TGFB signaling protein, genes and related uses

专利摘要:
The present invention relates to the discovery that proteins are encoded by a family of vertebrate genes referred to herein as signal-associated genes, relating to signal transduction induced by elements of the TGFβ superfamily. The present invention provides useful compositions and methods that can be used to create and / or maintain an array of other vertebrate tissues, for example, both in vitro and in vivo.
公开号:KR20000064501A
申请号:KR1019980704721
申请日:1996-12-20
公开日:2000-11-06
发明作者:조나단 엠. 그래프；토드 엠. 울프；핑 진；더글라스 에이. 멜톤
申请人:온토제니, 인코오포레이티드；조이스 브린톤；프레지던트 앤드 펠로우즈 오브 하바드 칼리지；
IPC主号:

专利说明:

TVFβ signaling protein, gene and related uses
The present invention relates to the discovery of new genes and gene products expressed in vertebrates, wherein the genes are hereafter referred to as the "signalin" gene family, whose products are considered signalin proteins. The signaline gene encodes an intracellular protein that acts downstream of the transforming Growth Factor β (TGFβ) of the ligand. The product of the signaline gene has clear and extensive improvements in mesoderm induction, tumor suppression, and the formation and maintenance of an orderly spatial arrangement of differentiated tissues in vertebrates, and in a variety of vertebrate tissues both in vitro and in vivo. It can be used or manipulated to create and / or maintain an array.
In general, the present invention describes a substantially pure preparation of an isolated vertebrate signal polypeptide, in particular one or more of the subject signal polypeptides of interest. The invention also provides a recombinantly produced signaline polypeptide. In a preferred embodiment, the polypeptide has the following biological activities: the ability to modulate the proliferation, survival and / or differentiation of mesodermal-derived tissues such as tissue derived from mesoderm of the spine; The ability to modulate the proliferation, survival and / or differentiation of ectoderm-derived tissues such as tissue derived from neural tubes, neural tombs, or head mesenchyme; The ability to regulate proliferation, survival and / or differentiation of endoderm-induced tissues, such as tissues derived from the primitive gut. Also in a preferred embodiment, the subject signaling protein has the ability to modulate intracellular signal transduction pathways mediated as receptors for the TGFβ phase and elements of molecules.
In one embodiment, the polypeptide is the same or similar to a signaline protein. Representative signaling proteins include SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26. Relevant elements of the vertebrate family of vertebrates are also observed, for example, a signal polypeptide may be observed even if a polypeptide with high sequence identity of, for example, 70, 80%, 90% is also observed. Preferably have at least 60% similar amino acids as the polypeptide represented by any of SEQ ID NOs: 14-26. The signaline polypeptide may comprise a full length protein as indicated by the sequence listing, or may be a specific site / domain, or any size, for example at least 5, 10, 25, 50, 100, 150 or 200 amino acids. May comprise fragments equal in length. In a preferred embodiment, the polypeptide or fragment thereof specifically acts as an agonist or antagonist to specifically regulate the signal transduction activity of the receptor for transforming growth factor β.
In one preferred embodiment, the invention describes a purified or recombinant signaline polypeptide having a molecular weight in the range of 45 kd to 70 kd. For example, α and β subfamily, which are preferred signal chain polypeptides, have a molecular weight in the range of 45 kd to 55 kd, and more preferably 50-55 kd, as described above. In another embodiment, the preferred signal chain γ subfamily γ subfamily has a molecular weight ranging from 60 kd to 70 kd, preferably 63-68 kd. Certain post-translational modifications, such as phosphorylation and similar action, obviously increase the molecular weight of the signaline polypeptide compared to the unmodified polypeptide chain.
In another embodiment, the signaline polypeptide comprises a signaline motif represented by the general formula shown in SEQ ID NO: 28. In one preferred embodiment, the signaline motif coincides with a signaline motif represented by one of SEQ ID NOs: 14-26. In another embodiment, the signaline polypeptides of the present invention comprise a v domain represented by Formula SEQ ID NO: 27. In one preferred embodiment, the v domain is identical to the v domain represented by one of the general formula SEQ ID NOs: 14-26. In another preferred embodiment, the signaline polypeptides of the present invention comprise a χ domain represented by the general formula SEQ ID NO: 29. In a more preferred embodiment, the χ region is identical to the χ domain represented by one of SEQ ID NOs: 14-26. In another preferred embodiment, the signaline polypeptide can be modulated by stimulating or reciprocating the intracellular pathway mediated by a receptor for TGFβ. In yet another embodiment, the polypeptide comprises an amino acid sequence represented by the general formula: LDGRLQVSHRKGLPHVIYCRVWRWPDLQSHHELKPXECCEXPFXSKQKXV. In yet another embodiment, the signaline polypeptides of the invention comprise an amino acid sequence represented by the general formula: LDGRLQVAGRKGFPHVIYARLWXWPDLHKNELKHVKFCQXAFDLKYDXV. In another embodiment, the signaline polypeptides of the invention comprise an amino acid sequence represented by the general formula: LDGRLQVXHRKGLPHVIYCRLWRWPDLHSHHELKAIENCEYAFNLKKDEV.
In another preferred embodiment, the present invention provides a purified or recombinant polypeptide fragment of a signalling protein that has the ability to modulate the activity of, for example, mimics or reverses the activity of a wild-type signalling protein. Explain. Preferably, the polypeptide fragment comprises a signaline motif.
In addition, as described below, preferred signal polypeptides may be naturally occurring agonists (eg, analogs) of naturally occurring proteins, or, optionally, antagonists, for example, It can regulate the proliferation and / or growth and / or survival of cells responsive to certain signaling proteins. Homologs of the subject signaline proteins are for example due to mutations that modify the site of modification (such as tyrosine, threonine, serine or aspazine residues) or inactivate the enzymatic activity associated with the protein, Modification of proteins that resist post-translational modifications.
The protein of interest may also be provided as a fictional molecule, such as in the form of a fusion protein. For example, the signaline protein may be provided as a recombinant fusion protein comprising a second polypeptide moiety, for example, the second polypeptide may comprise an (heterologous) amino acid sequence that is independent of the signaline polypeptide. For example, the second polypeptide moiety is an enzymatic activity, such as alkaline phosphatase, for example, the second polypeptide moiety is an epitope tag.
In one preferred embodiment, the signaline polypeptides of the invention modulate signal transduction from the TGFβ receptor. For example, it is possible to modulate the delivery of TGFβ receptors to one element of the signaline polypeptide such as dpp, such as dpp, BMP2 or BMP4. In another preferred embodiment, the signaline polypeptide modulates the signaling of dpp and urea and other TGFβ. For example, the signaline polypeptide is BMP5, BMP6, BMP7, BMP8, 60A, GDF5, GDF6, GDF7, GDF1, Vg1, dosulin, BMP3, DGF10, nodal, inhibin, activin, TGFβ1, TGFβ2, TGFβ3 , MIS, GDF9 or GDNE may be involved in signaling from one or more selected from the group consisting of.
In yet another embodiment, the invention encodes a signaline polypeptide, or similar polypeptide, having the ability to modulate at least an active portion of a naturally-type signaline polypeptide by, for example, mimicking or vice versa. Describe nucleic acids. Representative signaline polypeptides include SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 , SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26. In another embodiment, a nucleic acid of the invention hybridizes with one or more nucleic acid sequences of SEQ ID NOs: 1-13 under stringent conditions. In a preferred embodiment, the nucleic acid encodes a polypeptide that acts as an agonist or antagonist, thereby specifically regulating the signal transduction activity of the receptor for transforming growth factor β.
In another embodiment, the nucleic acid comprises a signaline motif represented by Formula SEQ ID NO: 28. In a preferred embodiment, the signaline motif coincides with a signaline motif represented by one of SEQ ID NOs: 14-26. In another embodiment, a nucleic acid of the invention encodes an amino acid sequence comprising a ν domain represented by the general formula SEQ ID NO: 27. In one preferred embodiment, the encoded ν region matches a ν domain represented by one of SEQ ID NOs: 14-26. In another embodiment, the nucleic acid encodes a signaline polypeptide of the invention comprising a χ domain represented by Formula SEQ ID NO: 29. In one preferred embodiment, the encoded χ region is identical to the χ domain represented by one of SEQ ID NOs: 14-26. In yet another embodiment, the nucleic acid sequence encodes a polypeptide comprising an amino acid sequence represented by the following general formula:
LDGRLQVSHRKGLPHVIYCRVWRWPDLQSHHELKPXECCEXPFXSKQKXV.
In another embodiment, a nucleic acid of the invention encodes a polypeptide comprising an amino acid sequence represented by the following general formula:
LDGRLQVAGRKGFPHVIYARLWXWPDLHKNELKHVKFCQXAFDLKYDXV.
In yet another embodiment, the nucleic acid encodes a polypeptide comprising an amino acid represented by the following general formula:
LDGRLQVXHRKGLPHVIYCRLWRWPDLHSHHELKAIENCEYAFNLKKDEV.
It is another object of the present invention to provide an independent nucleic acid having a nucleotide sequence encoding a signaline polypeptide. In a preferred embodiment, the encoded polypeptide specifically mimics or reverses the action of induction mediated by natural-type signaling proteins. The coding sequence of the nucleic acid may comprise a sequence identical to the coding sequence represented by one of SEQ ID NOs: 1-13, or it may be just similar to one or more of the sequences.
Further, in one preferred embodiment, the subject signaline nucleic acid can be, for example, a transcriptional control such as at least one of a transcriptional facilitating or transcriptional enhancer sequence operably linked to the signaling sequence. Sequence. The regulatory sequences are used to make a signaline gene sequence suitable for use as an expression vector. The invention also describes a method for producing a signaline protein by using the expression vector and a cell infiltrated with the expression vector, whether prokaryotic or eukaryotic.
In yet another embodiment, the nucleic acid is under stringent conditions, although at least 25 contiguous nucleotides of the sense or antisense sequence of one or more of SEQ ID NOs: 1-13 are preferred, more preferably at least 40, 50 or 75 contiguous. Although nucleotides are preferred, they hybridize to nucleic acid markers that match at least 12 consecutive nucleotides of one or more of the senses or antisenses of SEQ ID NO: 1-13.
Still another object of the present invention relates to an immunogen comprising a signaline polypeptide in an immunogen preparation, which is an immunogen capable of eliciting the following specific immune response to a signaline polypeptide: for example, wet response; For example, antibody response; For example, cellular response. In a preferred embodiment, the immunogen comprises an antigenic determinant such as, for example, a single determinant from one protein represented by one of SEQ ID NOs: 14-26.
Still another object of the present invention is to describe antibodies and antibody preparations that specifically react with epitopes of the signaline immunogen.
The invention also encompasses, for example, heterologous forms of the signalling genes described herein, or misexpresses endogenous signalling genes, for example in which one or more expressions of the subject signalnare protein Consider non-human transgenic animals, such as, for example, mice, rats, rabbits, chickens, frogs or pigs with transgenes such as destroyed animals. The transgenic animal can serve as an animal model for studying cell and tissue diseases, including mutated or mis-expressed signal traits, or for use in drug screening.
The present invention also provides a label / primer comprising substantially purified oligonucleotides, wherein the oligonucleotides are subjected to the sense or antisense of SEQ ID NO: 1-13 or their naturally occurring mutants under stringent conditions. A region of nucleotide sequence that hybridizes to at least 12 consecutive nucleotides. Nucleic acid markers specific for each vertebrate signal protein are observed by the present invention, and the markers can, for example, distinguish between nucleic acids encoding α, β, and γ signalins. In a preferred embodiment, the label / primer further comprises a label group that can be attached thereto and detected. The labeling group can be selected from the group consisting of radioisotopes, fluorescent compounds, enzymes and coenzymes, for example. The markers of the present invention are for detecting nucleic acid levels encoding a subject signaling protein in a cell sample isolated from a patient, as part of a diagnostic test kit for identifying dysfunction associated with mis-expression of the signaling protein. Can be acted upon, for example, by measuring signal levels of signaling mRNAs in cells, or by detecting that mutations or deletions in the signaling genes of the genome are made. So-called "labels / primers" of the present invention may also be used as part of an "antisense" therapy, which, depending on the dosage regime or situation, may be used to treat one or more of the subject signaling proteins under stringent intracellular conditions. Oligonucleotide labels or those that can inhibit the expression of the protein by specifically hybridizing (eg, binding) to, for example, binding to and encoding intracellular mRNA and / or genomic DNA Derivatives of Preferably, the oligonucleotide is at least 12 nucleotides, although primers of 25, 40, 50 or 75 nucleotides in length are also contemplated.
For still another object, the present invention relates to a method for regulating one or more of the growth, proliferation or survival of a mammalian cell in response to signal induction. In general, whether performed in vivo, in vitro or in situ, the method compares at least one of (i) growth rate, (ii) differentiation, or (iii) survival of the cell compared to cells without signalling treatment. Treating said cells with an effective amount of signaline polypeptide to alter. Thus, the method may be practiced with polypeptides that mimic the effects of naturally-occurring signalling proteins on the cells, as well as polypeptides that counteract the effects of naturally-occurring signalling proteins on the cells. Can be. In a preferred embodiment, the signaline polypeptide provided in the method is derived from a vertebrate source, for example it is a vertebrate signal polypeptide. For example, preferred polypeptides are SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21. Include an amino acid sequence that is the same as or similar to an amino acid sequence (eg, including bioactive fragments) designated as one of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 . The present invention also contemplates the use of other epigenetic (eg, non-vertebral) homologs of the same signaling polypeptide or bioactive fragments thereof as the vertebrate fragment.
In one embodiment, the method includes testicular cells that regulate spermatogenesis. In another embodiment, the method is used to modulate bone formation, including treating osteogenic cells with a signaline polypeptide. Similarly, if the treated cells are chondrogenic cells, the method is used to regulate chondrogenesis. In yet another embodiment, the signaline polypeptide can be used to regulate the differentiation of neurons, for example, the method causes differentiation of neurons, maintains neurons in the differentiated state, and / Or can be used to enhance the survival of neurons, for example, by inhibiting apoptosis or other forms of cell death. For example, the method can be used to influence differentiation of nerve cells such as motor neurons, cholinergic neurons, dopanergic neurons, serotenergic neurons, and peptide neurons. .
The method is applicable to cell culture techniques, such as, for example, the culturing of nerves and other cells whose survival or differentiation status depends on signaline function. In addition, signaling agonists and antagonists not only affect some mesodermal and endoderm differentiation processes, but also other vertebrate organ pathways such as other ectoderm patterning, as well as neurons in both the central nervous system and the peripheral nervous system. And therapeutic adjustments, such as to enhance the survival and maintenance of other neurons. In one embodiment, the method is used to modulate cell growth, cell differentiation, or cell survival in an animal, wherein (i) the growth rate of one or more cell-types of the animal in comparison to the absence of the signaline, ( ii) administering a therapeutically effective amount of a signaline polypeptide to alter at least one of differentiation, or (iii) survival.
Another object of the present invention is to provide a method for detecting whether a subject, such as, for example, a human patient, is at risk of a disease caused by excessive regulation of unwanted cell proliferation or differentiation. The method comprises a genetic variation encoding, for example, a signaline protein represented by one of (i) SEQ ID NOs: 14-26, or a homolog thereof, in the tissue of the subject, or (ii) a mistake in the signaline gene. Detecting the presence or absence of the genetic defect caused by at least one of the expressions. In a preferred embodiment, the step of detecting the genetic defect comprises the deletion of one or more nucleotides from a signaline gene, the addition of one or more nucleotides to the gene, the substitution of one or more nucleotides of the gene, the gene At least one of a large chromosomal rearrangement of the gene, a change in the messenger RNA transcript level of the gene, the presence of a non-natural type conjugation pattern of the messenger RNA transcript of the gene, or a non-natural type level of the protein. Checking the presence of the.
For example, detecting the genetic defect may comprise (i) a nucleic acid represented by one of SEQ ID NOs: 1-13, or a naturally occurring mutant thereof, or a 5 'or 3 naturally associated with the signalling gene. Providing a label / primer comprising an oligonucleotide containing a region of nucleic acid sequence that hybridizes to a sense or antisense sequence of a signaling gene, such as a flanking sequence; (ii) exposing the label / primer to nucleic acid of the tissue; (iii) detecting the presence or absence of a genetic defect by hybridization of the label / primer to nucleic acid, wherein detecting the defect comprises nucleotide sequence of the signalling gene and optionally And using the label / primer to detect the flanking nucleic acid sequence. For example, the label / primer may be used for polymerase chain reaction (PCR) or ligation chain reaction (LCR). In another embodiment, the level of signalling protein is detected by immunoassay using antibodies that specifically immunoreact with the signalling protein.
Embodiments of the present invention will use conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology, which will be known to those skilled in the art, unless otherwise indicated. The techniques are explained fully in the literature. See, eg, Molecular Cloning A Laboratory Manual, 2nd Edition, Sambrook Edit, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, I and II (D.N. Glover Publishing, 1985); Oligonucleotide Synthesis (M. J. Gait Published, 1984); U.S. Patent 4,683,195 to Mullis et al .; Nucleic Acid Hybridization (published by B.D. Hames & S. J. Higgins, 1984); Transcription And Translation (published by B. D. Hames & S. J. Higgins, 1984); Culture Of Animal Cells (R.I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); Paper, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (published by J. H. Miller and M. P. Calos, 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, 154 and 155 (Wu et al.), Immunochemical Methods In Cell And Molecular Biology (by Mayer and Walker, Academic Press, London, 1987); Handbook Of Experimental Immunology, Volume I-IV (published by D.M.Weir and C.C.Blackwell, 1986); Manipulating the Mouse Embryo, (Cold spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
Other features and advantages of the invention will be apparent from the following detailed description and claims.
