 14, wherein said compound is a therapeutic protein.   A method characterized by: 17． 15. The method of claim 14, wherein said compound is an endogenous G of the subject.   It is a drug that up-regulates the expression of LC1A gene   Method. 18． 18. The method of claim 17, wherein said compound is a strong promoter.   A method characterized in that: 19. 12. The method according to claim 11, wherein said compound is a mutant.   Characterized by comprising an antagonist of the LC1A gene or gene product   Method. 20. 20. The method of claim 19, wherein said compound is an antisense,   A method characterized by being a zym or a triple helix molecule. twenty one. 21. The method according to claim 20, wherein the compound is a small molecule, a peptide,   Or a peptidomimetic. twenty two. 22. The method of claim 21, wherein said compound is an antibody.   Features method. twenty three. A method for screening a GLC1A agonist or antagonist   What     a) GLC1A polypeptide or biologically active fragment thereof, GLC1A   And the test compound, in the absence of the test compound, the GLC1A   Under conditions that allow the protein to interact with the GLC1A binding partner   And binding; and     b) Binding of GLC1A protein / GLC1A to the presence of the test compound   Detecting the extent to which the partner complex is formed, wherein the absence of the test compound is detected.   Increased complex in the presence of the test compound as compared to that detected in the presence   The amount of body formation indicates that the test compound is an SGLC1A agonist   And the test compound is compared to that detected in the absence of the test compound.   The amount of reduced complex formation in the presence of the test compound is determined by the fact that the test compound is a GLC1A jaw.   Characterized by suggesting that the   A method comprising steps. twenty four. 24. The method according to claim 23, wherein the pharmaceutical composition is prepared from the test compound.   A method further comprising the step of preparing. twenty five. Determining whether human subjects have or are at risk of developing type II glaucoma   A way to     a) Obtain a sample containing a nucleic acid molecule containing the GLC1A gene from a tester   That; and     b) detecting the presence or absence of a genetic injury in the GLC1A gene of the test subject;   When,   A method comprising the steps of: 26. 26. The method according to claim 25, wherein prior to step b), the GLC1A remains.   Uses amplification primers that selectively anneal and amplify at least a portion of the gene   Further comprising amplifying a nucleic acid molecule comprising the GLC1A gene.   Method consisting of: 27. 26. The method according to claim 25, wherein said detecting step b) comprises GLC   Determining the sequence of at least a portion of the nucleic acid molecule containing 1A   A method characterized by the following. 28. 26. The method according to claim 25, wherein said detecting b) comprises:   Selectively hybridize an acid molecule to at least a part of the GLC1A gene   Contact with the nucleic acid probe under conditions that promote hybridization.   A method comprising: 29. 26. The method according to claim 25, wherein said detecting b) comprises:   Involves performing restriction endonuclease degradation of the acid molecule, thereby   Nucleic acid generated and selectively hybridizing to at least a part of the GLC1A gene   A method comprising contacting a probe with said digest. 30. How to determine if a human tester has glaucoma or is at risk for developing it   So,     a) obtaining a sample containing the GLC1A protein from the tester;   And     b) determining whether the GLC1A protein is normal or mutated   That   A method comprising: 31. 31. The method according to claim 30, wherein said detecting step b) comprises:   Mutated GLC that can bind protein to wild-type GLC1A protein   An antibody that cannot bind to 1A protein or a mutated GLC1A protein   Contact antibodies that can bind to quality but not to normal GLC1A protein   A method comprising: