JP2009118856A - Alk-7 (activin-like kinase) and serine threonine kinase receptor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new serine threonine kinase receptor, ALK-7, etc. <P>SOLUTION: Disclosed are a new serine threonine kinase receptor, an isolated nucleic acid molecule encoding a polypeptide containing an amino acid sequence corresponding to ALK-7, a substantially pure polypeptide containing an amino acid sequence corresponding to ALK-7, a nucleic acid probe for specifically detecting the presence of ALK-7 in a sample, a method for detecting ALK-7 nucleic acid in a sample, a kit for detecting the presence of ALK-7 nucleic acid in a sample, a recombinant nucleic acid molecule to efficiently initiate the transcription in a host cell, and a recombinant nucleic acid molecule containing a vector and the isolated nucleic acid molecule. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

(Background of the Invention)
(Field of Invention)
The present invention relates generally to a novel serine threonine kinase receptor, ALK-7. In particular, the present invention provides a nucleic acid molecule encoding ALK-7; a purified ALK-7 polypeptide; a recombinant nucleic acid molecule; a cell containing the recombinant nucleic acid molecule; a specific binding affinity for ALK-7. A nucleic acid probe for detecting ALK-7 nucleic acid; a method for detecting ALK-7 nucleic acid or polypeptide in a sample; and a kit containing the nucleic acid probe or antibody. The present invention further relates to a bioassay for diagnosing, evaluating or predicting a mammal suffering from a neurodegenerative disease using the nucleic acid sequence, receptor protein or antibody of the present invention. Also provided is a therapeutic use of AKL-7. The invention further relates to ligands, agonists and antagonists of the ALK-7 receptor and their diagnostic and therapeutic uses.

BACKGROUND INFORMATION Serine threonine kinase receptors are a family of growth factor signal transducers (He et al. (1993) Dev. Dyn. 196: 133-142). A series of serine threonine kinase receptors, activin receptor-like kinases 1-6 (ALK-1-6) were previously identified (tenDijke et al. (1993) Oncogene 8: 2879-2887; Franzen et al. (1993) Cell 75 : 681-692; Ebner et al. (1993) Science 260: 1344; Matsuzaki et al. (1993) J. Biol. Chem. 268: 12719; He
(1993) Dev.Dyn. 196: 133-142; Attisano et al. (1993) Cell 75: 681-692; ten Dijke et al. (1994) Science 264: 101-104; International Patent Publication No.94 / 11502 (Released on May 26, 1994)). The present invention provides a novel serine threonine kinase receptor, ALK-7.

SUMMARY OF THE INVENTION The present invention provides an isolated nucleic acid molecule that encodes a polypeptide comprising an amino acid sequence corresponding to a novel serine threonine kinase receptor, ALK-7.

The invention further provides a substantially pure polypeptide comprising an amino acid sequence corresponding to ALK-7.

The present invention also provides a nucleic acid probe for specifically detecting the presence of ALK-7 in a sample.

  The present invention further provides a method for detecting ALK-7 nucleic acid in a sample.

  The present invention also provides a kit for detecting the presence of ALK-7 nucleic acid in a sample.

  The invention further provides a recombinant nucleic acid molecule comprising 5 ′ to 3 ′ a promoter that efficiently initiates transcription in a host cell and said isolated nucleic acid molecule.

  The invention also provides a recombinant nucleic acid molecule comprising a vector and said isolated nucleic acid molecule.

The present invention further provides a recombinant nucleic acid molecule comprising a sequence complementary to an RNA sequence encoding an amino acid sequence corresponding to the polypeptide.

  The present invention also provides a cell comprising the recombinant nucleic acid molecule.

  The present invention further provides a non-human organism comprising the recombinant nucleic acid molecule.

The present invention also provides antibodies having specific binding affinity for ALK-7 polypeptides.

  The present invention further provides a method for detecting an ALK-7 polypeptide in a sample.

  The present invention also provides a method for measuring the amount of ALK-7 in a sample.

  The present invention further provides a diagnostic kit comprising a first container means comprising said antibody and a second container means comprising a conjugate comprising a monoclonal antibody binding partner and label.

  The present invention also provides a hybridoma that produces the monoclonal antibody.

  The present invention further provides methods for diagnosing human diseases, in particular neurovariable diseases, disorders and injuries.

The invention also provides methods of therapeutic use comprising all or part of the nucleic acid sequence encoding ALK-7 and the corresponding protein.

The present invention provides assays for isolating ALK-7 receptor ligands, agonists and antagonists and therapeutic uses of these molecules.

This study also provides assays for the evaluation and development of drugs that can activate or suppress the ALK-7 receptor and the therapeutic use of these drugs.

Further objects and advantages of the present invention will become apparent from the following description. The present invention also provides:
(Item 1) An isolated nucleic acid molecule encoding a polypeptide comprising an amino acid sequence corresponding to the serine threonine kinase receptor, ALK-7.
(Item 2) The isolated nucleic acid molecule of item 1, wherein the molecule has the nucleic acid sequence of SEQ ID NO: 1.
(Item 3) The isolated nucleic acid molecule according to item 1, wherein the molecule encodes the amino acid sequence set forth in SEQ ID NO: 2.
(Item 4) A substantially pure polypeptide comprising an amino acid sequence corresponding to ALK-7.
(Item 5) The polypeptide according to item 4, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2.
(Item 6) A nucleic acid probe for detecting the presence of ALK-7 in an individual-derived DNA sample, wherein the presence of the molecule according to item 1 in the sample is detected under stringent hybridization conditions. A probe comprising sufficient nucleic acid molecules for specific detection.
(Item 7) A method for detecting ALK-7 nucleic acid in a sample, comprising:
a) contacting the sample with the nucleic acid probe of item 6 under conditions such that hybridization occurs; and
b) detecting the presence of the probe bound to the ALK-7 nucleic acid;
Including the method.
(Item 8) A kit for detecting the presence of ALK-7 nucleic acid in a sample, comprising at least one container means having the nucleic acid probe according to item 6 disposed therein. 9. A recombinant nucleic acid molecule comprising 5 ′ to 3 ′ a promoter effective to initiate transcription in a host cell and the isolated nucleic acid molecule of item 1.
(Item 10) A recombinant nucleic acid molecule comprising a vector and the isolated nucleic acid molecule of item 1.
(Item 11) A cell comprising the recombinant nucleic acid molecule according to any one of items 9 or 10. (Item 12) A non-human organism comprising the recombinant nucleic acid molecule according to any one of items 9 or 10.
(Item 13) An antibody having binding affinity for the polypeptide according to item 4.
(Item 14) A method for detecting an ALK-7 polypeptide in a sample, comprising:
a) contacting the sample with the antibody of item 13 under conditions such that an immune complex is formed; and
b) detecting the presence of the antibody bound to the polypeptide;
Including the method.
(Item 15) Diagnostic kit:
a) first container means comprising the antibody of item 13, and
b) a second container means comprising a conjugate comprising a binding partner of said monoclonal antibody and a label;
A diagnostic kit comprising:
(Item 16) A hybridoma that produces the monoclonal antibody according to item 13.
(Item 17) A bioassay for evaluating a candidate drug or a ligand of an ALK-7 receptor,
a) contacting a candidate drug or ligand with a cell producing the polypeptide of item 4; and
b) assessing the biological activity mediated by the contact;
A bioassay.
(Item 18) A method for treating a neurological disease in a mammal, comprising a step of administering a therapeutically effective amount of the polypeptide of item 4 to the mammal in a gene delivery system.
(Item 19) The method according to item 18, wherein the disease is selected from the group consisting of a neurodegenerative disease, a neurodegenerative disorder, and a neurodegenerative injury.
(Item 20) A ligand capable of binding to the polypeptide of item 4.
(Item 21) A method for treating a neurological disease in a mammal, comprising a step of administering a therapeutically effective amount of the ligand according to item 21 to the mammal suffering from the disease.

FIG. Evolutionary relationship of the activin-like receptor kinase (ALK) family of serine-threonine kinase receptors. The relationship of ALK-7 to the receptor was shown. The amino acid sequence of rat ALK-1, rat ALK-2, mouse ALK-3, rat ALK-4, rat ALK-5, mouse ALK-6, and rat ALK-7 serine threonine kinase region, gene computer group of University of Wisconsin Aligned using the PILEUP program of (Genetic Computer Group). The alignment was then edited by LINEUP, and evolutionary relationships were calculated by DISTANCES using the default algorithm. The plot was prepared using GROWTREE. FIG. Analysis of ALK-7 protein. 3A and 3B. Expression of ALK-7 mRNA. 4A, 4B, and 4C. Expression of ALK-7 mRNA in the cerebellum. Figures 5A and 5B. Expression of ALK-7 mRNA analyzed by in situ hybridization.

Definitions In the description that follows, a number of terms used in recombinant DNA (rDNA) technology are used extensively. In order to provide a clear and consistent understanding of the specification and claims that include such terms within their scope, the following definitions are provided.

  Isolated nucleic acid molecule. An “isolated nucleic acid molecule” refers to a polymer of nucleotides, as commonly understood and used herein, and should not be limited to DNA and RNA.

  DNA segment. A DNA segment, as generally understood and used herein, refers to a molecule comprising linearly extended nucleotides. The nucleotide molecule is present in a sequence that can be encoded by the genetic code for a molecule comprising a linear sequence of amino acid residues. A molecule comprising a sequence of linear amino acid residues refers to a protein, protein fragment, or polypeptide.

gene. A DNA sequence relates to a single polypeptide chain or protein and includes 5 ′ and 3 ′ untranslated ends, as used herein. The polypeptide can encode a full-length sequence or any portion of the encoding sequence so long as it retains the functional activity of the protein.

  Complementary DNA (cDNA). Recombinant nucleic acid molecules were synthesized by reverse transcription of messenger RNA (mRNA).

Structural gene. The DNA sequence is transcribed into mRNA, which is then translated into an amino acid sequence having specific polypeptide characteristics.

Restriction endonuclease. A restriction endonuclease (or restriction enzyme) recognizes a specific base sequence (usually 4, 5, or 6 bp in length) within a DNA molecule and cleaves the DNA molecule at each occurrence of this sequence It can be an enzyme. For example, EcoRI recognizes the base sequence GAATTC / CTTAAG.

Restriction fragment. A restriction fragment refers to a DNA molecule produced by digestion with a restriction endonuclease. Any given genome can be digested into individual sets of restriction fragments by a particular restriction endonuclease.

Agarose gel electrophoresis. In order to detect restriction fragments of various lengths, an analytical method for fractionating double-stranded DNA molecules based on size is required. The most commonly used technique (although not the only method) to achieve such fractionation is agarose gel electrophoresis. The principle of this method is that the DNA molecules move through a gel like a sieve that slows the movement of the largest molecules through the gel to the greatest extent and the smallest molecules to the smallest extent. . It should be noted that smaller DNA fragments have greater mobility under agarose gel electrophoresis.

DNA fragments fractionated by agarose gel electrophoresis can be directly visualized by a staining procedure if the number of fragments contained in the pattern is small. Genomic DNA fragments can be brilliantly visualized. However, most genomes, including the human genome, contain too many DNA sequences to produce a simple restriction fragment pattern. For example, the human genome is digested into approximately 1,000,000 DNA fragments that differ by EcoRI.
To visualize a small subset of these fragments, a method called Southern hybridization procedure can be used.