Pattern formation is activity, whereby embryonic cells form an orderly spatial arrangement of differentiated tissue. The physical complexity of higher organisms arises through the interaction of cell-endogenous lineages with cell-exogenous signals during embryogenesis. Induction is essential for the generation of various cell types during patterning of embryos, patterning of organism systems, and tissue differentiation in the development of vertebrates from the initial establishment of the body plan (Davidson, E., (1990) Development 108: 365-389; Gurdon. JB, (1992) Cell 68: 185-199; Jessell, TM et al., (1992) Cell 68: 257-270). The effects of developing cell interactions vary. Typically, sensitized cells are changed (induced) from one pathway of cell differentiation by inducing a cell that is different from both the uninduced and induced state of the sensitized cell. Sometimes, cells induce their neighbors to differentiate like themselves (induced homologation); In other cases, the cell prevents its neighbor from differentiating like itself. Cellular interactions in early development can be continuous so that the initial induction between the two cell types leads to the developmental amplification of diversity. In addition, induction occurs not only in embryonic cells, but also in mature cells, and may induce differentiation as well as establish and maintain morphogenesis patterns (J.B. Gurdon (1992) Cell 68: 185-199).
Several hidden polypeptide groups are known to mediate cell-cell signaling during tissue development. An important group of such signaling proteins is the TGFβ superfamily of molecules, having a broad range of functions in many different species. Elements of this family are initially synthesized as larger precursor molecules with animal-terminal signal sequences and pro-domains of various sizes (Kingsley, D.M. (1994) Genes Dev. 8: 133-146). The precursor is then cleaved to release the mature carboxy terminal fragment of 110-140 amino acids. The active signaling moiety consists of a hetero- or homodimer of the carboxy-terminal fragment (Massague, J. (1990) Annu. Rev. Cell Biol. 6: 597-641). The active form of the molecule then interacts with its receptor consisting of two distance-related cross-linked serine / threonine kinases called type I and type II receptors for the molecule family (Massague, J. et al. 1992) Cell 69: 1067-1070; Miyazono, KA et al. EMBO J. 10: 1091-1101). TGFβ binds directly to type II receptors, thereby replenishing type I receptors and modifying them by phosphorylation. The type I receptor then transmits a signal to yet unknown downstream components (Wrana et al., (1994) Nature 370: 341-347).
Several elements of the TGFβ superfamily have been identified that play a prominent role during vertebrate development. Dorsalin is preferentially expressed on the spinal side of the developing chick neural tube (Basler et al. (1993) Cell 73: 687-702). This promotes the outgrowth of necrotic cells and inhibits the formation of motor neurons in vitro, assuming that it plays an important role in neurons patterning along the abdominal axis. Certain bone morphogenetic proteins (BMPs) can induce the formation of ectopic skeletons and cartilage when implanted subcutaneously or intramuscularly (Wozney, J.M. et al. (1988) Science 242: 1528-1534). In mice, mutations in BMP5 have been found to result in effects on many other skeletal elements, including reduced outer ear size and reduced recovery of skeletal cracks in the formation. Kingsley (1994) Genes Dev. 8: 133-146. Despite these effects on skeletal tissue, BMPs play different roles during normal development. For example, they are expressed in non-skeletal tissues (Lyons et al. (1990) Development 109: 833-844), and injection of BMP4 into developing Xenopus embryos promotes the formation of abdominal / posterior mesoderm (Dale et al. (1992) Development 115: 573-585). In addition, mice with mutations in BMP5 have an increased frequency of other soft tissue abnormalities in addition to the skeletal abnormalities described above (Green, M.C. (1958) J. Exp. Zool. 137: 75-88).
During Xenopus development, it was found that elements of the activin subfamily were important for mesoderm induction (Green and Smith (1990) Nature 47: 391-394; Thomsen et al. (1990) Cell 63: 485-493). inhibin) was first described as a gonad inhibitor of follicle-stimulating hormone from pituitary cells. In addition, antagonists of the signaling pathway can be used to convert embryonic tissues into ectoderm, which is a defective pathway of development in the absence of TGFβ-mediated signals. BMP-4 and activin have been shown to be potent inhibitors of neuronal activity (Wilson, P.A. and Hemmati-Brivanlou, A (1995) Nature 376: 331-333).
Another evidence of the importance of TGFβ and urea in early vertebrate development comes from retroviral insertion in the nodal gene of mice. This insertion leads to a failure of primitive gland formation in early embryonic formation, lack of axial mesoderm tissue and hyperplasia of ectoderm and extradermal mesoderm (Conlon et al. (1991) Development 111: 969-981; Iannaccone et al. (1992) Dev Dynamics 194: 198-208). The expected nodal gene product is consistent with previous studies showing that nodal is associated with activin and BMP (Zhou et al. (1993) Nature 361: 543-547). The role of TGFβ and its elements in the development of sexual organs has also been described; Mullerian inhibitors function to cause inhibition of the embryonic duct system that develops into the fallopian tubes and uterus during male development in vertebrates (Lee and Donahoe (1993) Endocrinol. Rev. 14: 152-164).
Elements of the family of signaling molecules also continue to function post-development. TGFβ has an antidifferentiating effect on many cell types, including endothelial cells, epithelial cells, soft muscle cells, fetal hepatocytes, and myeloid, erythropoietic and lymphocytic cells. Animals that cannot produce TGFβ1 (homozygous for invalid mutations in the TGFβ1 gene) are known to survive without birth with any apparent morphological abnormalities (Shull et al. (1992) Nature 359: 693-699; Kulkarni et al. ( 1993) Proc. Natl. Acac. Sci. 90: 770-774). However, these animals die near the baby food due to significant immune penetration in many different organs. These data are consistent with the inhibitory effect of TGFβ on lymphocyte cell growth (Tada et al. (1991) J. Immunol. 146: 1077-1082). In another system, expression of TGFβ transgenes in the breast tissue of mice has been shown to inhibit the development and unknown action of breast tissue during sexual maturation and pregnancy (Jhappan, C. et al. (1993) EMBO J. 12: 1835 -1845; Pierce, DF et al. (1993) Genes Dev. 7: 2308-2317). In addition to this inhibitory effect, TGFβ also enhances the growth of other cell types, as evidenced by its role in neovascularization and proliferation of connective tissue cells. Because of this activity, it plays an important role in wound healing (Kovacs, E.J. (1991) Immunol Today 12: 17-23).
1 is an illustration of a model system for testing the biological activity of the signaline proteins described herein.
FIG. 2 is a histological analysis of the cap explants of animals obtained from embryos or control embryos injected with either Signalin 1 or Signalin 2. FIG.
3 is a histological analysis of animal cap explants obtained from control, signalin1-treated, or signalin2-treated embryos.
4 is a radiograph showing the expression of the expression of various marker RNAs in the injected embryos detected by polymerase chain reaction. Brachiry is a common mesoderm marker, goosecoid is a mesoderm marker of the spine, Xwnt-8 is an abdominal-side mesoderm marker, globin is an abdominal mesoderm marker, and actin is a spine Mesoderm is a marker, NCAM is a marker of neural tissue, EF-1α is expressed ubiquitous, and has a regulating effect on the amount of RNA included in each reaction. The line labeled "E" contains total RNA from whole embryos and is a positive control. The line marked "-RT" is identical to the positive control line except that it does not contain reverse transcriptase and acts as a negative control. The lines designated "S1" and "S2" are samples from embryos injected with xe-signalin 1 and xe-signalin 2, respectively.
5 is a diagram illustrating possible classification of the signaline family into at least three different subfamily. Hatched boxes represent at least 10 inconsistencies across signaline motifs.
6 shows amino acid sequences of various human signal proteins (human-signalin 1-7; SEQ ID NO: 8-24) and Xenopus signaline protein sequences (xe-signalin 1-4; SEQ ID NO: 14-17) Is a sequence diagram comparing.
7A-7C are radiographs showing dose-dependent induction of mesoderm by Xe signaling.
FIG. 7A is a radiograph showing the expression of various marker RNAs in animal poles injected with Xe signaline 2 and cultured up to bag stage 11 (first half) or tadpole stage 38 (late). RNA expression was detected by polymerase chain reaction (PCR). The indicators and lines are the same as the description of FIG. 4 except that the negative control is marked with a minus sign (-).
FIG. 7B is a radiograph showing the expression of various marker RNAs in the poles of animals injected with Xe Signalin 1 and cultured up to tad 38. Total RNA was collected from animal poles expressing various concentrations of Xe signalin1 and detected by PCR. Xe signalin1 induces only the expression of ventral mesoderm, not spinal mesoderm. Note that there is no muscle actin expression (vertebral mesoderm) even at high doses.
FIG. 7C is a radiograph showing the expression of various marker RNAs in animal poles after co-expression of Xe Signalin 1 (or Xmad 1 below) and Xe Signalin 2 (or Xmad 2 below).
FIG. 8 is a radiograph showing RNA expression of Xe Signalin 1 (Xmad 1) and 2 (Xmad 2) during Xenopus development.
Top. Radiograph showing Xe signaline transcripts expressed singly in early Xenopus embryos. Stage 8 blastocysts were dissected into nearly identical third animals (animal, A), marginal (M), or plant growth (vegetal, V), and total RNA was harvested. In step 10, the dorsal (D) and ventral (V) edge regions were explanted and total RNA was harvested. The RNA was analyzed by RT-PCR for the presence of Xe Signaling 1, Xe Signaling 2 and EF-1α transcripts. Another control line is as described in FIG. 4.
bottom. Radiograph showing that expression of Xe signalin is not affected by mesoderm induction. The blastocyst animal cap was dissected and incubated in control buffer (C), 130M BMP-4 protein (B), or 2.3 nM activin protein (A). RNA was harvested at 40 minute intervals (last time point equals 10.5 of the first half) and Xe Signaling 1 (M1), Xe Signaling 2 (M2), Brachial (Bu), and EF-1α transcription The sieve was analyzed by RT-PCR while it was present. The other control lines are as described in the definition of FIG. 4 except that the negative control is marked with a minus sign (-).
9A-D are photographs showing that Xe signaling acts as a downstream of the receptor.
FIG. 9A shows the form (left column) or tissue (step column) of tissue from step 39 from embryos injected with predominantly negative BMP receptor (tBR) (2ng) in the presence or absence of Xe signaling 1 (M1) mRNA (2ng). Right column). The dominant negative BMP receptor does not block the Xe signalin1 of the ventral mesoderm as evidenced by the presence of vesicles (V), mesenchyme and mesoderm (Me).
9B is a radiograph showing the expression of various marker RNAs in animal poles injected with dominant negative BMP receptors. Embryos were injected with tBR (2 ng), Xe signalin 1 (Xmad 1; 2 ng) or Xe signalin 1 (M1) (2 ng each) mixed with tBR, step 39 animal cap RNA as described in FIG. Cultured until analyzed together.
FIG. 9C is a radiograph showing that Xe signalin 1 (Xmad 1) counteracts the effect of an edge cutting receptor. FIG. Embryos are dominant negative activin receptor (tAR) with or without the dominant negative BMP receptor (tBR) (4ng) in the presence or absence of Xmad 1 (M1) mRNA (2ng) or in the presence or absence of Xmad 1 (M1) mRNA (2ng). (2ng). Edge receptors result in expression of N-CAM by blocking TGF-β signaling. Coexpression of Xe signalin 1 (Xmad 1) counteracts this effect.
9D is a radiograph showing that the dominant negative activin receptor (tAR) does not block Xe signalin2 (Xmad 2) induction of spinal mesoderm. Embryos were injected with dominant negative activin receptor (tAR) (2 ng), Xe signalin 2 (2 ng) or Xe signalin 2 (M2) (2 ng each) mixed with tAR, and animal caps ) Or Tadpolgi (late).
10 is a radiograph showing that Xe signaline protein is present in the nucleus and cytoplasm.
Of particular importance in the development and maintenance of tissues in vertebrates is a type of extracellular communication, so-called induction, that occurs between neighboring cell layers and tissues (Saxen et al. (1989) Int J Dev Biol 33: 21-48; and Gurdon et al. (1987) Development 99: 285-306). In inducible interactions, the chemical signal hidden by one cell population affects the developmental fate of the second cell population. Typically, cells that respond to the inductive signal are converted from one cell fate to another cell whose neighbor is the same as the fate of the signaling cell. Inductive signals are carried by key regulatory proteins that act during development to determine tissue patterning. For example, signals mediated by TGFβ superfamily have been shown to play a variety of roles, including participating in vertebrate tissue induction.
The present invention is directed to a gene family of vertebrates called "signalins" herein that acts on intracellular signal transduction pathways initiated by the elements of the TGFβ-superfamily and plays a role in determining tissue fate and maintenance. It's about discovery. For example, the results provided below indicate that proteins encoded by vertebrate signal genes may participate in the regulation of the development and maintenance of various embryos and mature tissues. For example, during embryonic induction, some signals are involved in the patterning and differentiation of both spinal and ventral mesoderm.
Vertebrate and signaling genes or gene products provided by the present invention are apparently comprised of at least seven different elements that can be classified into at least three different subclasses within the signalling family. The vertebrate signal is clearly associated with the drosophila and C. elegans MAD genes both in sequence and in action (Sekelsky et al. (1995) Genetics 139: 1347). The cDNAs, consistent with the vertebrate signal gene transcripts, were originally cloned from Xenopus and are optionally defined as Xe-signalin 1-4. As described in the added examples, primers denatured from replication of the Xenopus signaline were also used to replicate human homologues with the gene. As a result, cDNAs for at least seven other human signal transcripts have been identified and are again optionally defined as Hu-signalin 1-7 herein. Instructions for designated SEQ ID NOs for nucleic acid and amino acid sequences for each signal replication are provided in Table 1 below.
Guidelines for Signaling Sequences in Sequence ListingNucleotide amino acid Xe-Signal 1 SEQ ID NO: 1 SEQ ID NO: 14 Xe-Signal 2 SEQ ID NO: 2 SEQ ID NO: 15 Xe-Signalin 3 SEQ ID NO: 3 SEQ ID NO: 16 Xe-Signal 4 SEQ ID NO: 4 SEQ ID NO: 17 Hu-Signalin 1 SEQ ID NO: 5 SEQ ID NO: 18 Hu-signal 2 SEQ ID NO: 6 SEQ ID NO: 19 Hu-Signalin 3 SEQ ID NO: 7 SEQ ID NO: 20 Hu-signal 4 SEQ ID NO: 8 SEQ ID NO: 21 Hu-signal 5 SEQ ID NO: 9 SEQ ID NO: 22 Hu-signal 6 SEQ ID NO: 10 SEQ ID NO: 23 Hu-signal 7 SEQ ID NO: 11 SEQ ID NO: 24
From apparent molecular weights, vertebrate and signaline proteins are clearly in the size range of about 45 kd to about 70 kd for unmodified polypeptide chains. For example, Xe-signalin 1 and 3 have an apparent molecular weight of about 52.2 kd, Xe-signalin 2 has an apparent molecular weight of about 52.4 kd, and Xe-signalin 4 has an apparent molecular weight of about 64.9 kd .
Analysis of the vertebrate signal sequence did not reveal any obvious similarities with any previously identified domains or motifs. However, with the observation that the signalling protein can be detected in both the nucleus and cytoplasm, the fact that each full-length replication lacks a signal sequence indicates that the vertebrate signalling genes encode internal cellular proteins. .
Notwithstanding the above, careful examination of the replicas presents at least two new domains, one or both of which have properties with the vertebrate signal family. The first clearly conserved structural element of the signaline family takes place in the N-terminal portion of the molecule and is defined herein as the "ν domain". With reference to xe-signalin-1, the ν domain coincides with amino acid residues Leu37-Val130. By regulation of vertebrate signal replicas, the elements are represented by the following sequence: LVKKLK-X (1) -CVTI-X (2) -RXLDGRLQVXXRKGXPHVIYXRWXWPDL-X (3) -VCXNPYHYXRV (SEQ ID NO: 27), wherein X (1) is about 17-25 residues, X (2) is about 1-35 residues, X (3) is about 20-25 residues, and each X is more preferably the corresponding Although it represents an amino acid residue in the vertebrate signal sequence, it represents any single amino acid.
Within the v domain, there is a motif that is conserved nearly between the vertebrate signaline, as well as between the related drosophila and C. elegans MAD polypeptides. In particular, the motif (hereinafter referred to as the "signalin-motif") comprises the consensus sequence LDGRLQVXXRKGXPHVIYXRWXWPDL (SEQ ID NO: 28). Repeatedly, each X independently represents any single amino acid, although more preferably represents an amino acid residue in the corresponding vertebrate signal sequence of the attached sequence listing.
Another apparent motif occurs at the C-terminal position with the signaline family. This is referred to herein as the "χ motif" and is consistent with the amino acid residues Leu405-Leu450 of xe-signalin-1. Repeatedly, by regulation of currently sequenced vertebrate replicas, the χ motif can be represented by the following consensus sequence: LXXXCXXRXSFVKGWGXXXXRQXXXXTPCWIEXHLXXXLQXLDXVL (SEQ ID NO: 29), where each X is a corresponding vertebrate signal in the attached sequence list, though Although more preferred to represent amino acids in the lean sequence, any single amino acid is represented independently.
If you do not wish to be bound by any particular theory, analysis of one of the explicitly conserved motifs (signaling motifs) suggests that the signaling protein family can be classified into at least three different sub-family. As FIGS. 5 and 6 illustrate, xe-signalins 1 and 3 and hu-signalins 1, 3, and 7 represent one sub-family of "signalins" ("α- subfamily" or "α-signalin"). Form clearly. Similarly, xe-signalin 4 and hu-signalin 4 and 2 form a second apparent sub-family ("β- subfamily" or "β-signalin"), and hu-signalins 5 and 6 Form a third sub-family ("γ- subfamily" or "γ-signalin"). Comparison of amino acids around the signaling motif between the elements of the α- subfamily shows that the consensus sequence for the signaling motif is represented as follows: LDGRLQVSHRKGLPHVIYCRVWRWPDLQSHHELKPXECCEXPFXSKQKXV (SEQ ID NO: 30). Similarly, the β and γ subfamily are each characterized by the signaline motif consensus sequence represented as follows: FPHVIYARLWXWPDLHKNELKHVKFCQXAFDLKYDXV (SEQ ID NO: 31) and LDGRLQVXHRKGLPHVIYCRLWRWPDLHSHH-ELKDEENCEYA (SEQ ID NO: 32).
In addition, as described in more detail below, human signalling gene portions have been identified with a list of expressed sequence tags (ESTs) based on the conservation of the one or more structural elements. Based on the analysis of groups to any of the above structural elements, the contiguous portion of the human signaling DNA sequence is linked by appropriate EST fragments and corrected for errors on the EST sequence (e.g., skeletal shift errors, etc.). Made.
In particular, the N-terminal fragment of human cDNA was collected from any such EST sequence and comprises the signaline motif of human cloned sequence hu-signalin 1. The 170 residue fragment represented by SEQ ID NO: 12 (nucleotide) and SEQ ID NO: 25 (amino acid) is an element of α- subfamily, with substantial homologues to other elements of α- subfamily even outside the signaline motif.
In a similar fashion, the C-terminal portion of human signalling replication was collected from the EST sequence based on the sequence for the Xenopus signalling copy. Analysis of the nucleotide (SEQ ID NO: 13) and amino acid (SEQ ID NO: 26) sequences of the fragment revealed that it most closely resembles xe-signalin 2 and, therefore, is clearly part of the transcript for the γ- subfamily element. .
Following identification of the fictitious human sequence using the EST sequence as a template, the full length human signal replica was isolated. The full length sequence is represented by SEQ ID NO: 5 (nucleotides) and SEQ ID NO: 18 (amino acids).
In addition, the experimental results showed that the signalling family was significantly longer than 6 Xenopus clones and 7 human clones. Thus, it is expected that the three defined sub- and each of the other elements still exist as if they were other sub- subs. In addition, the fact that there are substantial homologues between the signaling proteins of other vertebrate species, suggests that the signaling sequences provided herein replicate the signaling homologs obtained from other vertebrates, including fish, birds and other amphibians and mammals. Indicates that it can be used to
Experimental evidence indicates a functional role for the signalin in signal transduction mediated by elements of the TGFβ superfamily. As described in more detail below, certain roles of the signalin have been tested by ectopic expression in single-cell embryos. For example, in the blastocysts, the animal caps were eaten out and cultured until sibling control embryos developed either in stage 11 (vesicle, first half) or stage 38 (taddfold, late). After incubation, the explants were examined for morphology, histology and molecular markers. As described above in the accompanying examples, mRNA encoding xe-signalin1 converts ectoderm into abdominal mesoderm that expresses abdominal marker globin without expressing muscle actin or NCAM, a spinal marker. This data causes xe-signalin1 to act on the signaling cascade of the BMP. The role of xe-signalin2 was tested using the same method. As shown in the examples below, xe-signalin2 also converts the fate of animal poles from ectoderm to mesoderm. However, in contrast to xe-signalin1, the xe-signalin2-induced mesoderm is characterized by the spine. Xe-Signalin2 induces the expression of the following molecular markers: Brachiari, Xwnt-8. Goosecoid, and actin further indicating the presence of mesoderm. This causes xe-signalin2 to act on the signaling cascades of the TGFβs, Vg1 and activin. These data provide a basis for understanding the full extent of growth and patterning in developing vertebrate embryos, which may have important implications, for example, in the treatment of diseases occurring in mesodermal and / or ectoderm intact tissues. do.
Another class of experiments recorded below indicates that at least some signalins are post-translationally modified. For example, phosphorylated forms of protein have been detected. In addition, the nuclear-placed forms of the signaline protein appear to be slightly shifted in molecular weight, indicating a modification compared to the cytoplasmic form. The modification can be in the form of, for example, phosphorylation, ubiquitinylation, acrylated and the like. The post-translational modification of the signaline results in the observed configuration and may also contribute to protein-protein and / or protein-DNA interactions, or instead to the endogenous enzymatic activity of the signaline, or instead May contribute to the stability (eg, half-life) of the protein.
In addition, vertebrate signal gene products are clearly and diversely expressed in various tissues. Briefly, human cDNA samples were amplified from various tissues using denatured primers obtained from the signaline motifs. For the kidney, liver, lung, mammary gland, pancreas, spleen, testis and thymus angles, strong and dominant bands at the correct size for the signalling PCR product were observed by PCR reaction. An important aspect of this data is the observation that signaline gene products are expressed through a wide range of mature tissues.
The "A-tract" sequencing described below allows many different signal transcripts to be expressed in each tissue, and tissue-specific responses to individual elements of the TGFβ superfamily are at least partially different in the various tissues. The patterns of expression vary from one tissue type to the next, without contradicting the fact that they can be regulated by various expressions of.
As the data strongly suggests, the diversity of the signaling family is important for the diversity of responses to each element of the TGFβ family. That is, the ability of a cell to respond to a particular TGFβ, and the type of sensitizing cell present upon induction by said growth factor, may be at least partially a signal in which a signalling gene product is expressed in said cell and / or developed from a specific TGFβ receptor. It may depend on what is intended by. For example, enhancement of certain signaling proteins, or their stoichiometry, may be important for various signaling by elements of the TGF-β superfamily. Some signal proteins include TGFβ subfamily, activin subfamily, DVR subfamily (or even more specifically decapentaplegic or 60A subfamily), gross differentiation factor. 1, GDF-1), GDF-3 / VGR-2, dosulin, nodal, mullerian-inhibiting substance (MIS) or glia-derived neurotrophic growth factor, May be particularly involved in signaling by elements of GDNF).
Accordingly, one object of the present invention relates to a nucleic acid encoding a vertebrate signal protein, a signal protein itself, an antibody that immunoreacts with the signal protein, and a method for preparing the compositions. The present invention also provides diagnostic and therapeutic assays and reagents for detecting and treating diseases, including, for example, excessive expression (or loss thereof) of vertebrate signal homologs. In addition, drug discovery assays can modulate the biological function of the signaling protein, such as by modifying defects of vertebrate signal molecules downstream or upstream in the TGFβ signaling pathway, such as interaction with the TGFβ receptor. Provided to identify reagents. Such reagents may be useful therapeutically to alter the growth and / or differentiation of cells. Other objects of the present invention will be described below or will become apparent to those skilled in the art in light of the present specification.
For convenience, some terms used in the specification, examples, and claims are grouped below.
As used herein, the term “nucleic acid” refers to a polypeptide such as deoxyribonucleic acid (DNA) and suitably ribonucleic acid (RNA). The term should also be understood to include equally RNA or DNA analogues made from nucleotide analogs, and single (sensor or antisense) and double stranded polypeptides, as applicable to the examples.
As used herein, the term “gene” or “recombinant gene” refers to one of the vertebrate signal polypeptides of the present invention comprising exon and (optionally) intron sequences. Refers to a nucleic acid comprising an open reading frame. A "recombinant gene" encodes a vertebrate signal polypeptide, although it optionally comprises an intron sequence derived from a chromosomal vertebrate signal gene or from an unrelated chromosomal gene, and a vertebrate signal signal. -Refers to a nucleic acid comprising an encoded exon sequence. Representative recombinant genes encoding vertebrate signal polypeptides of the invention are shown in the attached sequence listing. The term “intron” refers to a DNA sequence that is not translated into protein and is present in a given vertebrate signalling gene commonly found between exons.
As used herein, the term “transfection” refers to the influx of nucleic acids, such as expression vectors, into recipient cells by nucleic acid-mediated gene transfer. "Transformation", as used herein, refers to an exogenous DNA or RNA whose genotype is exogenous, such as, for example, as a transformed cell expresses a recombinant version of a vertebrate signal polypeptide. Refers to a process in which the expression of the naturally-occurring form of the signalling protein is disrupted whereby the alteration or anti-sense expression resulting from the cellular uptake occurs from an infected gene.
As used herein, the term “specifically hybridize” refers to a spine such as a signaline sequence defined as one of SEQ ID NOs: 1-13, or a complementary sequence thereto, or naturally occurring mutants thereof. Less than 15% of the nucleic acid (eg, mRNA or genomic DNA) of a cell encoding a signal different from the signal protein as defined herein, hybridized to at least 15 consecutive nucleotides of an animal signal gene, Preferably the ability of the marker / primer of the invention to have a basic hybridization of less than 10%, and more preferably less than 5%. In a preferred embodiment, the oligonucleotide label specifically detects only one of the signalling paralogs of the invention, for example, which is not substantially hybridized to other signal homologs.
As used herein, the term "vector" refers to a nucleic acid molecule capable of carrying another linked nucleic acid. One type of preferred vector is an episome, ie a nucleic acid capable of replicating on the surrounding chromosome. Preferred vectors are those in which they can independently replicate and / or express the nucleic acid to which they are linked. Vectors that can influence the expression of genes to which they are operably linked are referred to hereinafter as “expression vectors”. In general, expression vectors of utility in the recombinant DNA art are often in the form of "plasmids" which refer to circular double-stranded DNA loops that do not typically bind to chromosomes in their vector form. In this specification, "plasmid" and "vector" are used interchangeably because the plasmid is the most commonly used form of vector. However, the present invention provides equivalent functionality and is intended to include such other forms of expression vectors that are successfully known in the art herein.
A "transcriptional regulatory sequence" is a genetic term used throughout the specification to refer to DNA sequences, such as initiation signals, enhancers and promoters, that induce or regulate the transcription of the proteins that they are operably linked to. In a preferred embodiment, the transcription of one of the recombinant vertebrate signalling genes is under the control of an promoter sequence (or other transcriptional regulatory sequence) whose expression regulates the expression of the recombinant gene in the cell-type intended. In addition, the recombinant gene is under the control of a transcriptional regulatory sequence that is the same as or different from the sequence that regulates the transcription of the naturally-occurring form of the signaling protein.
As used herein, the term “tissue-specific promoter” acts as a promoter, ie, modulates the expression of a selective DNA sequence operably linked to the promoter and, for example, hepatic, such as a nerve cell. Or a DNA sequence that affects the expression of said selected DNA in specific cells of a tissue, such as pancreatic cells. The term also includes so-called "leaky" promoters that regulate the expression of preferentially selected DNA in one tissue but cause expression in another tissue.
As used herein, a "transgenic animal" is any animal, preferably a non-human mammal, a bird or an amphibian, wherein one or more cells of the animal are by transgenic techniques well known in the art. As such, it contains heterologous nucleic acids derived by human regulatory methods. The nucleic acid is introduced into the cell either by direct or indirect entry into the precursor of the cell via careful genetic manipulation, such as by microinjection or infection with a recombinant virus. The term genetic manipulation does not include conventional cross-breeding or in vitro fertilization, but rather relates to the influx of recombinant DNA molecules. The molecule may be integrated within the chromosome, or may replicate DNA on the surrounding chromosome. In the conventional transgenic animals described herein, the transgenic allows the cell to express a recombinant form of one of a vertebrate signal protein, such as, for example, an agonist or antagonist form. However, transgenic animals in which the recombinant signaline gene is not active may also be considered, such as, for example, the FLP or CRE recombinase dependent constructs described below. Moreover, "transgenic animals" also include those recombinant animals in which gene disruption of one or more signalin genes occurs by human manipulation, including both recombinant and antisense techniques.
The "non-human animals" of the present invention include vertebrates, such as rodents, non-human primates, sheep, dogs, cattle, chickens, amphibians, reptiles and the like. Preferred non-human animals, although transgenic amphibians and transgenic chickens, such as members of Xenopus species, may also provide an important tool for understanding and identifying reagents that affect, for example, embryogenic and tissue formation. , Rodents and mice, more preferably rodents, including mice. The term "virtual animal" is used herein to refer to an animal in which the recombinant is expressed or in some cells, but not in all cells of the animal, or where the presynthetic gene is found. The term "tissue-specific virtual animal" indicates that one of the recombinant vertebrate signal genes is present in some tissue and / or expressed or destroyed and the other is not.
As used herein, the term “transgene” is partially or wholly heterogeneous, ie different, with respect to a transgenic animal or cell into which it is introduced into the animal's genome to change the genome of the cell into which it is inserted. A nucleic acid sequence similar to the endogenous gene of a transgenic animal or cell (eg, a spine) that is defined as or is inserted (ie, it is different from a native gene or is inserted at a position that results in a major strike) Encoding one of the animal signal polypeptides, or hearing an antisense transcript thereof). The transgene may comprise one or more transcriptional regulatory sequences and other nucleic acids necessary for optimal expression of the selected nucleic acid sequence, such as introns.
As is well known, the genes for a particular polypeptide may be present in a single or multiple copies in an individual genome. The replication genes can have any modification, including nucleotide substitutions, additions or deletions, which still encode for polypeptides that are identical or all have substantially the same activity. The term “DNA sequence encoding a vertebrate signal polypeptide” consequently refers to one or more genes in a particular individual. In addition, certain differences in nucleotide sequences may exist between individual organisms, which are referred to as alleles. Such allelic differences do not result in differences in the amino acid sequence of the encoded polypeptide still encoding proteins with the same biological activity.
"Homology" refers to a similar sequence between two nucleic acid molecules or between two peptides. Homology can be determined by comparing positions in each sequence placed for comparison. If the position in the compared sequence is filled by the same base or amino acid, the molecules are homologous at this position. The degree of homology between two sequences is a function of many coincident or similar positions divided by the sequences. An “unrelated” or “non-homologous” sequence shares less than 40% identity, although it is desirable to have less than 25% identity with one of the vertebrate signal sequences of the present invention.
"Cell", "host cell" or "recombinant host cell" are terms used interchangeably herein. The term refers not only to a particular subject cell but also to a descendant or potential descendant of the cell. Because some modifications are generated continuously due to mutations or environmental effects, the descendants are in fact not identical to the parental cells, but are still included within the purpose of the term as used herein.
A "virtual protein" or "fusion protein" refers to a first amino acid sequence that encodes one of the vertebrate signal polypeptides of the present invention and a domain that is substantially similar to or different from any domain of one of the vertebrate signal proteins. For example, a fusion of a second amino acid sequence defining a polypeptide portion). The hypothetical protein allows for the presence of an external domain found in an organ (although within the protein) that also expresses the first protein, or it is a protein expressed by other kinds of organs, such as "species", "intergenes", etc. It can be a fusion of structures. In general, the fusion protein may be represented by the general formula X-signalin-Y, where signalin means a portion of a protein derived from one of the vertebrate signalling proteins, where X and Y are each independently and naturally By an mutant that occurs, it means the presence or absence of an amino acid sequence that is independent of one of the vertebrate signal sequences in the organ.
As used herein, the terms “transformation growth factor-beta” and “TGFβ” refer to the growth and differentiation of structurally related paracrine polypeptides found ubiquitously in vertebrates and with large, round-tailed animals. And morphogenic factors (for review, see Massaque et al. (1990) Ann Rev Cell Biol 6: 597-641; Massaque et al. (1994) Trends Cell Biol. 4: 172-178; Kingsley (1994) Gene Dev. 8: 133-146; and Sporn et al. (1992) J Cell Biol 119: 1017-1021). As described in Kinsley, the TGFβ superfamily has at least 25 elements and can be divided into distinct sub-family with highly related sequences. The most obvious sub-family include: the TGFβ subfamily comprising at least four genes much more similar to TGFβ-1 than in the other elements of the TGFβ family; An activin subfamily comprising a homo- or hetero-dimer or two sub-units, inhibin β-A and inhibin β-B. The decapentaplegic subfamily includes the mammalian factors BMP2 and BMP4, which can induce the formation of ectopic skeletons and cartilage when implanted subcutaneously or intramuscularly. The 60A subfamily includes many mammalian homologues with osteogenic activity, including BMP5-8. Other elements of the TGFβ phase include macrophage differentiation factor 1 (GDF-1), GDF-3 / VGR-2, dosulin, nodal, Müller-inhibiting substance (MIS) and glycal-induced neurotropic growth factor (GDNF) It includes. The DPP and 60A sub-family were more closely related to one another than the other elements of the TGFβ superfamily, and were divided together as part of a larger collection of molecules, often called DVRs (dpp and vg1 related). Unless proven from the context used, the term TGFβ as used throughout the specification will generally be understood to be referred to as elements of the appropriate TGFβ superfamily. Reference to the elements of the TGFβ sub-family will be apparent or will be evidenced from the context in which the term TGFβ is used.
In addition, the term "isolated" as used herein for nucleic acids such as DNA or RNA, refers to molecules that are separated from other DNA or RNA, respectively, present in the natural source of the macromolecule. For example, an isolated nucleic acid encoding one of the vertebrate signal polypeptides of the present invention is preferably only 10 kilobases (kb) that naturally and directly flanks the vertebrate signal gene in genomic DNA. Nucleic acid sequences, more preferably only 5 kb of said naturally occurring flanking sequence, and most preferably less than 1.5 kb of said naturally occurring flanking sequence. As used herein, the term 'isolated' refers to an amino acid or peptide that is substantially free of cellular material, viral material or culture when produced by recombinant DNA technology or chemical precursors or other reagents when chemically synthesized. . "Isolated nucleic acid" is also meant to include nucleic acid fragments that do not occur naturally as fragments and are not found in a neutral state.
As described below, one object of the present invention relates to an isolated nucleic acid comprising a nucleic acid sequence encoding a vertebrate signal polypeptide and / or an equivalent thereof. The term nucleic acid as used herein is intended to include fragments as equivalents. The term equivalent is understood to include a nucleotide sequence encoding a functionally equivalent signal polypeptide or a functionally equivalent peptide having the activity of a vertebrate signal protein as described herein. Equivalent nucleotide sequences will include sequences distinguished by one or more nucleotide substitutions, additions, or removals, such as transgenic mutations, and therefore vertebrates represented by any of SEQ ID NOs: 1-13 due to denaturation of the genetic code. Sequences distant from the nucleotide sequence of the signaline cDNA sequence. Equivalents are also represented by one or more SEQ ID NOs: 1-13 under stringent conditions (ie, equal to about 20-27 ° C. below the melting point (Tm) of the DNA double helix structure formed in about 1M salt) It will include nucleotide sequences that hybridize to nucleotide sequences. In one embodiment, the equivalents will further comprise nucleic acid sequences derived from and evolutionarily associated with the nucleotide sequence represented by any of SEQ ID NOs: 1-13.