Southern transfer procedure. The purpose of the Southern Transfer procedure (also known as blotting) is to retain the relative position of the DNA fragments obtained from the fractionation procedure on nitrocellulose filter paper or other suitable surface by fractionating the DNA by agarose gel electrophoresis. The physical dislocation is performed as it is. The method used to accomplish the rearrangement from agarose gel to nitrocellulose involves aspirating DNA from the gel to the nitrocellulose paper by capillary action.

Nucleic acid hybridization. Nucleic acid hybridization is based on the principle that two single-stranded nucleic acid molecules having complementary base sequences regenerate a thermodynamically preferred double-stranded structure when mixed under appropriate conditions. This double-stranded structure is formed between two complementary single-stranded nucleic acids, even if one of them is immobilized on nitrocellulose. The Southern hybridization procedure is the latter case. As described above, the individual DNAs to be tested are digested with restriction endonucleases, fractionated by agarose gel electrophoresis, converted to single-stranded form, and transferred to nitrocellulose paper, resulting in high Available for re-annealing with hybridization probes.

Hybridization probe. In order to visualize specific DNA sequences in the Southern hybridization procedure, labeled DNA molecules or hybridization probes are reacted with fractionated DNA bound to a nitrocellulose filter. The region on the filter that has a DNA sequence that is complementary to the labeled DNA probe is itself labeled as a result of the re-annealing reaction. The area on the filter showing such labeling is visualized. In general, this hybridization probe is produced by molecular cloning of a specific DNA sequence.

Oligonucleotide or oligomer. A molecule comprising two or more deoxyribonucleotides or ribonucleotides (preferably three or more). Its exact size depends on many factors, and they depend on the ultimate function or use of the oligonucleotide. Oligonucleotides can be obtained synthetically or by cloning.

  Sequence amplification. A method that produces large amounts of target sequences. Generally, one or more amplification primers anneal to the nucleic acid sequence. Using appropriate enzymes, sequences found adjacent to or between primers are amplified.

Amplification primer. An oligonucleotide that can anneal to the vicinity of a target sequence and serve as an initiation point for DNA synthesis when subjected to conditions that initiate the synthesis of a primer extension product that is complementary to a nucleic acid strand.

vector. Plasmid or phage DNA or other DNA sequence that can be inserted such that the DNA is cloned. The vector can replicate autonomously in the host cell and can be further characterized by one or a few endonuclease recognition sites. At this recognition site such a DNA sequence can be cleaved in a deterministic manner and the DNA can be inserted. The vector can further include an appropriate marker used to identify cells transformed with the vector. The marker is, for example, tetracycline resistance or ampicillin resistance. The term “cloning vehicle” is sometimes used to mean “vector”.

  Expression. Expression is the process by which a structural gene produces a polypeptide. This includes transcription of the gene into mRNA and translation of the mRNA into polypeptide (s).

  Expression vector. A vector or carrier similar to a cloning vector, except that the transformed gene can be expressed after transformation into the host. A cloned gene is usually placed under the control of certain regulatory sequences, such as a promoter sequence (ie, operably linked).

Expression control sequences can vary depending on whether the vector is designed to allow the operably linked gene to be expressed in a prokaryotic or eukaryotic host, and it can be further enhanced by an enhancer. It may comprise elements, termination sequences, tissue specific elements and / or transcription elements such as transcription start sites, transcription stop sites.

Functional derivative. A “functional derivative” of a sequence (either protein or nucleic acid) is a biological activity (either functional or structural) that is substantially similar to the biological activity of the protein or nucleic acid sequence. It has molecules. Functional derivatives of proteins can contain post-translational modifications (eg, covalently linked carbohydrates), depending on the need for such modifications to perform specific functions. The term “functional derivative” is intended to include a “fragment”, “segment”, “variant”, “analog”, or “chemical derivative” of a molecule.

As used herein, a molecule is said to be a “chemical derivative” of another molecule if it contains additional chemical moieties that are not normally part of that molecule. Such moieties can improve the solubility, absorbability, biological half-life, etc. of the molecule. Alternatively, this moiety can reduce the toxicity of the molecule, eliminating or attenuating unwanted side effects, etc. of the molecule. Portions that can mediate such effects are disclosed by Remington's Pharmaceutical Sciences (1980). Procedures for attaching such moieties to molecules are well known in the art.

Mutant. A “variant” of a protein or nucleic acid is meant to refer to a molecule whose structure and biological activity are substantially similar to the original protein or nucleic acid. Therefore, if it is provided that two molecules possess common activities and can be substituted for each other, the composition of one molecule, or the secondary, tertiary, or quaternary structure is found in the other. Even if the amino acid sequence or nucleotide sequence is not the same, it is considered a variant as the term used herein.

  Allele. An “allele” is another form of a gene that occupies a given locus on a chromosome.

Mutation. A “mutation” is any detectable change in genetic material that can be transmitted to daughter cells and possibly even to the next generation, resulting in mutant cells or mutant individuals. If the progeny of a mutant cell in a multicellular organism only gives rise to somatic cells, a mutant spot or region of the cell is generated. Mutations in the germline of a sexually reproducing organism can be transmitted to the next generation by gametes, resulting in individuals with new mutational states in both somatic and germ cells. A mutation is a chemical or physical composition, variability, replication, phenotypic function, or any (or combination of) detectable non-natural changes that affect the recombination of one or more deoxyribonucleotides. possible. That is, a nucleotide can be added, deleted, substituted, inverted, or translocated to a new position with and without inversion. Mutations can occur spontaneously and can be induced experimentally by utilizing mutagens. Variations in nucleic acid molecule variants occur as a result of mutations. A mutated polypeptide can result from a mutated nucleic acid molecule.

  seed. A “species” is a group of natural populations that actually or potentially mate. Species variation within a nucleic acid molecule or protein is a change in nucleic acid or amino acid sequence that occurs between species, which can be determined by DNA sequencing of the molecule in question.

  Substantially pure. A “substantially pure” protein or nucleic acid is a protein or nucleic acid preparation that generally lacks other cellular components.

Ligand. Ligand refers to any protein or proteins that can interact with the binding region of the ALK-7 receptor. The ligand (s) can be soluble or membrane bound. The ligand (s) can be produced naturally occurring protein or synthetically or recombinantly. A ligand can also be a non-protein molecule that acts as a ligand when interacting with the ALK-7 receptor binding region. The interaction between the ligand and the receptor binding region includes, but is not limited to, a covalent or non-covalent interaction. A receptor binding region is any region of an ALK-7 receptor molecule that interacts directly or indirectly with an ALK-7 ligand. ALK-7 agonists and antagonists that can interact with the binding domain of the ALK-7 receptor are ligands.

Neurodegenerative disease. The term neurodegenerative disease includes, but is not limited to, mammalian conditions that may include chromosomal abnormalities, degenerative growth, and developmental disorders, viral infections, bacterial infections, brain contusions, or neoplastic conditions. Examples of neurodegenerative diseases that can be diagnosed, evaluated, or treated by the methods described herein include, but are not limited to, Alzheimer's disease, epilepsy, and schizophrenia. In preferred embodiments, specific diseases characterized by limbic neurodegeneration are diagnosed, evaluated, or treated by the methods disclosed herein. Examples of nervous system disorders include, but are not limited to, stroke or cardiac arrest and cerebral ischemia. Treatment of nervous system damage is also considered within this definition. In addition, neoplasms that contain neural tissue can be diagnosed, evaluated, or therapeutically treated by the methods set forth herein.

Drug. Drugs include, but are not limited to, proteins, peptides, modified peptides, drugs purified from prepared cell culture media, organic molecules, inorganic molecules, antibodies, or oligonucleotides. Other drug candidates include analogs of ALK-7 ligand (s). The drug can be naturally occurring or can be produced synthetically or recombinantly. One skilled in the art will appreciate that such drugs can be developed by the assays described below.

(Detailed description of the invention)
For purposes of clarity of disclosure, and not limitation, the detailed description of the invention is divided into the following subsections:

  I. An isolated nucleic acid molecule encoding an ALK-7 polypeptide.

  II. A substantially pure ALK-7 polypeptide.

  III. Nucleic acid probe for specific detection of ALK-7.

  IV. A method for detecting the presence of ALK-7 in a sample.

  V. A kit for detecting the presence of ALK-7 in a sample.

  VI. DNA constructs containing ALK-7 nucleic acid molecules and cells containing these constructs.

VII. An antibody having binding affinity for ALK-7 polypeptide and a hybridoma comprising the antibody.

  VIII. A method for detecting an ALK-7 polypeptide in a sample.

  IX. A diagnostic kit comprising an antibody against ALK-7.

  X. Diagnostic screening and treatment.

  XI. Transgenic ALK-7 “knockout” mice.

(I. Isolated nucleic acid molecule encoding ALK-7 polypeptide)
In one embodiment, the invention relates to an isolated nucleic acid molecule encoding a polypeptide having an amino acid sequence corresponding to a novel serine threonine kinase receptor, ALK-7. In one preferred embodiment, the isolated nucleic acid molecule comprises an ALK-7 nucleotide sequence having greater than 70% similarity to the ALK-7 nucleotide sequence present in SEQ ID NO: 1 (preferably greater than 80%; more Preferably greater than 90%). In another preferred embodiment, the isolated nucleic acid molecule comprises the ALK-7 nucleotide sequence present in SEQ ID NO: 1. In another embodiment, the isolated nucleic acid molecule encodes the ALK-7 amino acid sequence present in SEQ ID NO: 2.

Further, within the scope of the present invention are also functional equivalents of the isolated nucleic acid molecules described herein, and derivatives thereof. For example, the nucleic acid sequence shown in SEQ ID NO: 1 can be altered by substitutions, additions, or deletions that provide a functionally equivalent molecule. Due to the degeneracy of the nucleotide coding sequence, other DNA sequences that encode substantially the same amino acid sequence as shown in SEQ ID NO: 2 can be used in the practice of the invention. These are nucleotides comprising all or part of the ALK-7 nucleic acid set forth in SEQ ID NO: 1 that are altered by substitution of different codons encoding functionally equivalent amino acid residues within the sequence, thus producing a silent change. Including but not limited to sequences.

Such functional changes in a given nucleic acid sequence provide an opportunity to facilitate secretion and / or processing of a heterologous protein encoded by a foreign nucleic acid sequence fused to the given nucleic acid sequence. Thus, all variants of the nucleic acid sequence of the ALK-7 gene and ALK-7 gene fragments enabled by the genetic code are encompassed within the present invention.

In addition, the nucleic acid sequence comprises the 5 ′ end of
Or it may include a nucleotide sequence resulting from the addition, deletion, or substitution of at least one nucleotide relative to the 3 ′ end. Any nucleotide or polynucleotide in this regard can be used provided that the addition, deletion, or substitution does not change the amino acid sequence of SEQ ID NO: 2 encoded by the nucleotide sequence. Furthermore, the nucleic acid molecule of the present invention comprises
If necessary, it may have a restriction endonuclease recognition site added to its 5 'end and / or 3' end.