In addition, under certain circumstances, the signal of the invention acts on the ability defined as either a signaling agonist (similar) or a signaling antagonist to promote or inhibit only a subset of the biological activity of a naturally-occurring form of the protein. It will be generally contemplated that it may be advantageous to provide homologues of one of the lean polypeptides. As a result, specific biological effects are treated by homologs of limited function and by treatments with less side effects compared to treatment with agonists or antagonists involved in all of the biological activity of naturally occurring forms of the signaline protein. Can be derived.
Homologs of each signaline protein of the invention may be produced by mutagenesis, such as individual point mutagenesis or truncation. For example, a mutation can result in homologues that carry substantially the same or only a subset of the biological activity of the signalling polypeptide from which it is derived. Optionally, an antagonist form of the protein that can inhibit the function of the naturally occurring form of the protein, such as by competing defects in the downstream or upstream components of the signaling cascade comprising the signal protein, Can be generated. In addition, agonist forms of structurally active proteins can occur. As a result, the vertebrate signal proteins and their homologues provided herein can be positive or negative regulators of signal transduction by TGFβ.
In general, polypeptides referred to herein as having the activity (eg, "living") of a vertebrate signal protein are vertebrate signal signals represented by any one or more of SEQ ID NOs: 14-26. A polypeptide is defined that includes amino acids that match all or part of an amino acid sequence of a protein and that mimics or reverses all or part of the biological / biochemical activity of a naturally occurring signaline protein. Examples of such biological activity include the ability to induce (or otherwise regulate) the formation and differentiation of mesoderm or ectoderm tissue to develop vertebrate embryos. Therefore, the polypeptides of the present invention are stem cells including cells derived from chordamesoderm, vertebral (axial) mesoderm, intermediate mesoderm, lateral mesoderm, head mesenchymal, epithelial cell, neural tube or neural tort cells, etc. cell) or germ cells are characterized by their ability to induce and / or maintain survival. The signaline proteins of the present invention may also have biological activity, including the ability to modulate organogenesis, for example, through its ability to influence limb patterning by osteogenic activity. In addition, the signalin can be characterized by their ability to induce or inhibit proliferation of the cells as cells of the immune system and fibroblasts. Additional effects of signalling can be observed in tissue maintenance or post-denaturation recovery, such as skeletal repair or wound healing. Biological activities associated with the signaline proteins of the invention also include the ability to modulate sexual maturation or reproduction, including acting on suppression of Müller tracts, controlling lactation or production and follicle formation of follicle stimulating hormone. can do.
The bioactivity of the signaline proteins of the invention can also be determined by, for example, by engaging (activating or inhibiting) a transcript phase complex, such as one of homo- or hetero-oligomers, or of a protein of the complex. And the ability to change the rate of transcription of a gene, such as by modifying the ability and / or utility of the composition of the transcriptional complex. The signaline gene product may also be involved in regulating post-transcriptional modification of other cellular proteins, for example, by the action of endogenous enzyme activity, as regulatory subunits of enzyme complexes and / or as chaperons. have.
Yet another bioactivity of the signaline proteins of the present invention is the ability to interact with TGFβ receptor complexes or subunits thereof, especially receptor complexes having ligand binding thereto.
Other biological activities of the signaline proteins of the invention will be described herein or will be apparent to those skilled in the art. According to the present invention, a polypeptide has biological activity if it is a specific agonist or antagonist in a naturally-occurring form of vertebrate signal protein.
Preferred nucleic acids are at least 60%, more preferably 70%, most preferably 80 with the amino acid sequence of human or Xenopus α-signalin as selected from the group consisting of SEQ ID NOs: 14, 16, 18, 20 and 24 Encodes a vertebrate a-signal polypeptide comprising% similar amino acid sequences. Encodes a polypeptide that is at least about 90%, more preferably at least about 95%, most preferably at least about 98-99% similar to an amino acid sequence represented by one of SEQ ID NOs: 14, 16, 18, 20, and 24 Nucleic acids are also within the scope of the present invention. In one embodiment, the nucleic acid is a cDNA encoding a peptide having at least one activity of a vertebrate signal polypeptide of the invention. Preferably, the nucleic acid comprises all or part of a nucleotide sequence that matches the coding region of SEQ ID NO: 1, 3, 5, 7 or 11.
In one preferred embodiment, the invention characterizes a purified or recombinant signaline polypeptide having a molecular weight in the range of 45 kd to 70 kd. For example, α and β subfamily, which are preferred signal chain polypeptides, have a molecular weight in the range of about 45 kd to about 55 kd, more preferably 50-55 kd. In another embodiment, γ subfamily, which is a preferred signal chain polypeptide, has a molecular weight ranging from about 60 kd to about 70 kd, more preferably 63-68 kd. For example, it will be appreciated that some post-translational modifications, such as phosphorylation, may increase the apparent molecular weight of the signaline protein compared to unmodified polypeptide chains.
In another embodiment, the preferred nucleic acids are at least 50% of the amino acid sequence of human or Xenopus β- or γ-signalin, such as for example selected from the group consisting of SEQ ID NOs: 15, 17, 19, 21, 22 and 23 Bioactive fragments of vertebrate β- or γ-signal polypeptides comprising similar, more preferably 60% similar, more preferably 70% similar, most preferably 80% similar amino acid sequences Code it. A polypeptide that is at least about 90%, more preferably at least about 95% and most preferably at least about 98-99% similar to an amino acid sequence represented by one of SEQ ID NOs: 15, 17, 19, 21, 22 and 23 Nucleic acids encoding are also within the scope of the present invention.
Still other preferred nucleic acids of the invention correspond to all or a portion of amino acid residues 225-300 of SEQ ID NO: 14 or 230-301 of SEQ ID NO: 16, for example at least 5, 10, 25 or 50 amino acid residues. Encodes an α-signaling polypeptide comprising a polypeptide sequence. Similarly, preferred nucleic acids encoding γ-signaling polypeptides include sequences for polypeptide sequences that match all or part of amino acid residues 186-304 of SEQ ID NO: 15. More preferred nucleic acids encode a γ-signalin polypeptide comprising an amino acid sequence that matches all or part of the polypeptide sequence from 262-304 of SEQ ID NO: 15. In yet another preferred embodiment, the signalling nucleic acids encode a β-signaling polypeptide sequence comprising a polypeptide sequence that matches all or part of amino acid residues 170-332 of SEQ ID NO: 17. More preferred nucleic acids encode β-signalin polypeptides comprising amino acid sequences that match all or part of the polypeptide sequence from 260-332 of SEQ ID NO: 17.
Another object of the present invention is to provide nucleic acids that hybridize to nucleic acids represented by one of SEQ ID NOs: 1-13 under high or low stringency conditions. Appropriate stringent conditions that promote DNA hybridization reactions, such as, for example, 6.0 × sodium chloride / sodium citrate (SSC) at about 45 ° C., followed by 2.0 × SSC washes at 50 ° C., are known to those skilled in the art and are currently known in the art. , John Wiley & Sons, NY (1989), 6.3.1-6.3.6. For example, the salt concentration in the washing step can be selected from a low stringency of about 2.0 × SSC at 50 ° C. to a high stringency of about 0.2 × SSC at 50 ° C. In addition, the temperature in the washing step can be increased from room temperature, low stringency conditions of about 22 ° C. to high stringency conditions of about 65 ° C.
Nucleic acids having sequences different from the nucleotide sequence represented by one of SEQ ID NOs: 1-13 due to denaturation of the genetic code are also within the scope of the present invention. The nucleic acid encodes a functionally equivalent polypeptide (ie, a peptide having the biological activity of a vertebrate signal polypeptide) but differs in sequence from the sequence appearing in the sequence listing due to denaturation in the genetic code. For example, many amino acids are designated as one or more triplets. Codons specifying the same amino acid, or analog (eg, each of CAU and CAC encode histidine) result in a "inactive" mutation that does not affect the amino acid sequence of the vertebrate signal polypeptide. However, it is expected that DNA sequence polymorphisms resulting in changes in the amino acid sequence of the signaline polypeptides of the invention will exist between vertebrates. Those skilled in the art will appreciate that such modifications (up to about 3-5% of the nucleotides) in one or more nucleotides of a nucleic acid encoding a polypeptide having the activity of a vertebrate signal polypeptide are due to natural transformation. It will be appreciated that it may exist between species.
As used herein, a signaline gene fragment is more than a nucleotide sequence that encodes a fully mature form of vertebrate signal protein that (preferably) still encodes a polypeptide that retains some biological activity of the full length protein. It refers to a nucleic acid having a small nucleotide. Fragment sizes contemplated by the present invention include, for example, 5, 10, 25, 50, 75, 100 or 200 amino acid lengths.
As indicated in the examples described below, signaline protein-encoding nucleic acids can be obtained from mRNA present in any of many eukaryotic cells. It should also be possible to obtain nucleic acids encoding the vertebrate signal polypeptide of the invention from genomic DNA obtained from both maturity and embryos. For example, genes encoding signaline proteins can be cloned from either cDNA or genomic libraries, as well as those generally known to those skilled in the art, as well as the methods described herein. CDNA encoding the signal protein can be obtained by separating whole mRNA from cells, such as mammalian cells, such as human cells, including embryonic cells. Double stranded cDNAs can then be prepared from the whole mRNA and subsequently inserted into the appropriate plasmid or bacteriophage vector using any one of many known techniques. Genes encoding vertebrate signal proteins can also be replicated using polymerase chain reaction techniques completed according to the nucleotide sequence information provided by the present invention. The nucleic acid of the present invention may be DNA or RNA. Preferred nucleic acids are represented by sequences selected from the group consisting of SEQ ID NOs: 1-13.
Another object of the invention relates to the use of said isolated nucleic acid in "antisense" therapies. As used herein, an "antisense" therapy is one or more of the signaling proteins of the invention, under cellular conditions, to inhibit expression of the protein, for example by inhibiting transcription and / or translation. Dosage or in situ production of oligonucleotide labels or derivatives thereof that specifically hybridize with cellular mRNA and / or genomic DNA encoding the above. Such binding may be by conventional base pair complementarity or, for example, when binding to a DNA double helix, through specific interactions in the main groove of the double helix. In general, “antisense” therapies refer to the art commonly used in the art and include any therapy that relies on specific binding to oligonucleotide sequences.
The antisense constructs of the invention can be delivered, for example, as expression plasmids that produce RNA complementary to at least a portion of cellular mRNA that encodes a vertebrate signal protein when transcribed in a cell. In addition, the antisense constructs are oligonucleotide labels that are produced ex vivo and inhibit expression by hybridizing with mRNA and / or genomic sequences of vertebrate signal genes when introduced into the cell. The oligonucleotide markers are preferably modified oligonucleotides that are resistant to endogenous nucleases such as, for example, exonucleases and / or endonucleases, and are therefore stable in vivo. Do. Representative nucleic acid molecules for use as antisense oligonucleotides are phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (US Pat. Nos. 5,176,996; 5,264,564; and See also 5,256,775). In addition, general approaches to constituent oligomers useful for antisense therapies are described, for example, in Van der Krol et al. (1988) Biotechniques 6: 958-976; And Stein et al. (1988) Cancer Res 48: 2659-2668.
Thus, the modified oligomers of the present invention are useful for treatment, diagnosis and research. In therapeutic applications, the oligomers are generally used in a manner suitable for antisense treatment. For such treatments, oligomers of the present invention can be prepared for a variety of dosage drops including global and local or local administration. Techniques and preparations can typically be found in Remmington's Pharmaceutical Sciences, Meade Publishing Co., Easton, PA. For global dosing, the infusion preferably comprises intramuscular, intravascular, intraperitoneal and subcutaneous injections. For injection, the oligomers of the present invention can be prepared in a liquid solution, preferably in a physically acceptable buffer such as Hank's solution or Ringer's solution. The oligomers may also be prepared in solid form and redissolved or suspended immediately prior to use.
Overall dosing is also by a subcutaneous or focal membrane method, wherein the compound can be administered orally. For focal membranes or subcutaneous dosing, penetrants suitable for the barriers to be received may be used in the preparation. Such penetrants are commonly known and include, for example, gallbladder salts and fusidic acid derivatives for focal membrane administration. Also, permeants can be used to promote permeation. Focal membrane dosing can be via intranasal spray or using suppositories. For oral administration, the oligomers are prepared in conventional oral dosage forms such as capsules, tablets and tonics. For topical dosing, the oligomers of the invention are generally prepared in ointments, plasters, gels or creams as known in the art.
In addition to use in therapy, oligomers of the invention can be used as diagnostic reagents for detecting the presence or absence of target DNA or RNA sequences to which they specifically bind. The diagnostic test is described in more detail below.
Similarly, the antisense constructs of the present invention can be used to manipulate tissues such as tissue differentiation in both in vivo and ex vivo tissue culture by counteracting the normal biological activity of one of the signaline proteins.
In addition, the anti-sense technique (e.g., microinjection of an antisense molecule or infection with a plasmid whose transcript is anti-sense with respect to a signal mRNA or gene sequence) is a normal cellular function of the signal in mature tissues. In addition, it can be used to investigate the role of signaling in development. The technique can be used for cell culture, but can also be used for the creation of transgenic animals.
The present invention also provides an expression vector containing a nucleic acid encoding a vertebrate signalling polypeptide operably linked to at least one transcriptional regulatory sequence. Operationally linked means that the nucleotide sequence is linked to a regulatory sequence by expressing the nucleotide sequence. Regulatory sequences are known and are selected to induce expression of the vertebrate signal protein of the invention. Thus, the term transcriptional regulatory sequence includes promoters, enhancers and other expression control elements. Such regulatory sequences are described in Goeddel: Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). For example, one of the various expression control sequences that, when operably linked, regulates expression of the DNA sequence can be used as the vector expressing a DNA sequence encoding the vertebrate signal polypeptide of the invention. Such useful expression control sequences are, for example, LTR of Moloney murine leukemia virus, early and late promoters of SV40, adenoviruses or cytomegaloviruses that mediate general promoters. viral LTRs such as the lac system, trp system, TAC or TRC system. T7 promoter whose expression is advanced by T7 RNA polymerase, the main operating and promoter region of phage 1, the regulatory region for fd coat protein, 3-phosphoglycerate kinase or other Glycolytic enzymes such as promoters of acid phosphatase such as Pho5, yeast a-mating factors, polyhedron promoters of baculovirus systems and genes of prokaryotic or eukaryotic cells or these And other sequences known to modulate the expression of viruses and various mixtures thereof. It is to be understood that the design of the expression vector may depend on factors such as the choice of the host cell to be transformed and / or the type of protein to be expressed. In addition, the number of copies of the vector, the number of copies and expression of any other protein encoded by the vector, such as antibiotic markers, should also be considered. In one embodiment, the expression vector comprises a recombinant gene encoding a peptide having the agonist activity of the signaline polypeptide of the invention, or optionally a peptide which is an antagonist form of the signalling protein. The expression vectors can be used to infect cells and produce polypeptides, including fusion proteins encoded by nucleic acids as described herein.
In addition, the gene construct of the present invention may also be used as part of a gene therapy method for delivering nucleic acids encoding the agonist or antagonist forms of one of the vertebrate signal proteins of the invention. As a result, another object of the present invention is to reconstruct or eliminate the function of signaline-induced signaling in tissues in which the naturally-occurring form of the protein is misexpressed, or to change the differentiation of a tissue or to develop a tumorous trait. Characterize expression vectors for in vivo or in vitro infection and expression of vertebrate signal polypeptides in specific cell types to deliver protein types that inhibit modification.
Expression constructs of the vertebrate signal polypeptides of the invention and their mutants can be administered in a biologically effective carrier such as, for example, any formation or composition capable of effectively delivering the recombinant genes to cells in vivo. have. This includes the insertion of the gene into viral vectors, or recombinant bacteria or eukaryotic plasmids, including recombinant retroviruses, adenoviruses, adeno-associated viruses and herpes community virus-1. Viral vectors directly infect cells: plasmid DNA can be cationic liposomes (lipopeptins) or induced (eg, in addition to CaPO ₄ precipitation or direct injection of these gene constructs, for example, performed in vivo). For example, antibody conjugated) polylysine conjugate, gramasidine S, technical viral cover, or other such intracellular carriers may be delivered. Since delivery of appropriate target cells represents a critical first step in gene therapy, the choice of appropriate gene delivery system will depend, for example, on location or structurally, on factors such as the dosage route and the type of target intended. You can see the point. In addition, it can be seen that certain gene constructs provided for in vivo delivery of signal expression are also useful for in vitro delivery of cells, such as for use in the ex vivo tissue culture system described below.
There is a preferred approach for incorporating nucleic acids into cells in vivo, for example by the use of viral vectors containing nucleic acids such as cDNA encoding the specific signalling polypeptide intended. Infection of cells with viral vectors has the advantage that a large proportion of the targeted cells can accept the nucleic acid. In addition, molecules encoded in the viral vector, for example by cDNA contained in the viral vector, are effectively expressed in cells that take viral vector nucleic acids.
Retroviral vectors and adeno-associated viral vectors should generally understand the recombinant gene delivery system chosen for exogenous gene delivery in vivo, specifically to humans. The vectors provide for efficient delivery of the gene into the cell, and the delivered nucleic acid is stably integrated into the host's chromosomal DNA. An important necessity for the use of retroviruses ensures the stability of their use, particularly with regard to the spread of natural-type viruses in the cell population. The development of specialized cell lines (called "commercial cells") that produce only replication-defective retroviruses increases the use of retroviruses for gene therapy, and defective retroviruses are well characterized for use in gene delivery for gene therapy purposes. (For review, see Miller, AD (1990) Blood 76: 271). As a result, a recombinant retrovirus can be constructed that is replaced by a nucleic acid encoding one of the proteins of the invention such that a portion of the retroviral coding sequence (gag, pol, env) becomes a retrovirus replication defect. The replication defective retroviruses are eventually commercialized as virions that can be used to infect target cells through the use of helper viruses by standard techniques. Methods for producing recombinant retrovirusers and infecting cells with the viruses in vitro or in vivo are described in Current Protocols in Milecular Biology, Ausubel, F.M. (Published) Greene Publishing Associates, (1989). Section 9.10-9.14 and other standard experimental methods. Examples of suitable retrovirators include pLJ, pZIP, pWE and pEM, which are well known to those skilled in the art. Examples of commercially available virus lines suitable for producing both ecotropic and amphotropic retroviral systems include ψCrip, ψCre, ψ2 and ψAm. Retroviruses have been used to introduce a variety of genes into a variety of cell types, including neurons, in vivo and / or in vitro (eg, Eglitis et al. (1985) Science 230: 1395-1398; Danos and Mulligan). (1988) Proc. Natl. Acad. Sci. USA 85: 6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85: 3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87: 6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88: 8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA 88: 8377-. 8381; Chowdhury et al. (1991) Science 254: 1802-1805; van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89: 7640-7644; Kay et al. (1992) Human Gene Therapy 3: 641- 647; Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89: 10892-10895; Hwu et al. (1993) J. Immunol. 150: 4104-4115; US Pat. No. 4,868,116; US Pat. No. 4,980,286 PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application Let see WO 92/07573 call).
It has also been found that by modifying viral package proteins on the surface of viral particles, it is possible to limit the infection spectrum of retroviruses and consequently retrovirus-based vectors (eg, PCT Application WO93 / 25234). And WO94 / 06920). For example, strategies for modifying the infection spectrum of retroviral vectors include the steps of coupling antibodies specific for the cell surface to viral env proteins (Roux et al. (1989) PNAS 86: 9079-9083; Julan et al. 1992) J. Gen Virol 73: 3251-3255 and Goud et al. (1983) Virology 163: 251-254; Or coupling a cell surface receptor ligand to the viral env protein (Neda et al. (1991) J Biol Chem 266: 14143-14146). Coupling refers not only to the creation of a fusion protein (e.g., a single-chain antibody / env fusion protein) but also to chemical cross-linking with a protein or other substance (lactose which modifies the env protein with an asialoglycoprotein). It may be in the form of connecting. While the technique is useful for confining or manipulating the infection in certain tissue types, it can also be used to convert ecotropic vectors into ampotropic vectors.
In addition, the use of retroviral gene delivery may be further enhanced by tissue- or cell-specific transcriptional regulatory sequences that regulate the expression of the signalling genes of the retroviral vectors.
Another viral gene delivery system useful in the present invention uses adenovirus-derived vectors. The genome of adenoviruses can be manipulated to encode and express subject gene products, but this is inactivated by the ability to replicate in the viral survival cycle of normal cell lysis. See, eg, Berkner et al. (1988) Biotechniques 6: 616; Rosenfeld et al. (1991) Science 252: 431-434; And (1992) Cell 68: 143-155 by Rosenfled et al. Adenovirus lineages Suitable adenovirus vectors derived from Ad type 5 dl324 or other lineages of adenoviruses (eg, Ad2, Ad3, AD7, etc.) are known in the art. Recombinant adenoviruses include those that include airway epithelium (Rosenfeld et al. (1992), cited above), endothelial cells (Lemarchand et al. (1992), Proc. Natl. Acad. Sci. USA 89: 6482-6486), hepatocytes (Herz and Gerard ( 1993), Proc. Natl. Acad. Sci. USA 90: 2812-2816) and muscle cells (Quantin et al. (1992), Proc. Natl. Acad. Sci. USA 89: 2581-2584). It may be advantageous in some circumstances in that it may be used to infect. In addition, the virus particles can be purified and concentrated and are relatively stable and, as described above, can be modified to affect the spectrum of infectivity. In addition, the induced adenovirus DNA (and foreign DNA contained therein) is not integrated into the genome of the host cell, but retains the epidermal so that the induced DNA is integrated into the host genome (eg, retroviral DNA). Potential problems that can arise as a result of insertional mutagenesis in the context can be avoided. In addition, the ability of the adenovirus genome to transport foreign DNA is greater (up to 8 kilobases) than other gene transfer vectors (Berkner et al., Cited above; Haj-Ahmand and Graham (1986) J. Virol. 57: 267). Most of the replication-defective adenovirus vectors of interest in the present invention and therefore of interest in the present invention are deleted for all or part of the viral E1 and E3 genes, but retain as much as 80% of the adenovirus genetic material (eg See, for example, Jones et al. (1979) Cell 16: 683; Berkner et al., Supra; and Graham et al., Methods in Molecular Biology, EJ Murray (Humana, Clifton, NJ, 1991), vol. 7, pp. 109-127). . Expression of the inserted signal gene can be, for example, under the control of an E1A promoter, a major late promoter (MLP) and related leader sequence, an E3 promoter, or an exogenously added promoter sequence. have.
Yet another viral vector system useful for the delivery of one of the vertebrate signal genes of the present invention is adeno-associated virus (AAV). Adeno-associated viruses are naturally occurring defect viruses that require another virus, such as adenovirus or herpes virus, as an aid virus for effective replication and productive survival cycles (for review, Curr et al., Muzyczka et al. Topics in Micro. And Immunol. (1992) 158: 97-129). It is also one of the few viruses that integrate its DNA into non-isolated cells and shows a high frequency of stable integration (see, eg, Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol. 7 (349-356; Samulski et al. (1989) J. Virol. 63: 3822-3828; and J. Virol. 62: 1963-1973 by Mclaughlin et al. (1989)). Vectors containing as little as 300 base pairs of AAV can be packaged and integrated. The space for exogenous DNA is limited to about 4.5 kb. Tratschin et al. (1985) Mol. Cell. Biol. AAV vectors as described in 5: 3251-3260 can be used to introduce DNA into cells. Various nucleic acids have been introduced into various cell types using AAV vectors (eg, Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81: 6466-6470; Tratschin et al. (1985) Mol. Cell. Biol) 4: 2072-2081; Wondisford et al. (1988) Mol. Endocrinol. 2: 32-39; Tratschin et al. (1984) J. Virol. 51: 611-619; and Flotte et al. (1993) J. Biol. Chem. 268. See: 3781-3790).
In addition to viral delivery methods, as illustrated above, non-viral methods can also be used to express subject signaline polypeptides in animal tissues. Most nonviral methods of gene transfer rely on normal mechanisms used by mammalian cells for uptake and intracellular transport of macromolecules. In a preferred embodiment, the non-viral gene delivery system of the present invention relies on an endocytic pathway for uptake of the subject signaling polypeptide by the targeted cell. Representative gene delivery systems of this type include lipomalmal induction systems, polylysine conjugates, and artificial viral envelopes.
In medical applications, the gene delivery system of the therapeutic signalling gene can be introduced into a patient by one of many methods familiar in the art. For example, pharmaceutical preparation of the gene delivery system may be introduced entirely, such as for example, in a whitening injection, and specific delivery of the protein in the target cell may be necessary to control the expression of the gene delivery material, the receptor gene. Predominantly from the specificity of the infection provided by cell-type or tissue-type expression, or a combination thereof, due to filamentous regulatory sequences. In another embodiment, the initial delivery of the recombinant gene is further limited to influx into a fully localized animal. For example, the gene transfer material may be introduced by catheter (US Pat. No. 5,328,470) or by stereotactic injection (eg, Chen et al. (1994) PNAS 91: 3054-3057). Can be. Vertebrate signal genes, such as one of the copies represented by the group consisting of SEQ ID NOs: 1-13, can be prepared using, for example, techniques described by Dev et al. ((1994) Cancer Treat Rev 20: 105-115). It can be delivered to gene therapy constructs by electroporation.
Pharmaceutical preparation of such gene therapy constructs consists essentially of gene delivery systems in acceptable dilutions. In addition, where the complete gene delivery system can be produced directly from recombinant cells, such as, for example, retroviral vectors, the pharmaceutical preparation can include one or more cells producing the gene delivery system. .
Another object of the present invention relates to a recombinant form of the signaline protein. In addition to the negative signal protein, preferred recombinant polypeptides in the present invention are at least 60% similar, more preferably 70% similar, most preferably 80 to the amino acid sequence represented by any of SEQ ID NOs: 14-26. % similar. Has at least 90%, more preferably at least 95% and most preferably at least about 98 sequence activity of the signaline protein (ie, agonist or antagonist) and selected from the group consisting of SEQ ID NOs: 14-26 -99% similar polypeptides are also within the scope of the present invention.
The term "recombinant protein" refers to a polypeptide of the invention produced by recombinant DNA technology, wherein, typically, DNA encoding a vertebrate signal polypeptide is used to produce an appropriate expression vector, in other words a heterologous protein. Inserted by an appropriate expression vector used to transform the host cell. In addition, for recombinant signalling genes, the phrase “derived from.” Refers to those generated by mutations involving substitution and deletion (including cleavage) of the amino acid sequence of a naturally occurring signal protein or naturally occurring protein form. By "recombinant protein" having a similar amino acid sequence.
The present invention is directed to a gene derived from a vertebrate, in particular a mammal (eg, a human), having an amino acid sequence that is evolutionarily related to a signaline protein represented by SEQ ID NOs: 14-26. It relates to a recombinant of one of the signaline polypeptides. The recombinant signaline polypeptides may preferably function as one of at least one biologically active agonist or antagonist of a naturally-type ("true") signalin protein of the attached Sequence Listing. With respect to the amino acid sequence of a vertebrate signal protein, the term "evolutionally related" refers to both polypeptides having naturally occurring amino acids and variant variants of vertebrate signal polypeptides, e.g., induced by combinatorial mutagenesis. it means. The evolutionarily derived signal protein polypeptides noted by the present invention are at least 60% similar, more preferably 70% similar, most preferably similar to amino acids selected from the group consisting of SEQ ID NOs: 14-26. 80% similar. Polypeptides having at least about 90%, more preferably at least about 95%, and most preferably at least about 98-99% similarity with a sequence selected from the group consisting of SEQ ID NOs: 14-26 are also disclosed herein. It is in a category.
The invention also relates to a method of producing a subject signaline polypeptide. For example, a host cell infected with a nucleic acid vector expressing a nucleotide sequence encoding a subject polypeptide can be cultured under appropriate conditions to cause expression of the peptide. The cells are harvested, lysed, and proteins separated. Cell cultures include host cells, cultures and other byproducts. Suitable cultures for cell culture are well known in the art. The recombinant signaline polypeptide is ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification from an antibody specific for the peptide from cell culture culture, host cells, or both. Can be separated using law. In one preferred embodiment, the recombinant signaline polypeptide is a fusion protein containing a domain that facilitates its purification, such as a GST fusion protein or a poly (His) fusion protein.
The invention also relates to an infected host cell expressing a recombinant type of the subject signaline polypeptide. The host cell can be a prokaryotic or eukaryotic cell. As a result, nucleotide sequences derived from the replication of vertebrate signal proteins encoding all or selected portions of the full-length protein produce recombinant versions of vertebrate signal polypeptides via microbial or eukaryotic processes. It can be used to Ligation of the polynucleotide sequence with a gene construct, such as an expression vector, and transformation or infection with a host cell of either eukaryotic (east, avian, insect or mammal) or prokaryotic (bacterial cell) is for example MAP. It is a standard procedure used to produce other well-known proteins such as kinases, p53, WT1, PTP phosphatase, SRC and the like. Similar procedures or variations thereof can be used to prepare recombinant signaline polypeptides by microbiological methods or tissue-culture techniques in accordance with the present invention.
The recombinant signaling genes can be prepared by ligation of nucleic acids encoding signalling proteins or portions thereof into a vector suitable for expression in prokaryotic, eukaryotic or both. Expression vectors for the production of recombinant versions of the present signal polypeptides include plasmids and other vectors. For example, plasmid types such as pBR322-derived plasmid, pEMBL-derived plasmid, pEX-derived plasmid, pBTac-derived plasmid and pUC-derived plasmid for expression in prokaryotic cells such as E. Coli.
There are many vectors for the expression of recombinant proteins in yeast. For example, YEP24, YIP5, YEP51, YEP52, pYES2 and YRP17 are replicating and expressing materials useful for the entry of genetic constructs into S. cerevisiae (see, e.g., Broach et al. (1983), incorporated herein by reference). See Experimental Manipulation of Gene Expression, edited by M. Inouye Academic Press, page 83). The vectors can replicate in S. cerevisiae due to replication determinants of E. Coli and yeast 2 micron plasmids due to the presence of pBR322 origin. In addition, drug resistance indicators such as ampicillin may be used. In one embodiment, the signaline polypeptide is produced recombinantly using an expression vector produced by sub-cloning encoding the sequence of one of the signalin genes represented by SEQ ID NO: 1-13.
Preferred mammalian expression vectors contain both prokaryotic cell sequences and one or more eukaryotic transcriptional units expressed in eukaryotic cells to facilitate the development of said vector in bacteria. The pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg induction vectors are examples of mammalian expression vectors useful for infection of eukaryotic cells. Some of these vectors are modified with sequences obtained from bacterial plasmids such as pBR322 to promote replication and both drug resistance selection in prokaryotic and eukaryotic cells. In addition, derivatives of viruses such as bovine papillomavirus (BPV-1) or Epstein-Barr virus (pHEBo, pREP-induced and p205) can be used for transient expression of proteins in eukaryotic cells. . Various methods are known in the art for the production of plasmids and for the transformation of host organs. As well as general recombination methods, as well as other suitable expression systems for both prokaryotic and eukaryotic cells, the Molecular Cloning A Laboratory Manual by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989), 2nd Edition, 16 and 17 See chapter.
In some instances, it is desirable to express the recombinant signaline polypeptide by use of a baculovirus expression system. Examples of such baculovirus expression systems include pVL-derived vectors (eg pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (eg pAcUW1), and pBlueBac-derived vectors (eg β-gal containing pBlueBac III). Included.
If you want to express only a portion of a signaline protein, such as a mutant lacking a portion of the N-terminus, ie, a truncated variant lacking a signal peptide, add a start codon (ATG) to the oligonucleotide fragment containing the intended sequence to be expressed. It is necessary to let. It is known in the art that methionine at the N-terminal position can be enzymatically cleaved by the use of the enzyme methionine aminopeptidase (MAP). MAP has been cloned from E. Coli and Salmonella typhimurium (Ben-Bassat et al. (1987) J. Bacteriol. 169: 751-757) and in vitro activity has been revealed on recombinant proteins (Miller et al. (1987) PNAS 84: 2718-1722). Therefore, if desired, the removal of N-terminal methionine may be used in vivo, or by the use of purified MAP, by expressing a signaline-induced polypeptide in a host that produces the MAP (eg, E. Coli or CM89 or S. cerevisiae). In vitro (e.g., the method of Miller et al., Supra).
In addition, the coding sequence for the polypeptide can be inserted as part of a fusion gene comprising nucleotides encoding another polypeptide. Expression systems of this type can be used under conditions desired to produce immunogenic fragments of signaline proteins. For example, the VP6 capsid protein of rotavirus can be used as an immune carrier protein for a portion of the signaling polypeptide in the form of viral particles or in monomeric form. Nucleic acid sequences consistent with a portion of the signaline protein of the invention in which the antibodies are increased result in a late baccinia that produces a group of recombinant viruses expressing a fusion protein comprising a signaline epitope as part of the virion. ) Can be inserted into a fusion gene construct comprising the coding sequence for the viral structural protein. This has been argued with the use of immunogenic fusion proteins using a hepatitis B surface antigen fusion protein in which recombinant Hepatitis B virion can be used for this action. Similarly, virtual constructs encoded for fusion proteins containing portions of signaline proteins and poliovirus capsid proteins can be made to enhance the immunogenicity of a group of polypeptide antigens (eg EP See Publication No. 0259149 and Nature 339: 385 to Evans et al. (1989); J. Virol. 62: 3855 to Huang et al. (1988); and J. Virol. 66: 2 to Schlienger et al. (1992)).
Multiple Antigen Peptide Systems for Peptide-Based Immune Responses can also be used to generate immunogens, wherein the desired portion of the signaline polypeptide is onto an oligomeric sided lysine core. Obtained directly from organic-chemical synthesis of peptides (see, eg, JBC 263: 1719 by Posnett et al. (1988) and J. Immunol. 148: 914 by Nardelli et al. (1992)). Antigen determinants of signaline proteins may also be expressed and may be present by bacterial cells.
In addition to using fusion proteins to enhance immunogenicity, it is well known that fusion proteins can also promote expression of the protein and thus can be used for expression of vertebrate signaline polypeptides of the invention. The GST-fusion proteins may facilitate purification of the signaline polypeptide, for example, by the use of a glutathione-induced mattress (eg, Current Protocols in Molecular Biology, Ausubel et al. (John Wiley & Sons, 1991).
In another embodiment, a fusion gene encoded for a purified leader sequence, such as a poly- (His) / enterokinase cleavage site sequence, at the N-terminus of the desired portion of the recombinant protein. Enables the purification of the expressed fusion protein by affinity chromatography using Ni2 + metal resins. Purification leader sequences can be subsequently removed by treatment with enterokinase, resulting in purified proteins (eg, J. Chromatography 411: 177 by Hochuli et al. (1987); and PNAS 88: 8972 by Janknecht et al. See).
Techniques for making fusion genes are known in the art. Essentially, the binding of the various DNA fragments encoded for the various polypeptide sequences can be achieved by ligation, restriction enzyme digestion provided for appropriate ends, filling-in at appropriately terminated ends, and unwanted. Alkaline phosphatase treatment to avoid binding, and conventional techniques using blunt or twisted-end ends for enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques, including automated DNA synthesis. In addition, PCR amplification of gene fragments can be performed using an anchor primer that causes complementary overlap between two consecutive gene fragments that can be continuously annealed to generate a virtual gene sequence (eg, See, Current Protocols in Molecular Biology, Ausubel Publishing, John Wiley & Sons: 1992).
Signalin polypeptides can also be chemically modified to make signaline derivatives by forming covalent or mixed conjugates with other chemical moieties such as glycosyl groups, lipids, phosphates, acetyl groups and the like. Covalent derivatives of signaline proteins are prepared by binding the chemical moiety to a functional group on the amino acid side chain of the protein or at the N-terminus or C-terminus of the polypeptide.