In addition, to generate a polypeptide that is a structurally modified polypeptide but has substantially the same utility or activity as a polypeptide produced by an unmodified nucleic acid molecule, Alternatively, one or more codons can be replaced with codons other than degenerate codons. As recognized in the art, two polypeptides are functionally equivalent, as are the two nucleic acid molecules that produce them, even if the differences between the nucleic acid molecules are not related to the degeneracy of the genetic code. belongs to.

(A. Nucleic acid isolation)
In one aspect of the invention, an isolated nucleic acid molecule encoding a polypeptide having an amino acid sequence corresponding to ALK-7 is provided. In particular, the nucleic acid molecule can be isolated from a biological sample containing human RNA or DNA.

Nucleic acid molecules can be isolated from biological samples containing human RNA using cDNA cloning and subtractive hybridization techniques. Nucleic acid molecules can also be isolated from cDNA libraries using homologous probes.

  Nucleic acid molecules can be isolated from biological samples or genomic libraries containing human genomic DNA. Suitable biological samples include, but are not limited to blood, semen, and tissue. The method of obtaining a biological sample can vary depending on the state of the sample.

One skilled in the art can appreciate that the human genome can undergo slight allelic changes between individuals. Thus, an isolated nucleic acid molecule is also intended to contain allelic variations so long as the sequence is a functional derivative of ALK-7. If the ALK-7 allele does not encode the same sequence as found in SEQ ID NO: 1, then the ALK-7 allele is disclosed in the same techniques used herein and in particular herein. It can be isolated and identified as ALK-7 using PCR techniques that amplify the appropriate gene using primers based on the sequence.

One skilled in the art can understand that organisms other than rats also contain the ALK-7 gene (eg,
Eukaryotes; more particularly mammals, birds, fish and plants; more particularly humans, gorillas, rhesus monkeys and chimpanzees; preferably human ALK-7). The present invention is intended to include, but is not limited to, ALK-7 nucleic acid molecules isolated from the above organisms.

(B. Synthesis of nucleic acids)
Isolated nucleic acid molecules of the present invention are also intended to include chemically synthesized nucleic acid molecules. For example, nucleic acid molecules having a nucleotide sequence encoding the expression product of the ALK-7 gene can be designed and, if necessary, divided into suitable smaller fragments. Next, oligomers corresponding to the nucleic acid molecules or each fragmented can be synthesized. Such synthetic oligonucleotides can be prepared, for example, by the triester method of Matteucci et al., J. Am. Chem. Soc. 103: 3185-3191 (1981), or by using an automated DNA sequencer.

Oligonucleotides can be obtained synthetically or by cloning. If necessary, the 5 'end of the oligomer can be phosphorylated using T4 polynucleotide kinase. Single strand phosphorylation before annealing or phosphorylation for labeling can be accomplished using an excess of enzyme. If the phosphorylation is for probe labeling, ATP may contain a high specific activity radioisotope. Next, the DNA oligomer is annealed,
And can be subjected to ligation using T4 ligase and the like.

II. Substantially pure ALK-7 polypeptide
In another embodiment, the invention relates to a substantially pure polypeptide having an amino acid sequence corresponding to ALK-7, or a functional derivative thereof. In a preferred embodiment, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2, or a variant or species mutation thereof, or at least 70% identity or at least 85% similarity (preferably at least 90% identity). Or at least 95% similarity), or at least 43 contiguous amino acids thereof (preferably at least 50 or 100 contiguous amino acids thereof).

ALK-7 amino acid variants can be prepared by mutations in DNA. Such variants include, for example, deletions, insertions or substitutions of residues within the amino acid sequence shown in SEQ ID NO: 2. Any combination of deletions, insertions, and substitutions can also be made to arrive at the final construct, provided that the final construct has the desired activity. Obviously, mutations made in the DNA encoding the variant must not place its sequence outside of the reading frame and preferably do not create a complementary region capable of generating a secondary mRNA structure.

The site for introducing an amino acid sequence variation is predetermined, but the mutation per se need not be predetermined. For example, to maximize mutation potential at a given site, random mutagenesis can be performed at the target codon or region, and the expressed ALK-7 mutants screened for the optimal combination of desired activities. Can be done. A technique for performing substitution mutation at a predetermined site in DNA having a known sequence is well known (for example,
Site-directed mutagenesis).

Preparation of ALK-7 mutants according to the present invention is preferably accomplished by site-directed mutagenesis of DNA encoding an initially prepared mutant or non-mutant version of this protein. Site-directed mutagenesis is a specific oligonucleotide sequence that encodes the DNA sequence of the desired mutation, as well as sufficient size and sequence complexity to form a stable duplex on both sides of the traversing deletion junction ALK-7 variants are generated by using a sufficient number of contiguous nucleotides to provide a primer sequence of Typically, a primer about 20-25 nucleotides in length is preferred, with about 5-10 residues changed on either side of the sequence junction. In general, site-directed mutagenesis techniques are well known to those skilled in the art, as exemplified by publications such as Adelman et al., DNA2; 183 (1983).

As will be appreciated, site-directed mutagenesis techniques typically employ phage vectors that exist in both single-stranded and double-stranded forms. Typical vectors useful for site-directed mutagenesis include vectors such as the M13 phage disclosed in Messing et al., Third Cleveland Symposium on Macromolecules and Recombinant DNA, edited by A. Walton, Elsevier, Amsterdam (1981). These phage are readily available commercially and their use is generally well known to those skilled in the art. Alternatively, a plasmid vector containing a single-stranded phage origin of replication (Vieira et al., Meth. Enzymol. 153: 3 (1987)) can be used to obtain single-stranded DNA.

In general, site-directed mutagenesis according to the present invention is performed by first obtaining a single stranded vector containing a DNA sequence encoding the appropriate protein within the sequence. Oligonucleotide primers having the desired mutated sequence are generally prepared synthetically (eg, by the method of Crea et al., Proc. Natl. Acad. Sci. (USA) 75: 5765 (1978)). This primer is then annealed with a single stranded vector containing the protein sequence and subjected to a DNA polymerizing enzyme such as an E. coli polymerase I Klenow fragment to complete synthesis of the mutated strand. . Thus, the mutated sequence and the second strand have the desired mutation. This heteroduplex vector is then used to transform appropriate cells and clones containing a recombinant vector with a mutated sequence arrangement are selected.

  After such a clone is selected, the mutant protein region is excised and placed in a suitable vector (generally an expression vector of the type that can be used to transform a suitable host) for protein production. obtain.

  Amino acid sequence deletions generally range from about 1-30 residues, more preferably 1-10 residues, and are typically contiguous.

Amino acid sequence insertions include amino and / or carboxyl terminal fusions from one residue to essentially unlimited polypeptide length, as well as intrasequence insertions of single or multiple amino acid residues. Insertions within the sequence (ie insertions within the complete ALK-7 sequence) can generally range from about 1 to 10 residues, more preferably 1 to 5 residues.

The third group of variants are variants in which at least one amino acid residue in the ALK-7 molecule, preferably only one amino acid, has been removed and a different residue inserted in its place. Such substitutions are preferably made according to the following Table 1 when it is desired to finely adjust the characteristics of ALK-7.

Essential changes in functional or immunological identity are made by selecting substitutions that are less conservative than those in Table 1. That is, by selecting residues that have significantly different effects in maintaining the following: (a) the structure of the polypeptide backbone in the substitution region (eg, a sheet or helix structure) (b) the molecule at the target site Change or hydrophobicity, or (c) side chain size. Common substitutions are expected in the following: (a) replacing glycine and / or proline with another amino acid, or deleting or inserting; (b) a hydrophilic residue (eg, Serine or threonine) for (or) a hydrophobic residue (eg, leucine, isoleucine, phenylalanine, valine, or alanine); (c) a cysteine residue relative to any other residue (D) a positively charged residue (eg, lysine, arginine or histidine) relative to a negatively charged residue (eg, glutamic acid or aspartic acid) Or (e) replacing a residue having a large side chain (eg, phenylalanine) with (or into) a residue having no such side chain (eg, glycine) That.

Some deletions and insertions and substitutions are not expected to cause major changes in the properties of ALK-7. However, if the exact effect of a substitution, deletion, or insertion is difficult to estimate in advance, one skilled in the art can recognize that the effect can be assessed in routine screening assays. For example, the variant is typically site-directed mutagenesis of the nucleic acid encoding the original ALK-7, expression of the variant nucleic acid in recombinant cell culture, and optionally from the cell culture. (Eg, immunoaffinity adsorption through a column (adsorbing variants by binding to at least one residual immune epitope)). The activity of the cell lysate or purified ALK-7 molecular variant is then screened in a suitable screening assay for the desired property. For example, the immunological properties of the ALK-7 molecule (
For example, a change in affinity for a given antibody is measured in a competitive immunoassay. Changes in immunomodulatory activity are measured by appropriate assays. Modification of such protein properties (redox or temperature stability, hydrophobicity, susceptibility to proteolysis or tendency to aggregate with carriers or multimers) is assayed by methods well known to those skilled in the art.

A variety of methodologies known in the art can be used to obtain the peptides of the present invention. In one embodiment, the peptide is purified from tissues or cells that naturally produce the peptide. Alternatively, the isolated nucleic acid fragment described above could be used to express ALK-7 protein in any organism. Samples of the invention include cells, cellular protein extracts or membrane extracts, or biological fluids. The sample can vary based on the assay format, the detection method, and the nature of the tissue, cells, or extract used as the sample.

Any eukaryote can be used as a source of the peptides of the invention, so long as the biological source naturally includes such peptides. As used herein, “biological source” refers to the original organism from which the amino acid sequence of the subunit is derived, and relates to the organism in which the subunit is expressed and ultimately isolated. Without being told.

  One skilled in the art can readily perform known methods of isolating proteins to obtain peptides free of natural contaminants. These include, but are not limited to, immunochromatography, size exclusion chromatography, HPLC, ion exchange chromatography, and immunoaffinity chromatography.

III. Nucleic Acid Probes for Specific Detection of ALK-7 In another embodiment, the present invention binds to ALK-7 under stringent conditions for the above-described nucleic acid molecules or ALK-7. A nucleic acid probe for specific detection of the presence of ALK-7 in a sample comprising at least one fragment herein that does not bind ALK-1 to ALK-6. This probe is designed not to have 100% homology with the similarly located ALK probe.

Nucleic acid probes can be used to probe an appropriate chromosomal or cDNA library by conventional hybridization methods to obtain another nucleic acid molecule of the invention. Chromosomal DNA or cDNA libraries can be prepared from appropriate cells according to methods recognized in the art (see MolecularCloning: A Laboratory Manual, Second Edition, Sambrook, Fritsch, and Maniatis, Ed., Cold Spring Harbor Laboratory, 1989). ).

Alternatively, chemical synthesis can be performed to obtain a nucleic acid probe having nucleotide sequences corresponding to the N-terminal and C-terminal portions of the amino acid sequence of ALK-7. Thus, synthesized nucleic acid probes can be used as primers in polymerase chain reaction (PCR) performed according to recognized PCR techniques. In essence, according to PCR Protocols, A Guide to Methods and Applications, edited by Michael et al., Academic Press, 1990, an appropriate chromosome or cDNA library is used to obtain the fragments of the present invention.

  One skilled in the art can readily design such probes based on the sequences disclosed herein using computer alignment methods and sequence analysis known in the art (MolecularCloning: A Laboratory Manual, Second Edition). , Sambrook, Fritsch, and Maniatis, Cold Spring Harbor Laboratory, 1989).

The hybridization probes of the present invention can be labeled by standard labeling techniques (eg, radioactive labels, enzyme labels, fluorescent labels, biotin-avidin labels, chemiluminescence, etc.). After hybridization, the probe can be visualized using known methods.

  The nucleic acid probes of the present invention include RNA probes as well as DNA probes, and such probes are generated using techniques known in the art.

In one embodiment of the above method, the nucleic acid probe is immobilized on a solid support. Examples of such solid supports include, but are not limited to, plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, and acrylic resins such as polyacrylamide and latex beads. Techniques for binding nucleic acid probes to these solid supports are well known in the art.

Test samples suitable for the nucleic acid probe method of the present invention include, for example, cells or nucleic acid extracts of cells, or biological fluids. The sample used in the above methods can vary based on the assay format, detection method, and the nature of the tissue, cell, or extract being assayed. Methods for preparing cellular nucleic acid extracts are well known in the art and can be readily adapted to obtain a sample compatible with the method used.

IV. Method for Detecting the Presence of ALK-7 in a Sample In another embodiment, the present invention is a method for detecting the presence of ALK-7 in a sample, a) under conditions such that hybridization occurs. A method comprising contacting a sample with a nucleic acid probe as described above, and b) detecting the presence of a probe that binds to a nucleic acid molecule. One skilled in the art can select nucleic acid probes according to techniques well known in the art, as described above. Samples to be tested include, but should not be limited to, human tissue RNA samples.

ALK-7 has been found to be expressed in brain cells. Thus, the ALK-7 probe can be used to detect the presence of RNA from brain cells in the sample. Furthermore, a change in the expression level of ALK-7 RNA in an individual compared to the normal expression level may indicate the presence of a disease. In addition, ALK-7 probes can be used to assay general and specific cellular activity in brain tissue.

V. Kits for detecting the presence of ALK-7 in a sample In another embodiment, the invention relates to a kit for detecting the presence of ALK-7 in a sample. The kit includes at least one container means in which the nucleic acid probe is disposed. In a preferred embodiment, the kit further comprises another container comprising one or more of the following: a washing reagent and a reagent capable of detecting the presence of the bound nucleic acid probe. Examples of detection reagents include, but are not limited to, radioactive label probes, enzyme label probes (horseradish peroxidase, alkaline phosphatase), and affinity label probes (biotin, avidin, or streptavidin).

Specifically, compartmentalized kits include any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers, or plastic or paper pieces. Such containers allow for efficient transport of reagents from one container to another. Thereby, the sample and the reagent do not mix with each other. The drug or solution in each container can then be added from one container to another in a quantitative manner. Such containers include containers containing test samples, containers containing probes or primers used in the assay, containers containing wash reagents (eg, phosphate buffered saline, Tris buffer, etc.), and hybridized It includes a container that contains reagents used to detect probes, bound antibodies, amplification products, and the like.

  One skilled in the art will readily recognize that the nucleic acid probes described in the present invention can be readily incorporated into established kit forms well known in the art.

VI. DNA constructs comprising ALK-7 nucleic acid molecules and cells comprising these constructs In another embodiment, the present invention comprises a promoter effective to initiate transcription in a host cell from 5 ′ to 3 ′ and the above-described Recombinant DNA molecules, including nucleic acid molecules. In another embodiment, the present invention relates to a recombinant DNA molecule comprising a vector and a nucleic acid molecule as described above.

In another embodiment, the present invention provides a transcriptional regulatory region functional in a cell, a sequence complementary to an RNA sequence encoding an amino acid sequence corresponding to the above polypeptide, and a transcription termination region functional in a cell. A nucleic acid molecule comprising

  Preferably, the molecule is an isolated and / or purified DNA molecule.

  In another embodiment, the present invention relates to a cell or non-human organism comprising a nucleic acid molecule as described above.

  In another embodiment, the peptide is purified from cells that have been modified to express the peptide.

As used herein, when a cell is made to produce a protein that is not normally produced by genetic engineering or that the cell normally produces only at low levels, the cell Is modified to express the peptide of ". One skilled in the art can readily adapt the procedure to introduce and express either genomic, cDNA or synthetic sequences into eukaryotic or prokaryotic cells.

A nucleic acid molecule, such as DNA, contains a nucleotide sequence that includes transcriptional and translational regulatory information, and when such a sequence is “operably linked” to a nucleotide sequence that encodes the polypeptide, the polypeptide Is said to be “expressable”. An operable linkage is a linkage in which a regulatory DNA sequence and a DNA sequence that is required to be expressed are connected in such a way as to allow gene sequence expression. The exact nature of the regulatory regions required for gene sequence expression can vary from organism to organism. In general, however, in prokaryotes, a promoter (leads to initiate RNA transcription) and DNA that initiates signal synthesis when transcribed into RNA.
Contains a promoter region containing both sequences. Such regions usually include 5 ′ non-coding regions involved in transcription and translation initiation (eg, TATA box, cap sequence, CAAT sequence, etc.).

If desired, a non-coding region 3 ′ of the sequence encoding the ALK-7 gene can be obtained by the above method. This region can be maintained in its transcription termination regulatory sequence (eg, termination and polyadenylation). Thus, maintaining a 3 ′ region naturally adjacent to the DNA sequence encoding the ALK-7 gene can provide a transcription termination signal. If the transcription termination signal does not function satisfactorily in the expression host cell, a functional 3 ′ region in the host cell can be replaced.

Two DNA sequences (eg, promoter region sequence and ALK-7 sequence) are said to be operably linked if the nature of the bond between the two DNA sequences is as follows: (1) Frameshift mutation Does not interfere with the ability of the promoter region sequence to direct transcription of the ALK-7 gene sequence, or (3) does not interfere with the ability of the ALK-7 gene sequence to be transcribed by the promoter region sequence . Thus, a promoter region is operably linked to a DNA sequence if the promoter can affect the transcription of that DNA sequence.

  The present invention achieves expression of the ALK-7 gene (or functional derivatives thereof) in either prokaryotic or eukaryotic cells. Prokaryotic hosts are generally the most efficient and convenient for the production of recombinant proteins and are therefore preferred for the expression of the ALK-7 gene.

The most frequent prokaryotes are represented by various strains of E. coli. However, other microbial strains, including other bacterial strains, can also be used. In prokaryotic systems, plasmid vectors containing replication sites and control sequences derived from species compatible with the host may be used. Examples of suitable plasmid vectors include pBR322, pUC118, pUC119, etc .; suitable phage or bacteriophage vectors include λgt10, λgt11, etc .; and suitable viral vectors include pMAM-neo, pKRC Etc. Preferably, the selected vector of the present invention has the ability to replicate in the selected host cell.

Recognized prokaryotic hosts include bacteria such as E. coli, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia and the like. However, under these conditions,
The peptide is not glycosylated. Prokaryotic hosts must be compatible with the replicon and regulatory sequences in the expression plasmid.

In order to express ALK-7 in prokaryotic cells, it is necessary to operably link the ALK-7 sequence to a functional prokaryotic promoter. Such promoters can be either constitutive, or more preferably regulatable (ie inducible or disinhibitory). Examples of constitutive promoters include bacteriophage λ int promoter, pBR322 β-lactamase gene sequence bla promoter, pBR325 chloramphenicol acetyltransferase gene sequence CAT promoter, and the like. Examples of inducible prokaryotic promoters, the major right and left promoters (P _L and P _R) of bacteriophage lambda, trp of E. coli, recA, lacZ, lacI, and gal promoters, alpha-amylase (Ulmanen Et al., J. Bacteriol. 162: 176-182 (1985)), and B. subtilis
Ζ-28 specific promoter (Gilman et al., Genesequence 32: 11-20 (1984)), Bacillus bacteriophage promoter (Gryczan, The Molecular Biology of the Bacilli, Academic Press, Inc. NY (1982)), and Streptomyces promoter (Ward et al., Mol. Gen. Genet. 203: 468-478 (1986)). Prokaryotic promoters include Glick (J. Ind. Microbiol. 1: 277-282 (1987)); Centatiempo (Biochimie 68: 505-516 (1986)), and Gottesman (Ann. Rev. Genet. 18: 415-442 ( 1984)).

  Proper expression in prokaryotic cells also requires the presence of a ribosome binding site upstream of the gene sequence coding sequence. Such ribosome binding sites are disclosed, for example, by Gold et al. (Ann. Rev. Microbiol. 35: 365-404 (1981)).

The choice of control sequences, expression vectors, transformation methods, etc. depends on the type of host cell used to express the gene. As used herein, “cell”, “cell line”
And “cell culture” can be used interchangeably and all such names include progeny. Thus, the term “transformants” or “transformed cells” includes the first served cells and cultures derived from them, regardless of the passage number. It is also understood that all progeny cannot be exactly the same in DNA content due to intentional or accidental variants. However, as defined, mutant progeny have the same function as the originally transformed cell.

The host cells that can be used in the expression system of the present invention are not strictly limited as long as they are suitable for use in expressing the target ALK-7 peptide. Suitable hosts include eukaryotic cells.

  Examples of preferred eukaryotic hosts include, for example, yeast, fungi, insect cells, mammalian cells, either in vivo or in tissue culture. Preferred mammalian cells include HeLa cells, fibroblast-derived cells (eg, VERO or CHO-K1), lymphocyte-derived cells, and derivatives thereof.

  In addition, plant cells can also be utilized as hosts. Control sequences that are compatible with plant cells (eg, cauliflower mosaic virus 35S and 19S, and nopaline synthase promoter and polyadenylation signal sequences) can also be utilized.

Another preferred host is an insect cell (eg, Drosophila larvae). The Drosophila alcohol dehydrogenase promoter can be used using insect cells as the host (Rubin, Science 240: 1453-1459 (1988)). Alternatively, baculovirus vectors can be engineered to express large amounts of ALK-7 in insect cells (Jasny, Science 238: 1653 (1987); Miller et al., Genetic Engineering (1986), edited by Setlow, JK et al., Plenum 8: 277-297).

Different host cells have characteristic and specific mechanisms for protein translation and post-translational processing and modification (eg, glycosylation, cleavage). Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed. For example, expression in bacterial systems can be used to produce a non-glycosylated core protein product. Expression in yeast produces a glycosylated product. Expression in mammalian cells can be used to ensure “natural” glycosylation of heterogeneous ALK-7 proteins. Furthermore, different vector / host expression systems can result in processing reactions such as different degrees of proteolytic cleavage.

Any series of yeast gene sequence expression systems can be utilized. It incorporates a promoter derived from a gene sequence encoding an active glycolytic enzyme and a termination factor, and is produced in large quantities when grown on a medium rich in glucose. Known glycolytic gene sequences can also provide very efficient transcriptional control signals.

Yeast offers the substantial advantage that post-translational peptide modifications can also be made. There are many recombinant DNA strategies that utilize strong promoter sequences and multiple copy number plasmids that can be utilized to produce the desired protein in yeast. Yeast recognizes leader sequences on cloned mammalian gene sequence products and secretes peptides with leader sequences (ie, prepeptides). For mammalian hosts, several possible vector systems can be utilized for the expression of ALK-7.