The present invention also produces useful isolated signalling polypeptides that are isolated from, in particular, other signal transduction factors and / or transcription factors normally associated with the signalling polypeptide, or in other words, substantially free of other cellular proteins. The term "substantially free of other cellular proteins" (or referred to herein as "polluting proteins") or "substantially pure or purified preparation" refers to contaminating proteins of less than 20% (dry weight units), and preferably Is defined as the enveloping preparation of a signaline polypeptide having less than 5% contaminating protein. "Purified" when referred to a peptide or DNA or RNA sequence, means that the indicated molecule is in substantial absence of other biological macrophages, such as other proteins. As used herein, the term "purified" is preferably a dry weight of preferably at least 80% dry weight, more preferably 95-99% dry weight, most preferably at least 99.8% dry weight of biological macromolecules of the same type. It means weight. The term “pure” as used herein preferably has a numerical limit such as “purified” just above. "Isolated" and "purified" are separated into components (e.g. acrylamide gel), but neither is pure (e.g. contaminating proteins such as polymers and polymers such as acrylamide or agarose) or Natural substances not obtained as a solution or a solution) or in their natural state without chromatographic reagents. In a preferred embodiment, the purified signaline preparation is free of any contaminating protein from an animal, such as can be achieved by recombinant expression of human signalling proteins in non-human cells, for example, in which signalling is normally produced. will be.
As described above for the recombinant polypeptide, the isolated signal polypeptide is SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, matches a signaline polypeptide represented by one or more of these similar sequences It may include all or part of the amino acid sequence.
An isolated peptide portion of a signaline protein can be obtained by a screening peptide produced recombinantly from the corresponding fragment of nucleic acid encoding the peptide. The fragments can also be chemically synthesized using techniques known in the art, such as conventional Merrifield solid phase f-Moc or t-Boc chemistry. For example, the signaline polypeptides of the present invention may be optionally divided into fragments of desired length without overlapping the fragments, or may be preferably divided into overlapping fragments of desired length. The fragments can be prepared (recombinantly or chemically synthesized) and tested to identify such peptidic fragments that can act as agonists or antagonists of natural-type (eg, "true") signalling proteins.
The recombinant signaline polypeptides of the present invention may also be true signal such as modification of the protein that resists proteolytic cleavage, for example due to ubiquitination or variations that alter enzymatic targeting associated with the protein. Include allogenes of lean protein.
Modifications to the structure of the vertebrate signal polypeptides of the invention may be of therapeutic or prophylactic effect, stability (eg, ex vivo shelf life and resistance to proteolysis in vivo), or post-transcriptional modifications (eg, proteins). To change the phosphorylation pattern of the same). The modified peptides, when designed to retain at least one activity of a naturally-occurring form of the protein or to produce antagonists specific to them, are functional equivalents to the signaline polypeptides described in more detail herein. Is considered. The modified peptides can be prepared, for example, by amino acid substitution, deletion, or addition.
For example, independent substitution of leucine with isoleucine or valine, substitution of aspartate with glutamate, substitution of threonine with serine, or analogous substitution of amino acids with structurally related amino acids (eg, isotopes and And / or isopotent variations) do not have a major influence on the biological activity of the resulting molecule. Conservative substitutions occur within the amino acid families involved in their subchains. Genetically encoded amino acids can be divided into four families: (1) acidic = aspartate, glutamate; (2) basic = lysine, arginine, histidine; (3) nonpolar = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; And (4) uncharged polarity = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan and tyrosine are sometimes hybridized and classified as aromatic amino acids. In a similar manner, amino acid repertoires can be divided as follows: (1) acidic = aspartate, glutamate; (2) basic = lysine, arginine, histidine; (3) aliphatic = glycine, alanine, valine, leucine, isoleucine, serine, threonine, where serine and threonine can be selectively classified as aliphatic-hydroxyl groups; (4) aromatic = phenylalanine, tyrosine, tryptophan; (5) amide = asparagine, glutamine; And (6) sulfur-containing = cysteine and methionine. (See, eg, Biochemistry, 2nd edition, L. Street, WH Freeman & Co .; 1981). Whether the exchange of the amino acid sequence of the pentide generates a functional signaling homologue (eg, the functional polypeptide in the sense that it complements or reverses the native-type type) in a similar manner, for example, in cells It can be easily detected by generating a response to the natural-type protein or by identifying the forces of the variant peptides that competitively inhibit the response. Polypeptides with one or more substitutions can be easily tested in the same manner.
In addition, the present invention contemplates methods for making combinatorial variants of the signaline proteins of the present invention as well as corner cutting variants, which identify potential variant sequences (e.g. homologues) that function to modulate signal transduction from the TGFβ receptor. Essentially useful. The purpose of screening the combinatorial libraries (libraries) is to prepare new signaline homologues that can act, for example, as agonists or antagonists, and optionally have all of the new activity. For example, signaline homologues provide selective, essential activity of the TGFβ induction pathway by the method of the present invention as a similar induction by the TGFβ when the signaline homolog is expressed in a cell capable of responding to the TGFβ. It may be designed to. As a result, combinatorially-derived homologues can be prepared with increased capacity as compared to the naturally occurring form of the protein.
Similarly, signaline homologues can be prepared by the combinatorial approach of the present invention to selectively inhibit (opposite) induction by TGFβ. For example, mutagenesis can bind to other signal pathway proteins (or DNA), but can provide a signaling homologue that interferes with signal development, eg, the homologues can be dominant negative variants. have. Preferred dominant negative variants may comprise sufficient C-terminal fragments that counteract TGFβ signaling. In addition, manipulation of certain regions of the signal by the methods of the invention can provide a domain more suitable for use in a fusion protein.
For one purpose of the method, the amino acid sequence for the population of signaline homologues or other related proteins is preferably arranged to promote as high homology as possible. The variant population may include, for example, signaline homologs obtained from one or more species. The amino acids appearing at each position of the arranged sequences are selected to make a denaturing group of combination sequences. In one preferred embodiment, the altered library of signaline variants is made by combinatorial mutagenesis at the nucleic acid level and encoded by the altered gene library. For example, combinatorial oligonucleotide mixtures can be enzymatically ligated into a gene sequence such that a denaturing group of potential signaling sequences contains, as an individual polypeptide, or, optionally, a larger group of signaling sequences therein. It can be expressed as a group of proteins (eg for phage display).
As illustrated in FIG. 6, to analyze the sequences of a group of variants, the amino acid sequences of interest can be arranged in comparison to sequence homology. The presence or absence of amino acids from the arranged sequences of certain variants is proportional to the selected consensus length of the reference sequence, which may be true or artificial. In order to maintain high similarity in the arrangement of the sequences, deletions in the variant sequence compared to the reference sequence may be indicated by the amino acid space ( ^* ), while insertional variation in the variant compared to the reference sequence is ignored. The sequence of the variant may be omitted when arranged. For example, FIG. 6 includes an arrangement of signaline-motifs for several vertebrate signaline gene products. Analysis of the arrangement of the motifs from the signaline replicas can result in the generation of denatured libraries of polypeptides containing potential signaline sequences.
In one embodiment, the arrangement of signaline-motifs for Xenopus and human replicas can be used to prepare a modified group of signaline polypeptides comprising the signaline-motif represented by the following general formula:
VX (1) -X (2) -RKGX (3) -PHVIYX (4) -RX (5) -WRWPDLX (6) -X (7) -X (8) -X (9) -X (10)- LKX (11) -X (12) -X (13) -X (14) -CX (15) -X (16) -X (17) -FX (18) -X (19) -KX (20)- X (21) -X (22) -V.
Here, each denaturing position “X” may be an amino acid occurring at a position in either a human or Xenopus copy. For example, Xaa (1) represents Ser, Pro, or Ala, Xaa (2) represents His or Gly, Xaa (3) represents Leu or Phe, Xaa (4) represents Cys or Ala , Xaa (5) represents Val or Leu, Xaa (6) represents His or Gln, Xaa (7) represents Ser or amino acid gap, Xaa (8) represents His or Lys, Xaa (9) represents His or Asn, Xaa (10) represents Glu or Gly, Xaa (11) represents Pro, Ala, or His, Xaa (12) represents Leu, Ile, Val or Met, Xaa (13) represents Lys or Glu, Xaa (14) represents Cys, Asn or Phe, Xaa (15) represents Glu or Gln, Xaa (16) represents Tyr, Phe or Leu, Xaa ( 17) represents Pro or Ala, Xaa (18) represents Glu, Asn, Val or Asp, Xaa (19) represents Ser or Leu, Xaa (20) represents Gln, Lys or Tyr, and Xaa ( 21) represents Lys or Asp, and Xaa (22) is Glu or Asp. Indicates. In a more extended library, each denatured position X can be selected from any amino acid that conservatively substitutes with those amino acid residues occurring in Xenopus and human clones conserved, for example, isopotentially or by polarity. Even in a more extended library, each X can be selected from any amino acid.
There are many ways in which the library of potential signal homologs can be generated from denatured oligonucleotide sequences. Chemical combinations of degenerate gene sequences can be performed with automated DNA synthesizers, and the synthesized genes can in turn be ligated with the appropriate expression vector. The purpose of the denaturing group of genes is to provide all of the sequences that encode the desired group of potential signal sequences into one mixture. Synthesis of denatured oligonucleotides is well known in the art (eg, Narang, SA (1983) Tetrahedron 39: 3; Itakura et al. (1981) Recombinant DNA, Proc 3rd Edition Cleveland Sympos. Elsevier 273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53: 323; Itakura et al. (1984) Science 198: 1056; Ike et al. (1983) Nucleic Acid Res. 11: 477). Such techniques have been used in the direct development of other proteins (eg, US Pat. Nos. 5,223,409, 5,198,346 and 5,096,815, as well as Scott et al. (1990) Science 249: 386-390; Roberts et al. (1992). PNAS 89: 2429-2433; Devlin et al. (1990) Science 249: 404-406; Cwirla et al. (1990) PNAS 87: 6378-6382).
Similarly, libraries of encoded sequence fragments can be provided in signaline copies to make a varied group of signaline fragments for screening and continuous selection of bioactive fragments. Various techniques are known for making such libraries, including chemical combinations. In one embodiment, the library of coding sequence fragments comprises: (i) treating a double stranded PCR fragment of the signalling coding sequence with a nuclease under conditions where clearance cleavage occurs only about once per molecule; (ii) denaturing the double stranded DNA; (iii) regenerating the DNA to form double stranded DNA that may include sense / antisense pairs obtained from other gap cleavage products; (iv) removing the single stranded portion from the reformed duplex by treating with S1 nuclease; And (v) ligation of the resulting fragment library with an expression vector. By the above methods, expression libraries can be derived to encode N-terminal, C-terminal and intermediate fragments of various sizes.
Many techniques are known for screening gene products of combinatorial libraries made by point mutations or edge cutting, and for screening cDNA libraries for gene products with specific properties. The techniques will be applicable for rapid screening of gene libraries, which are generally generated by combinatorial mutagenesis of signaline homologues. The most widely used technique for screening large gene libraries involves replicating the gene library with a replicable expression vector, transforming the appropriate cells with the resulting library of vectors, and detecting the desired activity to detect the product. Expressing said combination gene under conditions that facilitate the isolation of the vector encoding the gene of interest. Each of the run assays described below can be analyzed with high throughput analysis, which is essentially necessary to screen the very large number of denatured signal sequences produced by combinatorial mutagenesis.
Still another technique that can be used to purify fragments of the signaline proteins of the invention, such as for example binding domains, is described in Roman et al. (1994) Eur J Biochem 222: 65-73. Roman et al. Describe the use of competitive-binding assays using short, overlapping synthetic peptides from large proteins. Roman et al. Have been applied to identify binding domains in proteins of approximately the same size as the signaline proteins of the invention.
In one embodiment, embryonic stem cells (ES) may be utilized to analyze the altered signal library. For example, a library of expression vectors can be infected with ES cell lines that typically respond to specific TGFβ. The infected cells are subsequently contacted with TGFβ and the effect of the signaline variant on the induction of morphotype markers by the paracrine factor can be detected, for example by FACS. Plasmid DNA can subsequently be recovered from cells aimed at inhibition, or optionally for enhancing TGFβ induction, and individual copies can be further characterized. Other cell lines can be substituted for ES cells, even from primitive animal cap cells to embryonic tumor cells, ie, cells from mature, differentiated tissue, such as, for example, chondrocytes or osteogenic cells.
Combination mutagenesis, for example, has the ability to make a very large library of variant proteins within the 10 ²⁶ molecule size. Combination libraries of this size are technically challenging to screen technically with high throughput screening assays. In order to overcome this problem, a new technology, recrusive ensemble mutagenesis (REM), has been developed, which avoids very high proportions of non-functional proteins in random libraries and functional proteins. By increasing the frequency of, the complexity required to achieve useful sampling of the sequence space is reduced. REM is an answer that increases the frequency of functional variants in libraries when appropriate selection or screening methods are used (Arkin and Yourvan, 1992, PNAS USA 89: 7811-7815; Yourvan et al., 1992, Parallel Problem Solving from Nature, 2nd Edition). , Edited in In Maenner and Manderick, Elservir Publishing Co., Amsterdam, pp. 401-410; Delgrave et al., 1993, Protein Engineering 6 (3): 327-331).
The invention also relates to the spine making a mimetic such as, for example, a peptide or non-peptide reagent, which may interfere with the binding of the vertebrate signal polypeptide of the invention with the upstream or downstream components of its signaling cascade. It provides a reduction in animal signal protein. As a result, the above-described mutagenesis techniques can also be applied to proteins or nucleic acids that can act downstream of the signalling polypeptide, for example whether they are positively or negatively controlled by the signalling polypeptide or Useful for mapping determinants of said signalling proteins involved in protein-protein interactions involved in binding the vertebrate signal signaling polypeptides of the invention to proteins capable of acting upstream (including activators and inhibitors thereof) Do. Illustratively, a signaline-derived analogue in which the critical residues of the signalling polypeptides of the invention involved in the molecular recognition of the upstream or downstream signalling component competitively inhibit binding of the portion with the true signalling protein. It can be determined and used to make it. For example, by employing a method of scanning for mutagenesis to map the amino acid residues of each of the signal proteins of the invention related to binding other extracellular proteins, Peptide-like compounds can be made that mimic the residue. As a result, the mimetics can be used to interfere with the normal functioning of the signal protein. For example, non-hydrolyzable peptide analogues of the residues include benzodiazepine (e.g., Freidinger et al., Peptides: Chemistry and Biology, GR Marshall, ESCOM Publisher: Leiden, Netherlands, 1988), azepine (E.g., Huffman et al., Peptides: Chemistry and Biology, GR Marshall Edit, ESCOM Publisher: Leiden, Netherlands, 1988), Substituted gamma lactam ring (Garvey et al. Peptides: Chemistry and Biology, GR Marshall Edit , ESCOM Publisher: Leiden, Netherlands, 1988), keto-methylene pseudopeptides (Ewenson et al. (1986) J Med Chem 29: 295; and Ewenson et al., Peptides: Structure and function (9th US) Presented at the Peptide Symposium) Pierce Chemical Co., Rockland, IL, 1985), β-turn dipeptide core (Nagai et al. (1985) Tetrahedron Lett 26: 647; and Sato et al. (1986) J Chem) Soc Perkin Trans 1: 1231), and β-aminoal Kool (see Gordon et al. (1985) Biochem Biophys Res Commun 126: 419; and Dann et al. (1986) Biochem Biophys Res Commun 134: 71).
Another object of the present invention relates to antibodies that specifically react with vertebrate signal proteins. For example, using immunogens derived from signaline proteins based on cDNA sequences, anti-protein / anti-peptide antiserum or monoclonal antibodies can be prepared by standard methods (e.g., Harlow and Lane ( Antibodies: A Laboratory Manual, compiled by Cold Spring Harbor Press: 1988). Mammals, such as mice, hamstars or rabbits, can be immunized with immunogenic forms of the peptides (eg, vertebrate signal polypeptides or antigen fragments capable of eliciting antibody response). Techniques for identifying immunogenicity on proteins or peptides include conjugation to a carrier or carriers or other techniques known in the art. The immunogenic portion of the signal protein may be administered in the presence of an adjuvant. The development of immunization can be monitored by the detection of antibody titers in plasma or serum. Standard ELISA or other immunoassays can be used with the immunogen as an antigen to confirm the level of the antibody. In one preferred embodiment, the antibodies of the invention are antigenic determinants or highly related homologs of a signaline protein of a vertebrate, such as a mammal, such as, for example, an antigenic determinant of a protein represented by SEQ ID NOs: 14-26. Eg, at least 85% similar, preferably at least 90% similar, more preferably at least 95% similar). In still another preferred embodiment of the invention, the anti-signalin is provided for providing immuno-selective antibodies against isolated signal homologues such as, for example, hu-signalin 1 or hu-signalin 2 Polypeptide antibodies do not substantially cross react (ie, do not specifically react) with, for example, less than 85%, 90% or 95% similar proteins with the selected signaline. For "substantially no cross-reacting", this means that the antibody is at least 1 class, more preferably at least 2 class, most preferably at least 3 class small non- than the binding affinity of the antibody for the intended target signaline. It has a binding affinity for homologous proteins.
After immunization of the animal with an antigenic preparation of the signaline polypeptide, anti-signalin antiserum can be obtained and, if desired, polyclonal anti-signalin antibodies can be isolated from the serum. To produce monoclonal antibodies, antibody-producing cells (lymphocytes) can be harvested from immunized animals and standardized with immortal cells, such as myeloma cells, that produce hybridoma cells. Fusion may be by somatic cell fusion procedure. Such techniques are well known in the art and include, for example, hybridoma technology (mostly developed by Kohler and Milstein (1975), Nature, 256: 495-497), human B cell hybridoma technology (Kozbar Et al. (1983) Immunology Today, 4:72), and EBV-hybridoma technology to produce human monoclonal antibodies (Cole et al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. 77 P. 96). Hybridoma cells can be immunochemically screened for the production of antibodies that specifically react with the vertebrate signal polypeptide of the invention and monoclonal antibodies isolated from embryos comprising the hybridoma cells.