A wide variety of transcriptional and translational regulatory sequences can be used, depending on the nature of the host. Transcriptional and translational regulatory signals may be derived from viral sources such as adenovirus, bovine papilloma virus, monkey virus, etc., where the regulatory signal is associated with a specific gene sequence having a high level of expression To do. Alternatively, promoters derived from mammalian expression products (eg, actin, collagen, myosin, etc.) can be used. A transcription initiation regulatory signal that allows repression or activation can be selected. As a result, the expression of the gene sequence can be altered. The regulatory signal in question is a temperature sensitive regulation such that expression can be suppressed or initiated by changing the temperature, or a regulatory signal subject to chemical (eg, metabolite) regulation.

As mentioned above, expression of ALK-7 in a eukaryotic host requires the use of a eukaryotic regulatory region. Such a region generally includes a promoter region sufficient to direct the initiation of RNA synthesis. Preferred eukaryotic promoters include, for example, the promoter of the mouse metallothionein I gene sequence (Hamer et al., J. Mol. Appl. Gen. 1: 273-288 (1982)); the herpesvirus TK promoter (McKnight, Cell 31: 355-365 (1982)); SV40 early promoter (Benoist et al., Nature (London) 290: 304-310 (1981)); yeast gal4 gene sequence promoter (Johnston et al., Proc. Natl. Acad. Sci. (USA) 79 : 6971-6975 (1982); Silver et al., Proc. Natl. Acad. Sci. (USA) 81: 5951-5955 (1984)).

As is widely known, translation of eukaryotic RNA begins with a codon that encodes the first methionine. To this end, it is preferable to ensure that the linkage between the eukaryotic promoter and the DNA sequence encoding ALK-7 does not contain any intervening codon (ie AUG) that can encode methionine. The presence of such a codon is either a fusion protein (if the AUG codon is in the same reading frame as the ALK-7 coding sequence) or a frameshift mutation (if the AUG codon is not in the same reading frame as the ALK-7 coding sequence). Bring.

The ALK-7 nucleic acid molecule and the operably linked promoter are either linear or more preferably covalently closed circular molecules to the recipient prokaryotic or recipient eukaryotic cell, It can be introduced as any non-replicating DNA (or RNA) molecule. Since such molecules cannot self-replicate, gene expression results from transient expression of the introduced sequence. Alternatively, permanent expression can occur by integration of the introduced DNA sequence into the host chromosome.

In one embodiment, a vector is used that is capable of integrating the desired gene sequence into the host cell chromosome. Cells in which the introduced DNA is stably integrated into the cell's chromosomes
It can also be selected by introducing one or more markers that allow the selection of host cells containing the expression vector. The marker may provide resistance to bactericides (eg, antibiotics, heavy metals such as copper) for prototrophy to autotrophic hosts. The selectable marker gene sequence can either be directly linked to the DNA gene sequence to be expressed or introduced by co-transfection into the same cell. Another factor may also be required for optimal synthesis of single-stranded binding protein mRNA. These factors include splice signals, as well as transcription promoters, enhancers, and termination signals. CDNA expression vectors incorporating such factors include those described by Okayama, Molec. Cell. Biol. 3: 280 (1983).

In a preferred embodiment, the introduced nucleic acid molecule is incorporated into a plasmid or vector capable of self-replication in the recipient host. Any of a wide variety of vectors can be used for this purpose. The key elements for selecting a particular plasmid or viral vector are: the ease with which recipient cells containing the vector are recognized and selected from recipient cells that do not contain the vector; desired within a particular host Whether it is desirable to be able to “shuttle” the vector between host cells of different species. Preferred prokaryotic vectors include plasmids such as plasmids that can replicate in E. coli (eg, pBR322, ColE1, pSC101, pACYC 184, πVX). Such plasmids are disclosed, for example, by Sambrook (cf. Molecular Cloning: A Laboratory manual, 2nd edition, edited by Sambrook, Fritsch & Maniatis, Cold Spring Harbor Laboratory, 1989). Bacillus plasmids include pC194, pC221, pT127 and the like. Such a plasmid is disclosed by Gryczan (The Molecular Biology of the Bacilli, Academic Press, NY (1982, pages 307-329)). Suitable Streptomyces plasmids are pIJ101 (Kendall et al., J. Bacteriol. 169: 4177-4183 (1987)) and φC31 (Chater et al., Sixth International Symposiumon Actinomycetales Biology, Akademiai Kaido, Budapest, Hungary (1986), pages 45-54). Including streptomyces bacteriophages. The Pseudomonas plasmid has been reviewed by John et al. (Rev. Infect. Dis. 8: 693-704 (1986)) and Izaki (Jpn. J. Bacteriol. 33: 729-742 (1978)).

Preferred eukaryotic plasmids include, for example, BPV, vaccinia, SV40, 2 μm circles, etc., or derivatives thereof. Such plasmids are well known in the art (Botstein
MiamiWntr.Symp. 19: 265-274 (1982); Broach, The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 445-470 (1981); Broach, Cell 28: 203-204 (1982); Bollon et al., J. Clin. Hematol. Oncol. 10: 39-48 (1980); Maniatis, Cell Biology: A Comprehensive Treatise, Volume 3, GeneSequenceExpression, Academic Press, NY, 563-608 Page (1980)).

Once the vector or nucleic acid molecule containing the construct is prepared for expression, the DNA construct can be transformed into any of a variety of suitable means (ie, transformation, transfection, conjugation, protoplast fusion, electroporation, particle It can be introduced into an appropriate host cell by cancer techniques, calcium phosphate precipitation, direct microinjection, etc. After the introduction of the vector, the recipient cell is grown in a selective medium, thereby selecting the growth of the cell containing the vector. Expression of the modified gene molecule results in the production of ALK-7, which may be in transformed cells or after induction of these cells for differentiation (eg bromodeoxyuracil into neuroblastoma cells, etc.) Can occur).

VII. Antibodies having binding affinity for ALK-7 polypeptides and hybridomas comprising such antibodies In another embodiment, the present invention is specific for an ALK-7 polypeptide as described above, or an ALK thereof. -7 relates to an antibody having specific binding affinity for a polypeptide binding fragment. If the antibody does not bind to ALK-1 to ALK-6, it will specifically bind to the ALK-7 polypeptide or binding fragment thereof. An antibody that selectively binds to ALK-7 is selected for use in a method that may (but should not be limited to) the analysis of altered ALK-7 expression in tissues containing ALK-7. Is done.

The ALK-7 proteins of the invention can be used in a variety of procedures and methods (eg, for the production of antibodies, for the identification of pharmaceutical compositions, and for the study of DNA / protein interactions).

The ALK-7 peptides of the present invention can be used to make antibodies or hybridomas. It will be appreciated by those of skill in the art that if antibodies are desired, such peptides are made as described herein and used as immunogens.

The antibodies of the present invention include monoclonal and polyclonal antibodies, as well as fragments of these antibodies. The present invention further includes single chain antibodies. Antibody fragments that contain the idiotype of the molecule can be generated by known techniques. For example, such fragments include, but are not limited to, F (ab ′) ₂ fragments, Fab ′ fragments, Fab fragments, and Fv fragments.

Of particular interest to the present invention are antibodies that have been produced in humans or “humanized” (ie, non-immunogenic in humans) by recombinant or other techniques. Humanized antibodies can be made, for example, by replacing the immunogenic portion of an antibody with a corresponding non-immunogenic portion (ie, a chimerized antibody) (Robinson, RR et al., International Patent Publication PCT / US86). / 02269; Akira, K. et al., European Patent Application No. 184,187; Taniguchi, M., European Patent Application No. 171,496; Morrison, SL et al., European Patent Application No. 173,494; Neuberger, MS et al., PCT Application WO86 / 01533 ; Cabilly, S. et al., European Patent Application No. 125,023; Better, M. et al., Science 240: 1041-1043 (1988); Liu, AY et al., Proc. Natl. Acad. Sci. USA 84: 3439-3443 (1987) Liu, A.
Y. et al., J. Immunol. 139: 3521-3526 (1987); Sun, LK et al., Proc. Natl. Acad. Sci. USA 84: 214-218 (1987); Nishimura, Y. et al., Canc. Res. : 999-1005 (1987); Wood, CR et al., Nature 314: 446-449 (1985); Shaw et al., J. Natl. Cancer Inst. 80: 1553-1559 (1988)).
A general review of “humanized” chimeric antibodies is provided by Morrison, SL (Science 229: 1202-1207 (1985)) and Oi, VT et al. (BioTechniques 4: 214 (1986)). Suitable “humanized” antibodies can be made by CDR or CEA substitution (Jones, PT et al., Nature 321: 552-525 (1986); Verhoeyan et al., Science 239: 1534 (1988); Beidler, CB et al., J .Immunol.
141: 4053-4060 (1988)).

  In another embodiment, the present invention relates to a hybridoma producing the above monoclonal antibody. Hybridomas are immortalized cell lines that can secrete specific monoclonal antibodies.

  In general, techniques for preparing monoclonal antibodies and hybridomas are well known in the art (Campbell, “Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology”, Elsevier Science Publishers, Amsterdam, The Netherlands (1984); St. Groth et al., J. Immunol. Methods 35: 1-21 (1980)).

Any animal (mouse, rabbit, etc.) known to produce antibodies can be immunized with the selected polypeptide. Methods for immunization are well known in the field. Such methods include subcutaneous or intraperitoneal injection of the polypeptide. It will be appreciated by those skilled in the art that the amount of polypeptide used for immunization will vary based on the animal to be immunized, the antigenicity of the polypeptide, and the site of injection.

The polypeptide can be modified or administered in an adjuvant to increase the antigenicity of the peptide. Methods for increasing the antigenicity of a polypeptide are well known in the art. Such procedures include conjugation of antibodies and heterologous proteins (eg, globulin or β-galactosidase), or through inclusion of an adjuvant during immunization.

  For monoclonal antibodies, spleen cells from the immunized animal are removed, fused with myeloma cells, and made into monoclonal antibody-producing hybridoma cells.

Any one of a number of methods well known in the art can be used to identify hybridoma cells that produce antibodies having the desired characteristics. These include screening for hybridomas using ELISA assays, Western blot analysis, or radioimmunoassays (Lutz et al., Exp. Cell Res. 175: 109-124 (1988)).

Hybridomas secreting the desired antibody are cloned and the class and subclass are determined using procedures known in the art (Campbell, Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology, supra (1984)).
.

  For polyclonal antibodies, antisera containing antibodies are isolated from the immunized animal and screened for the presence of antibodies with the desired specificity using one of the procedures described above.

In another embodiment of the invention, the antibody described above is detectably labeled. By using radioactive isotopes, affinity labels (eg, biotin, avidin, etc.), enzyme labels (eg, horseradish peroxidase, alkaline phosphatase, etc.), fluorescent labels (eg, FITC or rhodamine), paramagnetic atoms, etc. Can be detectably labeled. Procedures for achieving such labeling are well known in the art (eg, Sternberger et al., J. Histchem. Cytochem. 18: 315 (1970); Bayer et al., Meth. Enzym. 62: 308 (1979 Engval et al., Immunol. 109: 129 (1972); Goding, J. Immunol. Meth. 13: 215 (1976).
The labeled antibodies of the invention can be used for in vitro, in vivo, and in situ assays to identify cells or tissues that express a particular peptide.

In another embodiment of the invention, the antibody described above is immobilized on a solid support. Examples of such solid supports are plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, acrylic resins such as polyacrylamide, and latex beads. Techniques for attaching antibodies to such solid supports are well known in the art (Weir et al., “Handbook of Experimental Immunology”, 4th edition, Blackwell Scientific Publications, Oxford, England, Chapter 10 (1986); Jacoby. Et al., Meth. Enzym. 34, Academic Press, NY (1974)). The immobilized antibodies of the invention can be used for in vitro, in vivo, and in situ assays, and in immunochromatography.

Furthermore, one of ordinary skill in the art can readily apply the techniques, methods, and kits disclosed above with respect to currently available procedures and antibodies, and reasonably designed anti-peptide peptides (
See, eg, Hurby et al., “Application of Synthetic Peptides: Antisense Peptides”, Synthetic Peptides, A User's Guide, WHFreeman, NY, pages 289-307 (1992), and Kaspczak et al., Biochemistry 28: 9230-8 (1989). In order to make a peptide, one can make a peptide that can bind to a specific peptide sequence.

Anti-peptide peptides can be made in one of two ways. First, anti-peptide peptides can be made by replacing basic amino acid residues found in ALK-7 peptide sequences with acidic residues while maintaining hydrophobic and uncharged polar groups. For example, lysine, arginine, and / or histidine residues are substituted with aspartic acid or glutamic acid, and glutamic acid residues are substituted with lysine, arginine, or histidine.

VIII. Method for Detecting ALK-7 Polypeptide in a Sample In another embodiment, the present invention provides a method for detecting an ALK-7 polypeptide in a sample, comprising the following steps: a) an immune complex comprising Contacting the sample with the antibody described above under the conditions to be formed, and b) detecting the presence of the antibody bound to the polypeptide. Specifically, the method includes incubating a test sample with one or more antibodies of the present invention and assaying whether the antibody binds to the test sample. A change in the level of ALK-7 in the sample as compared to the normal level may indicate the presence of a particular disease.

The conditions for incubating the antibody with the test sample will vary. Incubation conditions depend on the format used in the assay, the detection method used, and the type and nature of the antibody used in the assay. Those skilled in the art will readily adapt commonly available immunological assay formats (eg, radioimmunoassay, enzyme-linked immunosorbent assay, diffusion-based octalony method, or rocket immunofluorescence assay) to use the antibodies of the invention. Understand what can be done. Examples of such assays include Chard, An Introduction to Radioimmunoassay and Related Technologies, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock et al., Techniquesin Immunocytochemistry, Academic Press, Orland, FL, Volume 1 (1982), Volume 2. (1983), Volume 3 (1985); Tijssen, Practice and Theory of Enzyme Immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

Test samples for immunological assays of the present invention include cells, or protein or membrane extracts of cells, or biological fluids (eg, blood, serum, plasma, or urine). The test sample used in the above methods will vary based on the assay format, the nature of the detection method, and the tissue, cells, or extract used as the sample to be assayed. Methods for preparing cellular protein extracts or membrane extracts are well known in the art and can be readily adapted to obtain samples that are resistant to the systems utilized.

IX. Diagnostic Kit Containing Antibody Against ALK-7 In another embodiment of the invention, a kit is provided that includes all of the reagents necessary to carry out the detection method described above. The kit may include: i) a first containing the antibody described above.
And ii) a second container means comprising a conjugate comprising the antibody's binding partner and label. In another preferred embodiment, the kit further comprises one or more other containers including one or more of the following: a wash reagent, and a reagent capable of detecting the presence of bound antibody. Examples of detection reagents include a labeled secondary antibody, or alternatively a chromophore reagent, enzyme reagent, or antibody binding reagent that can react with the labeled antibody when the primary antibody is labeled. However, it is not limited to these. The compartmentalized kit can be as described above for the nucleic acid probe kit.

  One skilled in the art will readily appreciate that the antibodies described in the present invention can be readily incorporated into one of the established kit formats well known in the art.

X. Diagnostic Screening and Treatment Although the following discussion relates specifically to human patients, it should be understood that the teachings are also applicable to any animal expressing ALK-7.

According to the diagnosis and screening method of the present invention, a patient who is presumed to be at risk of developing a disease associated with a change in the expression level of ALK-7, or a diagnosis of a disease associated with ALK-7 is based on the family. It is particularly useful for the desired patient.

According to the present invention, presymptmatic screening of individuals in need of such screening is possible using DNA encoding the ALK-7 protein of the present invention. The screening methods of the present invention allow pre-diagnostic diagnosis (including fetal diagnosis) of the presence of an ALK-7 gene deletion or abnormality in an individual, and thus such an individual can detect ALK-7 related diseases. Enables an opinion on the onset or likelihood of developing. This is of particular value for the identification of carriers of altered or deleted ALK-7 genes, eg, from individuals with a family of diseases associated with ALK-7. Early diagnosis is also desirable to maximize intervention at the right time.

In a preferred embodiment of the screening method, a tissue sample is taken from such an individual and (1) the presence of a “normal” ALK-7 gene, (2) the presence of ALK-7 mRNA, and / or (3) Screen for the presence of ALK-7 protein. Normal human genes are
For example, detection of restriction digestion patterns of “normal” DNA on patient DNA using DNA probes prepared against the ALK-7 sequence (or functional fragment thereof) taught in the present invention (including RFLP analysis) Can be characterized on the basis of Similarly, ALK-7 mRNA can be characterized and using a similar probe (a) of normal ALK-7 mRNA as found in a human population at risk of developing a disease associated with ALK-7 It can be compared to the level and / or (b) size. In conclusion, ALK-7 protein can be (a) detected and / or (b) quantified using biological assays for ALK-7 activity, or using immunological assays and ALK-7 antibodies. When assaying ALK-7 protein, an immunological assay is preferred because of its speed. (1) Abnormal ALK-7 DNA size pattern, and / or (2) Abnormal ALK-7 mRNA size or level, and / or (3) Abnormal ALK-7 protein level, the patient has ALK-7 It is an indicator of the risk of developing a related disease.

The screening and diagnostic methods of the present invention do not require that the entire ALK-7 DNA coding sequence be used for the probe. Rather, the presence of the ALK-7 gene in DNA preparations from normal or diseased individuals, the loss of such genes, or altered physical properties of such genes (eg, changes in the migration pattern of electrophoresis) It is only necessary to use sufficient nucleic acid fragments or nucleic acid lengths to detect.

Fetal diagnosis can be performed using any known method for obtaining fetal cells, including amniocentesis, chorionic villi sampling (CVS), and fetal mirror, if desired. Analysis of fetal chromosomes can be used to determine whether a chromosomal portion having a normal ALK-7 gene is present in a heterozygous state.

In a method of treating a disease associated with ALK-7 in a patient in need of such treatment, functional ALK-7 DNA is present in such a time and amount sufficient to treat such a patient. Such patient cells can be provided in a manner and in an amount that allows expression of the ALK-7 protein supplied by the gene. Many vector systems are known in the art to provide such delivery to human patients in need of genes or proteins that are deficient from cells. For example, retroviral systems (especially modified retroviral systems and especially herpes simplex virus systems) can be used. Such methods are provided, for example, in the teachings of Breakfield, XA et al., TheNew Biologist 3: 203-218 (1991); Huang, Q. et al., Experimental Neurology 115: 303-316 (1992), WO93 / 03743 and WO90 / 09441. Is done. Delivery of a DNA sequence encoding a functional ALK-7 protein effectively replaces a defective or mutated ALK-7 gene.

In another embodiment, either neuronal or glial stem cell populations can be genetically engineered to express functional ALK-7 receptors. Such cells that recombinantly express the ALK-7 receptor can be transplanted into diseased or damaged areas of the mammalian neurological system (Neural Transplantation. A Practical Approach, Donnet and Djorklund, Oxford University Press, New York, NY (1992)). In yet another embodiment, embryonic tissue or fetal neurons can be genetically engineered to express functional ALK-7 receptors and transplanted into a diseased or damaged area of the mammalian limbic system. The feasibility of transplanting fetal dopamine neurons into patients with Parkinson's syndrome has been demonstrated (Lindvall et al., Archives of Neurology 46: 615-631 (1989)).

Studies of molecular interactions between ligands and their receptors have shown that only the extracellular domain of the receptor is involved in specific physical interactions between molecules (Riedel et al., Nature 324: 68-70 (1986) Riedel et al., EMBO J. 8: 2943-2945 (1989)). Thus, the extracellular domain of the receptor can be used as a probe to screen an expressed cDNA library for ALK-7 ligand. In one approach for the detection of receptor probes, placental alkaline hostase is fused to the extracellular domain of the receptor and positive clones are detected by the presence of alkaline hostase activity.

Another approach to isolating ALK-7 ligand is to take advantage of the finding that co-expression of a receptor and its ligand in the same cell results in uncontrolled proliferation and malignant transformation (Klein Cell 66: 395-403 (1991); Gazit et al., Cell 39: 89-97 (1984)). A eukaryotic cDNA expression library can be transfected into cells expressing the receptor, and the presence of the ligand generates an autocrine loop, resulting in a transformed phenotype. This approach has been successfully used by Miki et al., Science 251: 72-75 (1991) to isolate KGF receptors using cells that express keratinocyte growth factor (KGF). In yet another approach, ALK-7 receptor protein can be expressed in cell lines or Xenopus oocytes by the recombinant techniques described above and as detected by anti-phosphotyrosine antibodies (UBI, Happauge, New York), Activation of tyrosine kinase activity stimulated by the ligand can be used to assay and purify the ligand. For example, cells expressing recombinant ALK-7 receptor can be exposed to a mammalian brain extract. Brain extracts can be fractionated by chromatography and used to assay for the presence of ligand activity. Once activity is identified in a particular fraction, it can be further purified by conventional biochemical techniques.

In another approach, the ALK-7 extracellular domain can be used to screen random peptide libraries (Cull et al., Proc. Natl. Acad. Sci. USA 89: 1865-1869 (1982); Lam et al., Nature 354: 82-84 (1991)). Isolated peptides can be assayed for their ligand activity.

In another embodiment of the invention, the ALK-7 ligand is expressed as a recombinant gene in the cell,
The cells can therefore be transplanted into a mammal, preferably a human in need of gene therapy. In order to provide gene therapy to an individual, a gene sequence encoding all or part of an ALK-7 ligand is inserted into a vector and introduced into a host cell. Examples of diseases that may be suitable for gene therapy include, but are not limited to, neurodegenerative diseases or disorders, Alzheimer's disease, schizophrenia, epilepsy, neoplasia, and cancer. Examples of vectors that can be used for gene therapy include, but are not limited to, defective retroviral vectors, adenoviral vectors, or other viral vectors (Mulligan, RC, Science 260: 926-932 (1993)). Not. Means that can introduce the vector carrying the gene into the cell include microinjection, electroporation, transduction, or transfection using DEAE dextran, lipofection, calcium phosphate, or other procedures known to those skilled in the art. However, it is not limited to these (Molecular Cloning, A Laboratory Manual, edited by Sambrook et al., Cold Spring Harbor Press, Plainview, New York (1989)).