The term antibody as used herein is meant to include fragments thereof which also specifically react with one of the vertebrate signal polypeptides of the invention. Antibodies can be fragmented using conventional techniques and the fragments can be screened for use in the same method as described above for the whole antibody. For example, F (ab) ₂ fragments can be prepared by treating the antibody with pepsin. The resulting F (ab) ₂ fragment can be processed to reduce the disulfide bridge that produces the Fab fragment. Antibodies of the invention may be further intended to include bispecific and imaginary molecules having affinity for a signaline protein identified by at least one CDR region of the antibody.
Both true signal polypeptides or monoclonal and polyclonal antibodies that act against signal fragments and antibody fragments such as Fab and F (ab) ₂ both block the action of one or more signalin proteins and For example, it can be used to study the role of these proteins in embryogenesis and / or maintenance of various tissues. In a similar manner, hybridomas that produce biodegradable gels in which anti-signaling monoclonal Abs or anti-signaling Abs are suspended can be implanted in or near the region where the signaling action is blocked. Such characterization experiments can help to decipher the action of these and other factors that may be involved in limb patterning and tissue formation.
Antibodies that specifically bind to a signaline epitope can also be used for immunochemical staining of tissue samples to assess the respective expression patterns and abundances of the signaline polypeptides of the invention. Anti-signalin antibodies can be used for diagnostics in immuno-precipitation and immuno-blotting to detect and assess signal levels of signaling proteins in tissues as part of medical test procedures. For example, the above measures can be used for predictive assessment of the onset or development of skeletal disease. Similarly, the ability to monitor signal protein levels in a patient allows to confirm the effectiveness of a specific therapeutic regimen on a patient suffering from the disease. The level of signaline polypeptide can be measured from cells in body fluids, such as cerebral spinal fluid or amniotic fluid samples, or can be measured in tissue made by biopsy. Diagnostic assays using anti-signalin antibodies may include, for example, immunoassays designed to assist in the early diagnosis of specific pathological degenerative diseases manifested at birth. Diagnostic assays using anti-signalin polypeptide antibodies may also include immunoassays designed to help phenotypic and early diagnose tumorous or aberrant proliferation.
Another application of the anti-signalin antibodies of the invention is in immunological screening of cDNA libraries formed with expression vectors such as λgt11, λgt18-23, λZAP and λORF8. Messenger libraries of this type with coding sequences inserted into the correct reading backbone and direction can make fusion proteins. For example, [lambda] gt11 can be made into a fusion protein whose amino terminus consists of the β-galactosidase amino acid sequence and its carboxy terminus consists of an external polypeptide. For example, an antigenic epitope of a signal protein, such as another artholog of a specific signal protein or another paralog from the same species, may be, for example, a nitrocellulose filter raised from an infected plate. By reacting a nitrocellulose filter with an anti-signalin antibody, it can be detected using the antibody. Positive phage detected by the assay can subsequently be separated from the infected plate. As a result, the presence of signaline homologues can replace heterozygotes (including conjugated variants) obtained from humans and therefore can be detected and replicated from other animals.
In addition, nucleotide sequences detected from replication of the signaline genes from vertebrates identify and / or replicate signaline homologues from other vertebrates, as well as other cell types, such as other tissues. To allow for the generation of deceased markers and primers for use. For example, the present invention also provides a label / primer comprising a substantially purified oligonucleotide, wherein the oligonucleotide is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, sequence SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or naturally occurring ones thereof And regions of nucleotide sequences that hybridize under stringent conditions to at least 10 consecutive nucleotides of sense or antisense selected from the group consisting of variants. For example, primers based on the nucleic acid represented by SEQ ID NO: 1-13 can be used in a PCR reaction that replicates signaling analogs. Similarly, labels based on the signaline sequences of the present invention can be used to detect transcriptional or genomic sequences encoding the same or similar proteins. In a preferred embodiment, the labels comprise a label group attached thereto and detectable, for example the label group is selected from radioisotopes, fluorescent substances, enzymes and coenzymes.
The markers may also be detected by detecting the levels of signaline-encoded nucleic acid in a cell sample obtained from a patient, for example by detecting signal mRNA levels or by checking whether the signaline genes in the genome have been mutated or deleted. It can be used as part of a diagnostic test kit to identify cells or tissues that misexpress a signaling protein.
By way of example, nucleotide labels can be made from the signaline genes of the present invention that facilitate histological screening of adjacent tissues and tissue samples for the presence (or absence) of signaline-encoded transcripts. Similar to the diagnostic use of anti-signalin antibodies, the use of a signal directed to a signal, or a genomic signal sequence, may be used, for example, for tumorous or hyperproliferative diseases (eg, unwanted cell growth) or tissue. It can be used for both the prophylactic and therapeutic assessment of trait mutations that result from abnormal differentiation of. The oligonucleotide markers used in conjunction with the immunoassay described above can facilitate the detection of a molecular basis for evolutionary diseases, including some abnormalities associated with the expression (or lack thereof) of signaline proteins. . For example, modifications of polypeptide synthesis can be differentiated from variations in the coding sequence.
Thus, the subject provides a method for determining whether a method of the present invention is at risk of a disease due to excessive cell proliferation and / or differentiation. In a preferred embodiment, the method comprises (i) alteration affecting the nature of the gene encoding the signal-protein, typically in a sample of cells from a subject, or (ii) the error of the signaline gene. And detecting the presence or absence of a genetic defect characterized by at least one of the expressions. For example, the genetic defect may include (i) removal of one or more nucleotides from the signalling gene, (ii) addition of one or more nucleotides to the signalling gene, (iii) one or more nucleotides of the signalling gene Substitution of, (iv) large chromosomal rearrangements of the signaling gene, (v) large replacement at the level of the messenger RNA transcript of the signaling gene, (vi) signaling signals such as genomic DNA methylation patterns Excessive modification of (vii) the presence of a non-natural type conjugation pattern of the messenger RNA transcript of the signaline gene, (viii) the non-natural type level of the signaline-protein, (ix) the allele of the signal gene Loss, and (x) the presence of one of the inappropriate post-transcriptional modifications of the signaline-protein. As described below, the present invention provides a number of assay techniques for detecting defects in the signaline gene, and more importantly between signaline-dependent and various molecular causes that affect cell growth, proliferation and / or differentiation. Provides the ability to identify from
In one exemplary embodiment, the 5 'or 3' flanking naturally associated with the sense or antisense sequence of the signalling gene represented by SEQ ID NO: 1-13 or naturally occurring variants thereof, or the signalling gene of the present invention. Nucleic acid compositions are provided comprising (purified) oligonucleotide labels comprising regions of nucleotide sequences capable of hybridizing with sequences or naturally occurring variants thereof. The nucleic acid of the cell is easy to access for hybridization, the label is exposed to the nucleic acid of the sample, and hybridization of the label to the sample nucleic acid is detected. The technique can be used not only to detect mRNA transcript levels, but also to detect defects in the genomic or mRNA, including deletions, substitutions and the like.
In one embodiment, the detection of the defect is polymerase chain reaction (PCR) such as Anker PCR or RACE PCR (see, eg, US Pat. Nos. 4,683,195 and 4,683,202), or optionally ligation chain reaction (LCR) ( Landegran et al. (1988) Science 241: 1077-1080; and Nakazawa et al. (1944) PNAS 91: 360-364), the latter of which detect point mutations in the signaline genes. Can be used in particular. In one embodiment, the method comprises (i) collecting a sample of cells from a patient, (ii) isolating nucleic acid (eg, genomic, mRNA, or both) from cells of the sample, (iii) (if Contacting said nucleic acid sample with one or more primers specifically hybridizing to a signalling gene under conditions such that hybridization and amplification of said signalling gene occurs, and (iv) Detecting the size and comparing the length with the control sample.
As noted above, one object of the present invention relates to diagnostic assays for detecting whether in the context of cells isolated from a patient a variation has occurred in one or more signalsin of a sample cell. The method of the present invention provides a method for detecting whether a subject is at risk of a disease due to excessive cell proliferation and / or differentiation. In a preferred embodiment, the method comprises detecting the presence or absence of a genetic defect characterized by an exchange that affects the properties of a gene encoding a signaline typically in a cell sample obtained from a subject. Is characterized. For example, the genetic defects may include (i) removal of one or more nucleotides from the signaline-gene, (ii) addition of one or more nucleotides to the signaline-gene, (iii) one of the signaline-genes Or substitution of more nucleotides, and (iv) the presence of at least one of the presence of a non-natural type conjugation pattern of the messenger-gene's messenger RNA transcript. As described below, the present invention provides a number of assay techniques for detecting defects in the signaline genes, and more importantly, various molecular causes that affect signaline-dependent transient cell growth, proliferation and / or differentiation. Provide the ability to identify.
In one embodiment, the detection of the defect is polymerase chain reaction (PCR) such as Anker PCR or RACE PCR (see, eg, US Pat. Nos. 4,683,195 and 4,683,202), or optionally ligation chain reaction (LCR) ( Landegran et al. (1988) Science 241: 1077-1080; and Nakazawa et al. (1944) PNAS 91: 360-364), the latter of which detect point mutations in the signaline genes. Can be used in particular. In one embodiment, the method comprises (i) collecting a sample of cells from a patient, (ii) isolating nucleic acid (eg, genomic, mRNA, or both) from cells of the sample, (iii) (if Contacting said nucleic acid sample with one or more primers specifically hybridizing to a signalling gene under conditions such that hybridization and amplification of said signalling gene occurs, and (iv) Detecting the size and comparing the length with the control sample. PCR and / or LCR are expected to be preferred for use as a preliminary amplification step in conjunction with any of the techniques used to detect mutations described herein.
In one preferred embodiment of the assay, the signaline genes obtained from the sample cells are detected by exchange in restriction enzyme cleavage patterns. For example, sample and control DNA were isolated, (optionally) amplified, digested with one or more restriction endonucleases, and fragment length size was measured by gel electrophoresis. In addition, the use of sequence specific ribozymes (see, eg, US Pat. No. 5,498,531) can be used to record the presence of specific variations by the development or loss of ribozyme cleavage sites.
In yet another embodiment, any of the various sequencing reactions known in the art can be used to detect mutations by directly sequencing the signalling gene and comparing the native-type (control) sequence with the sample signalling sequence. have. Representative sequencing reactions include methods based on techniques developed by Maxim and Gilbert (Proc. Natl Acad Sci USA (1977) 74: 560) or Sanger (Sanger et al. (1977) Proc. Natl. Acad. Sci 74: 5463). do. It is contemplated that any of a variety of automated sequencing methods may be used when performing the assays of the present invention, including sequencing by mass spectrophotometry (eg, PCT Publication WO 94/16101; Cohen et al. (1996) Adv Chromatogr). 36: 127-162 and Griffin et al. (1993) Appl Biochem Biotechnol 38: 147-159). In one embodiment, it will be apparent to those skilled in the art that the generation of only one, two or three nucleic acid bases should be determined by sequencing reaction. For example, an A-track or similar may be performed where only one nucleic acid is detected.
In another embodiment, protection from cleavage reagents (along with nucleases, hydroxylamine or osmium tetroxide, and piperidine) is mismatched in RNA / RNA or RNA / DNA heteroduplexes. Can be used to detect nucleotides (Myers et al. (1985) Science 230: 1242). In general, the field of “mismatched cleavage” provides heteroduplexes formed by hybridizing (labeled) RNA or DNA containing a naturally occurring type signaling sequence with a potential variant RNA or DNA obtained from a tissue sample. Start by The double-stranded duplex is treated with a reagent that cleaves a single-stranded region of the duplex due to mismatched base pairs between the control and sample strands. For example, RNA / DNA duplexes can be treated with RNases, and DNA / DNA hybrids can be treated with S1 nucleases that enzymatically digest mismatched regions. In other embodiments, the DNA / DNA or RNA / DNA duplexes can be treated with hydroxylamine or osmium tetroxide and piperidine to digest mismatched regions. After digestion of the mismatched regions, the resultant material eventually separates by crevice on a modified polyacrylamide gel to determine the site of mutation. For example, Cotton et al. (1988), Proc. Natl. Acad. Sci USA 85: 4397; Saleeba et al. (1992) Methods Enzymod. See 217: 286-295. In one preferred embodiment, the control DNA or RNA can be labeled for detection.
In yet another embodiment, the mismatch cleavage reaction uses one or more proteins (so-called "DNA mismatch recovery" enzymes) that recognize mismatched base pairs in double-stranded DNA. For example, E. Coli's mutY enzyme cleaves A at G / A mismatch, and thymidine DNA glycoslase from HeLa cells cleaves T at G / T mismatch (Hsu Et al. (1994) Carcinogenesis 15: 1657-1662). According to an exemplary embodiment, a label based on a signalling sequence, such as, for example, a natural-type signalling sequence, is hybridized to cDNA or other DNA product obtained from test cells. The duplex is treated with a DNA mismatch repair enzyme and the cleaved product, if present, can be detected from electrophoresis or the like. See, for example, US Pat. No. 5,459,039.
In other examples, changes in electrophoretic mobility will be used to identify variations in the signaline genes. For example, single strand conformation polynorphism (SSCP) can be used to detect differences in electrophoretic mobility between variants and native type nucleic acids (Orita et al. (1989) Proc. Natl. Acad. Sci. USA 86: 2766, Cotton (1993) Mutat Res. 285: 125-144; and Hayashi (1992), Genet Anal Tech Appl 9: 73-79). Single-stranded DNA fragments of the sample and the control signal nucleic acid will be denatured and regenerated. Secondary structures of single-stranded nucleic acids vary depending on their sequence, with the resulting change in electrophoretic mobility allowing detection of even single base changes. The DNA fragment is labeled or detected with a labeled label. The sensitivity of this assay can be enhanced by using RNA whose secondary structure is more sensitive to changes in sequence (rather than DNA). In a preferred embodiment, the method utilizes a heteroduplex analysis that separates double stranded heteroduplexes based on changes in electrophoretic phase mobility (Keen et al. (1991) Trends Genet 7: 5).
In still other examples, transfer of variant or natural-type fragments to polyacrylamide gels containing denaturant gradients is detected using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313: 495 ). When DGGE is used as a detection method, for example, by adding approximately 40 bp of GC clamp of high-melt GC-containing DNA by PCR, the DNA will be modified to not denature completely. In another example, temperature gradients are used instead of denaturing reagent gradients to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265: 12753).
Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers can be prepared that hybridize to the target DNA under conditions permitting hybridization only if such known variations occur centrally and consequently only complete consensus is found (Saiki et al. (1986) Nature 324: 163; Saiki et al. (1989) Proc. Natl. Acad. Sci USA 86: 6230). The trait specific oligonucleotide hybridization technique can be used to test for many different variations when the oligonucleotide is hybridized to a PCR amplified target DNA or when attached to a hybridization membrane and hybridized with a labeled target DNA. Can be.
In addition, trait specific amplification techniques that rely on selective PCR amplification can be used with the present invention. Oligonucleotides that are used as primers for specific amplification can be used at the center of the molecule (amplification will depend on other hybridizations) (Gibbs et al. (1989) Nucleic Acids Res. 17: 2437-2448) or under appropriate conditions, Subject mutations can be made at the 3 'end of the primer (Prossner (1993) Tibtech 11: 238) as long as mismatch can prevent or reduce polymerase expansion. In addition, it may be desirable to derive new restriction sites in the variant regions that make cleavage-based detection methods (Gasparini et al. (1992) Mol. Cell Probes 6: 1). In one embodiment, amplification can also be performed using Tag ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci. USA 88: 189). In such cases, ligation will occur if there is complete consensus at the 3 'end of the 5' sequence that allows for the detection of the presence of known mutations at specific sites by observing the presence or absence of amplification.
In another embodiment of the invention, the sense or antisense sequences of the signalling genes or naturally occurring variants thereof, or 5 'or 3' flanking sequences or intronic sequences naturally associated with the signaline-genes of the invention Or nucleic acid compositions comprising (purified) oligonucleotide labels comprising regions of nucleotide sequences capable of hybridizing to naturally occurring variants thereof. The nucleic acid of the cell can be in close proximity for hybridization, the label being exposed to the nucleic acid of the sample, and the hybridization of the label to the sample nucleic acid is detected. The technique can be used to detect defects on the genomic or mRNA level, including deletion of mRNA transcript levels, as well as deletions, substitutions and the like. The oligonucleotide markers can be used for the prophylactic and therapeutic assessment of trait variations, for example, in neoplastic or hyperproliferative diseases (eg, excessive cell proliferation).
In yet another embodiment, the level of signaline-protein can be detected by immunoassay. For example, cells of a biopsy specimen can be lysed and the levels of signaline-proteins present in the cells can be quantified by standard immunoassay techniques. In yet another embodiment, the excessive methylation pattern of the signalling gene is sensitive to the methylation reaction, and the recognition site for it is with one or more restriction endonucleases in the signalling gene (including flanking and intronic sequences). It can be detected by digesting genomic DNA from patient samples. See, eg, Buiting et al. (1994) Human Mol Genet 3: 893-895. Digested DNA is isolated by gel electrophoresis and hybridized with a label derived, for example, from genomic or cDNA sequences. The methylation status of the signaline gene can be determined by comparing restriction patterns generated from sample DNA with those for standard known methylation.
For still another object of the present invention, the signaline polypeptide is used to separate the coding sequence for another cellular protein ("signalin-binding protein" or "signalin-bp") that binds to signalin. Hybridization ”assays or“ interaction trap ”assays (eg, US Pat. No. 5,283,317; Zervos et al. (1993) Cell 72: 223-232; Madura et al. (1993) J Biol Chem 268: 12046- 12054; Bartel et al. (1993) Biotechniques 14: 920-924; Iwabuchi et al. (1993) Oncogene 8: 1693-1696; and Brent WO 94/10300.) The signaline-binding protein is for example the top of the signaling pathway. It is easily involved in the development of the TGFβ signal by the signaline protein as a stream or downstream element, or as a side modulator of signal bioactivity.