The ability of ALK-7 antagonists and agonists to interfere with or enhance ALK-7 activity can be assessed using cells containing ALK-7. The assay for ALK-7 activity in cells is:
Agents that can be used to determine the functionality of ALK-7 protein in the presence of agents that can act as antagonists or agonists, and thus, agents that interfere with or enhance the activity of ALK-7 are identified.

Agents screened in this assay include, but are not limited to, peptides, carbohydrates, vitamin derivatives, or other pharmaceutical substances. These agents can be selected and screened 1) randomly, 2) by rational selection, or 3) by design using, for example, protein or ligand modeling techniques.

For random screening, drugs (eg, peptides, carbohydrates, pharmaceutical agents, etc.) are randomly selected and assayed for their ability to bind to or stimulate / block its activity on ALK-7 protein.

  Alternatively, agents can be rationally selected or designed. As used herein, an agent is said to be “rationally selected or designed” if the agent is selected based on the ALK-7 protein or known ligand configuration. .

In one embodiment, the present invention is a screening method for antagonists or agonists that stimulate or block the activity of ALK-7, wherein the following steps are tested: (a) ALK-7 expressing cells are tested. And (b) assaying the cells for the activity of the ALK-7 protein by measuring the effect of the agent on the ALK-7 binding of the ALK-7 ligand. .

Any cell can be used in the above assay as long as the cell expresses a functional form of ALK-7 protein and its ALK-7 activity can be measured. Preferred expression cells are eukaryotic cells or eukaryotic organisms. Such cells can be modified to include a DNA sequence encoding ALK-7 protein using routine procedures known in the art. Alternatively, those skilled in the art can introduce mRNA encoding ALK-7 protein directly into cells.

Using ALK-7 ligands (including antagonists and agonists as described above), the present invention further provides methods for modulating the activity of ALK-7 protein in cells. In general, agents that have been identified to block or stimulate ALK-7 activity (antagonists and agonists) are formulated such that the agent can contact in vivo cells that express the ALK-7 protein. obtain. Contact of such cells with such agents results in modulation of the activity of ALK-7 protein in vivo. As long as there are no formulation or toxicity barriers, the agents identified in the above assay are effective for in vivo use.

In another embodiment, the present invention provides ALK-7 or ALK-7 ligand (including ALK-7 antagonists and agonists) to an animal, preferably a mammal (especially a human), and ALK-7 in that animal. It relates to a method of administration in an amount sufficient to effect a level modification. An administered ALK-7 or ALK-7 ligand may in particular provide a function associated with ALK-7. In addition, ALK-7
Is expressed in brain tissue, administration of ALK-7 or ALK-7 ligand can be used to alter ALK-7 levels in the brain.

One skilled in the art will recognize that the amount to be administered for any particular treatment protocol can be readily determined. Dosage should not be so great as to cause adverse side effects (eg, unwanted cross-reactions, anaphylactic reactions). In general, dosage will vary depending on age, condition, sex, and extent of the patient's disease, counterindication (if any), and other such variables to be adjusted by each physician To do. Dosages can vary from 0.001 mg / kg to 50 mg / kg of ALK-7 or ALK-7 ligand for one or several days with one or more daily doses. ALK-7 or ALK-7 ligand can be administered parenterally by injection or by gradual perfusion for a predetermined time. This can be administered intravenously, intraperitoneally, intramuscularly or subcutaneously.

Preparations for parenteral administration include sterile or aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil (eg olive oil), injectable organic esters (eg ethyl oleate). Aqueous carriers include water, alcoholic / aqueous solutions,
Emulsions or suspensions are included, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer dextrose and sodium chloride, lactated Ringer's solution or fixed oil. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (eg, Ringer dextrose-based replenishers), and the like. Preservatives and other additives also include, for example, antibacterial agents, antioxidants, chelating agents, inert gases, and the like. See generally Remington's Pharmaceutical Science, 16th edition, edited by Mack (1980).

In another embodiment, the invention provides an amount of ALK-7 or an ALK-7 ligand sufficient to alter the activity associated with ALK-7, and a pharmaceutically acceptable diluent, carrier, or excipient. The present invention relates to a pharmaceutical composition containing an agent. Appropriate concentrations and dosage unit sizes can be readily determined by those skilled in the art as described above (eg, Remington's Pharmaceutical Sciences (16th edition, Osol, A. Ed., Mack, Easton PA (1980) and WO 91/19008).

XI. Transgenic ALK-7 “knockout” mouse transgenic non-human animal production method The non-human animal of the present invention is a transgene interruption of endogenous ALK-7 gene.
Or an animal having a change (knockout animal) and / or its genome in human ALK-7
Including animals into which one or more transgenes that detect the expression of are introduced.

Such non-human animals include vertebrates such as rodents, non-human primates, sheep, dogs, cows, amphibians and reptiles. Preferred non-human animals are selected from animals of the non-human mammalian species, most preferably animals selected from vaginal dentistry including rats and mice, most preferably mice.

The transgenic animals of the present invention have been engineered by one or more genes that are not naturally present in the animal, such as foreign genes, genetically engineered, by non-natural means (eg, artificial manipulation). An animal into which an endogenous gene has been introduced. A non-naturally introduced gene, known as a transgene, may be derived from the same or different species as the animal, but naturally occurs in the animal in an arrangement and / or chromosomal locus called the transgene. It may not be found. The transgene may contain foreign DNA sequences, ie sequences that are not normally found in the genome of the host animal. Alternatively, or in addition, the transgene is rearranged or mutated in vitro to alter the normal in vivo pattern of gene expression or to alter or eliminate the biological activity of the endogenous gene product encoded by the gene. May contain non-normal endogenous DNA sequences (Watson, JD et al., Recombinant DNA, 2nd edition, WH Freeman & Co., New York (1992), pages 255-272; Gordon, JW, Intl. Rev. Cytol. 115: 171-229 (1989); Jaenish, R., Science 240: 1468-1474 (1989); Rossant, J., Neuron 2: 323-334 (1990).

  The transgenic non-human animals of the present invention can be obtained by introducing a transgene into the germ line of a non-human animal. Target cells of embryos at various stages of development are used to introduce the transgene of the present invention. Different methods are used depending on the stage of development of the embryonic target cells.

1. Conjugate microinjection is a preferred method for incorporating a transgene into the genome of an animal in the course of practicing the present invention. Conjugates (fertilized eggs that have not undergone pronuclear fusion or subsequent cell division) are preferred target cells for microinjection of transgenic DNA sequences. The male pronucleus of mice reaches about 20 microns in diameter and is a feature that allows reproducible injection of 1-2 picoliters of solution containing transgenic DNA sequences. Using a zygote to perform the transgene introduction often results in the injected transgenic DNA sequence before the first cell division,
(Brinster et al., Proc. Natl. Acad. Sci. (USA) 82: 4438-4442 (1985). As a result, the resulting transgenic animal (founder animal, founder All cells of animals) stably retain a transgene incorporated at a specific locus called a transgenic allele, which exhibits Mendelian inheritance: transgenic and non-transgenic animals. Half of the offspring resulting from the crossing of the inherited transgenic alleles according to the Mendelian rule of random combinations.

2. Viral integration can also be used to introduce the transgenes of the invention into animals. Developing embryos are cultured in vitro until the developmental stage known as the blastocyst.
At this time, the blastomeres can be infected with an appropriate retrovirus (Jaenich, R., Proc. Natl. Sci. (USA) 73: 1260-1264 (1976)). Infection of blastomeres is enhanced by enzymatic removal of the zona pellucida (Hogan et al., Manipulating the Mouse Embryo, Cold Spring Harbor Press, Cold Spring Harbor, NY (1986)). Transgenes are typically replication-deficient, but by virtue of viral vectors that remain competent for integration of viral binding DNA sequences, including transgenic DNA sequences that bind to such viral sequences, Introduced into the genome (Jahner et al., Proc. Natl. Acad. Sci. (USA) 82: 6927-6931 (1985); Van der Putten et al., Proc. Natl. Acad. Sci. (USA) 82: 6148-6152 (1985)). Transfection is easily and efficiently obtained by culturing blastomeres on a monolayer of cells producing a transgene-containing viral vector (Vander Putten et al., Proc. Natl. Acad. Sci. ( USA) 82: 6148-6152 (1985); Stewart et al., EMBO Journal 6: 383-388 (1987)). Alternatively, infection can be performed at a later stage such as the blastocoel (Jahner, D et al. Nature 298: 623-628 (1982)). In all events, most of the transgenic founder animals obtained by viral integration are mosaics of transgenic alleles; that is, the transgene is taken up by only a subset of the total cells that form the transgenic founder animal. Furthermore, multiple viral integration events can occur in a single founder animal. This generates multiple transgenic alleles that segregate in future generations of offspring. Introduction of transgenes into germline cells by this method is possible but probably occurs at a low frequency (Jahner, D. et al., Nature 298: 623-628 (1982). When a transgene is introduced into a cell, the transgenic allele can produce offspring that are present in all of the animal cells (ie, both somatic and germline cells).

3. Embryonic stem (ES) cells can also serve as target cells for introducing the transgene of the present invention into animals. ES cells are obtained from pre-transplanted embryos cultured in vitro (Evans, MJ et al., Nature 292: 154-156 (1981); Bradley, MO et al., Nature 309: 255-258 (1984); Gossler
Proc. Natl. Acad. Sci. (USA) 83: 9065-9069 (1986); Robertson et al., Nature 322; 445-448 (1986); Robertoson, EJ, Teratocarcinomasand Embryonic Stem Cells: A Practical Approach, Robertoson, EJ, IRL Press, Oxford (1987) 71-112). Commercially available ES cells (eg, from GenomeSystems Inc., St. Louis, MO) have been established (Lovell-Badge, RH Teratocarcinomas and Embryonic Stem Cells: APractical Approach
, Edited by Robertson, EJ, IRL Press, Oxford, (1987), pages 153-182) and can be transformed with one or more transgenes. Transformed ES cells can be combined with animal blastocysts. ES cells then colonize the embryo and become the germline of the resulting animal. It is a chimera (consisting of cells from two or more animals) (Jaenisch, R., Science 240: 1468-1474, (1988), Bradley, A., Teratocarcinomasand Embryonic Stem Cells: APractical Approach, Robertson, EJ, IRL Press, Oxford (1987), pages 113-151). Again, once the transgene has been introduced into germline cells in this manner, the transgenic allele can give rise to progeny that are present in all of the animal cells (ie, both somatic and germline cells). .

Even if that happens, the initial introduction of the transgene is a Lamarck (non-Mendel) event. However, the transgene of the present invention can be stably integrated into germline cells and transmitted as the Mendelian locus to the offspring of the transgenic animal. Other transgenic techniques result in mosaic transgenic animals. In mosaic transgenic animals, the cells may or may not have a transgene. In mosaic transgenic animals whose germ line cells do not have a transgene, transgene transmission to offspring does not occur. However, mosaic transgenic animals may exhibit a phenotype associated with the transgene.

Transgenes can be introduced into non-human animals to provide an animal model of human disease. Such animal models are, for example, transgenes encoding mutant gene products with congenital metabolic disorders of human genetic diseases, and human pathogens (ie, bacteria, viruses or other pathogenic microorganisms). Including a transgene encoding the human factor necessary to confer sensitivity (Leder et al., US Pat. No. 5,175,383 (December 29, 1992); Kindt et al., US Pat. No. 5,183,949 (February 2, 1993)); Small et al., Cell 46: 13-18 (1986); Hooper et al., Nature 326: 292-295 (1987); Stacey et al., Nature 332: 131-136 (1988); Windle et al., Nature 343: 665-669 (1990); Katz Cell 74: 1089-1100 (1993) Transgenically introduced mutations are gene sequences in which the DNA sequence encoding a selectable and / or detectable marker is usually endogenous to non-human animals. Is replaced with null (null) ("knock Uto ") alleles. The resulting transgenic non-human animal, which is prone to disease or whose transgene causes disease, identifies the composition that induces the disease and induces the disease. To assess the virulence capacity of known or anticipated compositions (Berns, AJM, US Pat. No. 5,174,986 (1992 12
29th)), or alternatively to evaluate compositions that can be used to treat disease or improve its symptoms (Scott et al., WO94 / 12627 (1994)).

Progeny that have inherited the transgene of the present invention can be analyzed by analysis of genetic material derived from the progeny for the presence of a biomolecule that corresponds to or contains a characteristic sequence encoded by the transgene of the present invention, Distinguished from litters who have not inherited the transgene. For example, a biological fluid that uniquely contains an encoded polypeptide can be immunoassayed for the presence of that polypeptide by the selectable marker of the transgene of the present invention. A simpler and more reliable method of identifying transgenic offspring is to obtain a tissue sample from the end of an animal, eg, the tail, and a characteristic part of the transgene of the invention (eg, its selectable marker). Analyzing the sample for the presence of a nucleic acid sequence corresponding to the DNA sequence. The presence of such nucleic acid sequences can be determined, for example, by hybridization (“Southern”) analysis using a DNA sequence corresponding to a characteristic portion of the transgene, PCR reaction products and transgenes using the DNA sequence in the sample as a substrate. It can be measured by analysis of oligonucleotides derived from DNA sequences.

  The present invention will be described in more detail, but is not limited to the following examples.

Example 1
Isolation of ALK-7
The ALK-7 PCR fragment was synthesized from the motif VAVKIF (SEQ ID NO: 4) having a BamHI restriction enzyme site
) -Derived primers, 5'-GCGGATCCGT (C / G / T) CG (A / C / T) GT (C / G / T) AA (A / G) AT (A / C / T) TT-3 '(SEQ ID NO: 3) and primer derived from motif YMAPE (SEQ ID NO: 6) with EcoRI restriction site, 5'-CGGAATTC (A / G / T) GG (A / G / T) GCCAT (A / G) TA Isolated using -3 '(SEQ ID NO: 5). PCR amplification was performed for 5 cycles consisting of 94 ° C (1 minute), 50 ° C (1 minute) and 72 ° C (1 minute), then 94 ° C (1 minute), 55 ° C (1 minute) and 72 ° C. A cycle consisting of ° C. (1 minute) was performed 35 times.

Using this PCR fragment, full-length cDNA was cloned from a 7 day old whole rat brain library of λgt10 and washed with high stringency (60 ° C, 0.1% SDS, 0.1XSSC). MolecularCloning: ALaboratory Manual 2nd edition, edited by Sambrook, Fritsch and Maniatis, Cold Spring Harbor Laboratory, 1989). Full length ALK-7 cDNA
The clone has been deposited with the American Type Culture Collection (12301 Parklawn Drive, Rockville, MD 20852) and has been assigned the deposit number ATCC 75945. The nucleotide and amino acid sequences of the coding region of ALK-7 clone are shown in SEQ ID NOs: 1 and 2, respectively.

Example 2
Isolation of ALK-7 Ligand Screening cDNA expression library for ALK-7 ligand using extracellular domain-alkaline phosphatase fusion protein as probe ALK-7 extracellular domain and secreted placental alkaline phosphatase (SEAP) In order to construct a fusion protein, a vector called APtag-1 constructed by Flannagan and Leder, Cell 63: 185-194 (1990) is used. APtag-1 contains a set of restriction sites to insert a region of ALK-7 cDNA encoding the extracellular domain. Downstream of the insertion site is the full-length sequence of SEAP, which is fused to the upstream sequence.

To create an ALK-7 receptor fusion protein, an ALK-7 cDNA sequence containing the ALK-7 secretion signal peptide and the sequence encoding the entire extracellular domain, ending immediately before the first hydrophobic amino acid in the transmembrane region Insert the 5 'end into APtag-1. The resulting plasmid encodes a fusion protein with ALK-7 extracellular domain bound to SEAP. This fusion protein is expressed from the Moloney Murine Leukemia virus LTR promoter. This fusion construct is transfected into NIH / 3T3 cells that have been shown to express high levels of APtag-Kit fusion protein (Flannagan and Leder, Cell 63: 185-194 (1990)). This fusion construct is cotransfected with a selectable marker plasmid, pSV2neo, and selected with G418 (400-800 μg / ml). New resistant colonies are grown in 96 well plates and screened for secretion of SEAP activity into the medium.
The fusion protein is concentrated, purified, and used as a probe to screen a cDNA expression library derived from a mammalian brain, preferably a human brain.

Three types of positive clones are expected: (1) clones with background ant-kariphosphatase activity; (2) clones that bind non-specifically with the fusion protein; and (3) clones that encode an ALK-7 ligand. Background phosphatase clones are positive in the presence of alkaline phosphatase substrate, even without added probe. To distinguish specific ones from clones with non-specific interactions, extracts from bacteria expressing these clones were used for ALK-7 kinase activity in NIH / 3T3 cells expressing ALK-7. Used to stimulate. Only the ligand can stimulate activation of ALK-7 kinase activity.

To undergo proper glycosylation of the ALK-7 extracellular domain, it is preferable to produce receptor probes in NIH / 3T3 cells rather than bacteria. However, glycosylation of growth factors has often been shown not to be necessary for their activity. For example, M-CSF (Metcalf, Blood 67: 257-267 (1986)) and NGF (available from Boehringer Mannheim) obtained in bacteria are biologically active, so glycosylated receptor probes are In the course of screening, it should interact properly with its ligand synthesized by E. coli in phage plaques.

In addition to using an Ap-tagged ALK-7 probe for screening ALK-7 ligands in vitro, this probe also allows histological staining in mammalian brain sections to locate ligand expression. Can be used. Measurement of the expression position of the ALK-7 ligand allows biochemical purification of the ligand obtained from the tissue cell source for further analysis.

Functional screening of ALK-7 ligand
Another approach to isolate ALK-7 ligand is to use a functional screening approach. ALK-7 full length cDNA is cloned into the expression vector pMEX under the MMLV LTR promoter. NIH / 3T3 with ASV-7 expression vector along with pSV2Hygro containing hygromycin β-phosphotransferase gene conferring hygromycin resistance
Cotransfect into cells (Gritz and Davis, Gene 25: 179-188 (1983)). Transfected cells are selected with hygromycin B at a concentration of 350 μg / ml. Resistant clones are grown in 12-well plates and screened for expression of ALK-7 using anti-ALK-7 antibody by Western blot analysis.

  A direct eukaryotic cDNA library derived from rat brain mRNA is constructed using a vector system developed by Miki et al., Gene 83: 137-146 (1989). This vector has the MMLVLTR promoter for expression of the cDNA insert and the Neo gene driven by the SV40 early promoter as a selectable marker. In addition, the vector contains the pBR322 origin of replication, and the cDNA insert of interest can be obtained by NotI digestion and ligation of the crude λDNA preparation and then transfected into bacterial cells. This cDNA library is constructed as described in more detail by Miki et al., Gene 83: 137-146 (1989).

The cDNA library is transfected into ALK-7 expressing NIH / 3T3 mouse embryo fibroblasts. A population (foci) from the transfected cells is isolated and tested for Neo resistance to remove background transformants in NIH / 3T3 cells. Genomic DNA obtained from each Neo resistant transformant is cut with NotI releasing the plasmid. Digested DNA is ligated under dilute conditions and used to transform competent bacteria. Plasmid DNA obtained from each population is purified and transfected into NIH / 3T3 cells that express or do not express ALK-7. Transformants with ALK-7 ligand but not with other proteins are expected to depend on the presence of ALK-7 expression. The clones are then further analyzed by sequencing, the encoded protein is purified and assayed for ALK-7 binding.

Example 3
ALK-7 protein
The ALK-7 cDNA fragment was labeled with a hemagglutination (HA) epitope at the C-terminus and subcloned into pBS KS (Stratagene). This plasmid was used for in vitro translation using a reagent kit purchased from Promega according to the manufacturer's instructions. ^{The 35} S-Cys labeled product was immunoprecipitated with anti-HA monoclonal antibody (12CA5) and separated by SDS / PAGE, followed by autoradiography (FIG. 2, left lane). The ALK-7 protein consisted of a dominant 55K band, with additional bands arising from internal Met codons.

ALK-7 HA-tagged cDNA was expressed in COS cells from the pRc / MMV expression vector (Invitrogen). Transfected cells were labeled with ¹²⁵ I, lysed, immunoprecipitated with 12CA5 antibody, and analyzed by SDS / PAGE and autotriographed (FIG. 2, right lane). A 55K protein, the expected size of ALK-7, was detected after ¹²⁵ I labeling of the protein on the surface of the transfected cells.

Example 4
ALK-7 mRNA expression
ALK-7 mRNA expression was examined by RNase protection analysis (RPA) in different regions of the adult rat brain (FIG. 3A) and in developing peripheral tissues (FIG. 3B). ALK-7 mRNA was selectively restricted to the central nervous system. Predominant expression was seen in the adult cerebellum but also in the hippocampus, midbrain, pons, medulla, hills and thalamus. COS cells did not express ALK-7 mRNA. Low expression of ALK-7 mRNA was seen in 2 weeks old, but not adult rat ovaries, and adult kidneys. Moderate expression of ALK-7 mRNA was also detected in the superior cervical ganglion (SCG) at 1 day of life (P1).

ALK-7 mRNA expression analyzed by RPA during cerebellar development showed increased expression during cerebellar maturation, the highest level in the adult cerebellum (FIG. 4A). A model of hyperthyroidism was induced in neonatal rats using propylthiouracil (PTU). It delays the development of granule cells and Purkinje cells in the cerebellum. The treatment also delayed the development of ALK-7 mRNA during development. This indicates the role of ALK-7 during maturation of different neuronal cell types in the cerebellum (FIG. 4B). In vitro, ALK-7 mRNA
Was readily detected in granule cell cultures (FIG. 4C).

A cross section through the adult cerebellum is shown in FIG. 5 showing ALK-7 mRNA expression in Purkinje cells (arrowheads) and granule cells (arrows). FIG. 5B shows ALK-7 mRNA expression in motor neurons of facial motor cell nuclei (arrows).

  All publications mentioned above in this specification are hereby incorporated by reference in their entirety.

  Although the foregoing invention has been described in some detail for purposes of clarity and understanding, various changes in form and detail may be made without departing from the true scope of the invention and the appended claims. Those skilled in the art will appreciate from reading this disclosure that can be done.

  (Sequence Listing)

Claims (1)

Serine threonine kinase receptor.

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