Briefly, the interaction trap relies on reconstituting a functional transcriptional active agent in vivo from two separate fusion proteins. In particular, the method allows the use of hypothetical genes that express hybridization proteins. By way of example, the first hybridization gene comprises a coding sequence for the DNA-binding domain of a transcriptal activator fused in framework to the coding sequence for a signaline polypeptide. The second hybridization protein encodes a transcriptional active domain fused within the backbone to the sample gene obtained from the cDNA library. If hypothetical and sample hybridization proteins can form interactions, eg, signaline-dependent complexes, they result in two very close domains of transcriptional activators. This proximity is sufficient to cause transcription of the reporter gene operably linked to a transcriptional regulatory site responsive to a transcriptional activator, the expression of the reporter gene being detected and used to record the interaction of the signaline and the sample protein. Can be.
In addition, by making useful purified and recombinant signalling polypeptides, the present invention provides for the pathogenesis of the normal cellular function or cellular differentiation and / or proliferation of the signalling polypeptides of the invention and their role in diseases associated therewith. It can be used to screen for drugs containing signaline homologues that are agonists or antagonists. In one embodiment, the assay assesses the ability of a compound to modulate the binding between a signalling polypeptide and a molecule, which is a protein or DNA that acts as an upstream or downstream of the signalling polypeptide in the TGFβ signaling pathway. For example, the assay can be used to identify compounds that inhibit or enhance the interaction of TGFβ receptor complexes or their subunits with signaline polypeptides. Various assay formats will be met and will be understood by those skilled in the art in light of the present invention.
In many drug screening programs that test libraries of compounds and natural extracts, high throughput assays are desirable to maximize the number of compounds tested within a certain time. As derived with purified or semi-purified proteins, assays performed in cell-free systems often make them possible to detect rapid development and relatively easy exchange within molecular targets mediated by test compounds. It is referred to as a "raw" screen in that it can be lost. In addition, the effects of cytotoxicity and / or bioavailability of the test compound may generally be neglected in in vitro systems, and the assay may instead be directed to molecular targets that appear in changes in binding affinity with upstream or downstream scrim elements. The primary focus is on the drug's effectiveness. Thus, in an exemplary screening assay of the present invention, a subject compound may be upstream or upstream of a protein or nucleic acid that functions downstream of a signaline peptide, whether they are negatively regulated positively by the peptide or the active agent of that activity. Or any inhibitor). As a result, a composition containing the signaline polypeptide is added to the mixture of the compound and the upstream or downstream elements. Detection and quantification of the signaline complexes with upstream or downstream elements provides a means for detecting the potency of the compound when inhibiting (or enhancing) complex formation between the signaline and signaline-defective elements. The ability of the compound can be investigated by making an appropriate dose curve from the data obtained using various concentrations of the test compound. In addition, control assays can also be performed to provide a baseline for comparison. In the control assay, the isolated and purified signal polypeptide is added to the composition containing the signal-binding element and the formation of the complex is quantified in the absence of the test compound.
Complex formation between the signaline polypeptide and the signal binding element can be detected by various techniques. Manipulation of the preparation of the complex is quantitated by immunoassay or chromatogram detection, using detectably labeled proteins such as, for example, radiolabeled, fluorescently labeled or enzymatically labeled signal polypeptides. Can be.
Typically, it will be desirable to immobilize the signaline or binding protein thereof to facilitate the automation of the assay, as well as to facilitate separation of the complex from the uncomplexed form of one or both of the proteins. In the presence or absence of candidate reagents, binding of the signaline to the upstream or downstream elements can be carried out in a container suitable for containing the reactants. Examples include microtitre plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein may be provided that adds a domain that allows the protein to bind to the matrix. For example, the glutathione-S-transferase / signalin (GST / signalin) fusion protein is absorbed on glutathione sepharose beads (Chemat. St. Louis, MO) or glutathione induced microtitre plates. Consequently combined with, for example, ³⁵ S-labeled cell lysates and test mixtures, the mixtures being for example biotic conditions for salts and pH, although slightly more stringent conditions are preferred. Incubated under conditions that contribute to complex formation. After incubation, the beads are washed to remove any unbound label, the matrix is fixed and the radiolabel is determined either directly or in the supernatant after the complex is continuously degraded. In addition, the complexes can be cleaved from the matrix, separated by SDS-PAGE, and signaline-binding protein levels found in the bead fraction using standard electrophoretic techniques as described in the accompanying examples. This is quantified from the gel.
Other techniques for immobilizing proteins on the mattress are also useful for use in this assay. For example, a signaline or a conjugate binding protein thereof can be immobilized using the conjugation of biotin and streptavidin. For example, biotinylated signal molecules may be biotin-NHS (N-hydroxy-succinimide (N-hydroxy) using techniques known in the art (e.g., biotinylation kits, Pierce Chemicals, Rockford, IL). -succinimide) and can be immobilized in the wells of streptavidin-gotten 96 well culture plates. In addition, antibodies that react with the signalline but do not interfere with the binding of the upstream or downstream elements can be transferred to the culture well, where the signal is captured into the well by antibody conjugation. As above, preparations of signaline-BP and test compounds can be cultured in the signaline-existing wells of the culture plate, and the amount of complex trapped in the wells can be quantified. In addition to those described above for the GST-immobilized complexes, representative methods for detecting the complexes include the signaline as well as enzyme-linked assays existing in methods for detecting enzymatic activity associated with binding elements that are endogenous or exogenous activity. Immunoassay of a complex using an antibody that reacts with or binds to a signaling protein and competes with the binding element. In the latter case, the enzyme may be chemically conjugated and provided as a fusion protein with the signaline-BP. For example, the signaline-BP can be chemically cross-linked or genetically fused with horseradish peroxidase, and the amount of polypeptide trapped in the complex can be, for example, 3,3 It can be identified as a colorant substrate of an enzyme such as' -diamino-benzadine terachloride) or 4-chloro-1-naphthol. . Similarly, a fusion protein can be provided comprising the polypeptide and glutathione-S-transferase, and complex formation is performed using 1-chloro-2,4-dinitrobenzene (1- It is quantified by detecting GST activity using chloro-2,4-dinitrobenzene) (Habig et al. (1974) J Biol Chem 249: 7130).
For methods that rely on immunodetection to quantify one of the proteins captured in the complex, antibodies such as anti-signalin antibodies against the protein can be used. In addition, the protein detected in the complex may be "epitope tagged" in the form of a fusion protein comprising a second polypeptide in which antibodies are readily available in addition to the signaline sequence. For example, the GST fusion proteins described above can also be used for quantification of binding using antibodies against the GST moiety. Other useful epitope representations include the pFLAG system (International Biotechnologies, Inc.) or the pEZZ-protein A system (Pharamacia, NU), as well as the myc-epitope (dP, including 10 residue sequences obtained from c-myc). Ellison et al. (1991) J Biol Chem 266: 21150-21157).
In addition to cell-free assays, as described above, readily available feedstocks of vertebrate signal proteins provided by the present invention also provide for the generation of cell-based assays to identify small molecule agonists / antagonists and the like. To promote. Cells susceptible to signalling-mediated induction by TGFβ are recombinant signalling proteins, with or without assays, with the assay aimed at adjusting in signalin-induced response by target cells mediated by the test reagent, in the presence or absence of the test reagent. Can cause overexpression. In conjunction with the cell-free assay, reagents can be identified that make a generally meaningful change in signallin-dependent induction (inhibition or enrichment). In one embodiment, the embryo or ES cell causes ectopic expression of the signaling polypeptide, and the effect of the subject compound on tissue pattern induction is measured.
For example, as described in the appended examples, overexpression of signalin in embryonic cells can lead to tissue induction of differentiation in a manner apparently similar to the induction mediated by various TGFβ factors. Thus, the recombinant cells can be used to identify inhibitors of specific TGFβ factors by the ability of compounds that inhibit signal transduction activity downstream of the signaling protein. For example, the recombinant xe-signalin 1 animal cap of Example 2 can be contacted with a panel of test compounds and the inhibitor is documented by its ability to inhibit the conversion of the ectoderm cells to the ventral mesoderm fate. Compounds that generally produce significant reductions in abdominal mesodermal induction can be selected for further testing. The assay can be further simplified by recording the expression of genes that are up- or down-regulated in response to signaline-dependent signal cascades. In a preferred embodiment, the regulatory region of said gene, such as, for example, the 5 'flanking promoter and enhancer region, is operably linked to a detectable indicator (luciferase luciferase) that encodes a easily detectable gene product. .
In another embodiment of the drug screen, two hybridization assays can be made with signalin and signalin-binding proteins. Drug dependent inhibition or fortification of the interaction can be recorded.
In cases where the signaline protein is capable of binding DNA and modifying the transcription of the gene in itself or in complexes with other proteins, for example, a transcriptional based assay using a signaline sensitive regulatory sequence is detectable indicator gene. Is operatively connected to.
In addition, each of the assay systems described above may be created in a "other" format. In other words, the assay format can provide information regarding specificity as well as capability. For example, a side-by-side comparison of the effect of a test compound on other signaling may provide information on selectivity, allowing identification of compounds that selectively modulate the sub-group-specific bioactivity with the signalling. .
Another object of the present invention is to contact the cells with a reagent that modulates signaline-dependent signaling by the growth factor, thereby inducing and / or maintaining differentiated states, enhancing survival and / or TGF. A method of promoting (or selectively inhibiting) proliferation of cells in response to a -β factor. For example, in light of the discovery of the present invention, which is a clear improvement of the signaled protein by the formation of an ordered spatial arrangement of differentiated tissue in vertebrates, the method provides for the arrangement of various vertebrate tissues both in vitro and in vivo. It can be used to make and / or maintain. “Signalin treatment”, whether inducible or anti-induced for signaling by TGF-β, is suitably used as an isolated polypeptide, gene therapy construct, antisense molecules, peptide analog or herein It may be one of the preparations described above including the reagents identified in the provided drug assays. In addition, based on the observation of the activity of vertebrate signal proteins in drosoophila, for therapeutic and diagnostic purposes, signalling therapeutics include Drosophila and C.elegans MAD proteins and analogs thereof. There are a wide variety of histopathological cell proliferation conditions in which the signaline therapeutics of the invention may be used for treatment. For example, such reagents can provide therapeutic advantages where the general method is the inhibition of non-sperm cell proliferation. Diseases that act advantageously from the method include, but are not limited to, chronic infectious diseases, as well as various cancers and leukemias, psoriasis, skeletal diseases, connective tissue, fibroproliferative diseases and other soft muscle differentiation diseases. In particular, it is expected that mutations or deletions of both alleles of the signaline gene of the present invention will result in excessive proliferation, ie the signaline will act as a tumor suppressor gene. In this regard, about 90% of human pancreatic cancers have been shown to exhibit allele loss at chromosome 18q (Hahn et al. (1996) Scence 271: 350). DPC4, a gene similar to Mad and sma-2, sma-3, and sma-4, was found to be a homozygous deletion of approximately 30% of pancreatic cancers tested.
In addition to proliferative diseases, the present invention is directed to the treatment of differentiated diseases resulting from non-differentiation of tissues (optionally) accompanied by (optionally) developmental insufficiency into mitosis such as apoptosis. Promote the use of therapeutic agents. Such degenerative diseases include chronic neurodegenerative diseases of the nervous system such as Alzheimer's disease, Parkinson's disease, Huntington's chorea, muscular dystrophy, as well as spinal cord degeneration. Other differentiated diseases include gastric ulcer binding characterized by dedifferentiation of vascular diseases, including, for example, chondrocytes, or osteoblasts, denatured changes in gland cells, as well as vascular diseases including non-differentiation of endothelial and flexible tissue cells. Include diseases associated with tissue.
By temporary use as a modulator of the signaling pathway, in vivo tissue remodeling can be performed, for example, in the development and maintenance of organs. By controlling the proliferative and differentiation potential for various cells, the genetic constructs of the present invention can be used to rebuild wound tissue or to improve the shape and transplantation of transplanted tissue. For example, signaline agonists and antagonists have been used in various ways to control various stages of organ repair after physical, chemical or pathological injury. For example, the diet can be used to promote cartilage repair, increased skeletal density, hepatic recovery following partial hepatocytes, or regeneration of lung tissue in the treatment of emphysema.
For example, the method is applicable to cell culture techniques. In vitro neural culture systems are used to identify nutritional and growth factors such as nerve growth factor (NGF), ciliary trophic factor (CNTF) and brain derived neurotrophic factor (BDNF). Rather, it proved to be a basic and necessary tool for the study of neural development. When a neuron is finally-differentiated, it will not change to another finally-divided cell-type. However, nerve cells can nevertheless easily lose their differentiated state. This is commonly observed when they are grown in culture from sexual tissue and when they form embryos during regeneration. The method provides a method for identifying a suitable restrictive environment for maintaining nerve cells at various stages of differentiation, and can be used, for example, in cell culture designed to test the specific activity of other nutritional factors. In this embodiment of the method, the cultured cells are induced by the TGF-β factor activin to maintain preculture of finally-differentiated neurons by inducing differentiation loss or to induce neuronal differentiation. Can be contacted with a reagent that inhibits the signalling-mediated signal that is present. As described in Melton and Hemmati-Brivanlou PCT Application PCT / US94 / 11745, the defect fate of ectoderm tissue is a neural unit rather than mesoderm and / or ectoderm. In particular, it has been found that inhibiting or counteracting signaling by activin can lead to differentiation along the neural unit-fate pathway.
In one embodiment, for example, the role of the signalin therapy of the present method of culturing hepatocytes may induce differentiation of non-committed progenitor cells, resulting in committed or finally-differentiated neurons. To further limit the fate of initiation of commissioned species to become cells. For example, the present invention can be used in vitro to induce peptidic and serotonergic neurons, as well as glial cells, schwann cells, chromaffin cells, cholinergic sympathetic nervous system or paranergic neurons. have. Said signalling therapy may be used alone or in combination with other neurotropic factors that serve to specifically enhance the special differentiation fate of the progenitor cells on neurons. In a similar manner, relatively undifferentiated hepatocytes or primitive neuroblasts can be maintained in culture and cause differentiation in the treatment of signalin therapeutics. Representative primitive cell cultures include cells harvested through the embryonic neural tube or neural plate before much obvious differentiation occurs.
Still another object of the present invention is to provide signaling for regulating morphogenesis signals related to organogenic pathways in other vertebrates, in addition to neuronal differentiation such as, for example, TGF-β action in both mesodermal and ectoderm differentiation processes. It relates to the use of therapeutic agents. As a result, it is contemplated that a composition comprising a signaline therapeutic may also be used in both cell culture and therapeutic methods, including the production and maintenance of non-neural tissue.
In one embodiment, the present invention has found that signaline proteins can be easily involved in controlling the development and formation of other organs derived from the digestive tract, liver, pancreas, lung and primitive gut. As described in the Examples below, signalin proteins are presumably related to cellular activity in response to TGF-β induced signals. Thus, signaline agonists and / or antagonists may be used for the development and maintenance of artificial livers, which may have various metabolic functions in normal livers. In one embodiment, signalin therapeutics may be used to manipulate extracellular mattresses or may be encapsulated in a water-soluble polymer to form an implantable and extracorporeal artificial liver.
In another embodiment, the composition of the signaline therapeutic agent may be used with embryonic liver structure, as well as implantation of the artificial liver, to promote intraperitoneal transplantation, angiogenesis and in vivo differentiation and maintenance of transplanted liver tissue.
Similar uses of signaline therapeutics are contemplated for pancreatic culture and the production and maintenance of artificial pancreatic tissue and organs.
In another embodiment, in vitro cell culture can be used for the identification, isolation and study of genes and gene products expressed in response to disruption of signaline-mediated signal transduction, and thus facilitate the development and / or maintenance of tissue. Related. These genes are the "substream" of the signaline gene product. For example, if new transcription is required for signalling-mediated induction, a negative cDNA library prepared with cells overexpressing the signalling gene and control cells can be used to isolate genes that are turned on or off by this process. The powerful minus library method incorporating the PCR technique described by Wang and Brown is an example of a method useful with the present invention for isolating the gene (Wang et al. (1991) Proc. Natl. Acad. Sci. USA 88: 11505-11509). For example, this approach has been used to successfully isolate more than sixteen genes involved in tail resorption with and without thyroid hormone treatment in Xenopus. Using the control and treated cells, the induced pool can be subtracted from the uninduced pool to isolate the turned on gene, resulting in a pool not derived from the derived pool for the turned off gene. For this reason, two mRNA groups can be identified. Group I RNA includes RNA expressed in untreated cells and is reduced or eliminated in induced cells, ie, the down-regulated manipulation of RNA. Group II RNA is upregulated in response to induction and consequently is more abundant in treated cells than untreated cells. RNA extracted from treated cells versus untreated cells can be used as a primary test for classification of replicates isolated from the library. Replicas of each group are further characterized by sequencing and constructional distribution determined in embryos by in situ full mount and improved northern blot analysis.
In yet another embodiment, the signaline therapeutic agent may be used to modulate the tissue after physical, chemical or pathological injury. For example, a therapeutic composition comprising a signaline therapeutic may be used for partial liver resection followed by liver recovery. Similarly, therapeutic compositions comprising a signaline therapeutic may be used to promote regeneration of lung tissue in the treatment of emphysema.
In yet another embodiment of the present invention, a composition comprising a signaline therapeutic agent may be used for in vivo treatment of skeletal tissue defects as well as for in vivo treatment of skeletal tissue defects. The present invention contemplates the use of signaline therapeutics that upregulate or mimic the inducing activity of skeletal morphogenic protein (BMP) or TGF-β, as can be used to modulate cartilage and / or bone formation. A "skeletal tissue defect" refers to the induction of TGF-β regardless of the cause of the defect, for example, as a result of surgery, cancer, tumor removal, transplantation, fracture, or other trauma or as a result of degenerative conditions. As long as the regulation of the response is appropriate, it means a defect in the skeletal or other skeletal connective tissue at the position where the skeletal or connective tissue is to be restored.
For example, the present invention provides useful therapeutic methods and signaline therapeutic compositions for restoring cartilage function in connective tissue. The method includes, for example, replacement of torn meniscus tissue, knee meniscus chondrectomy, relaxation of the joint by torn ligaments, severe injury of the joint, trauma to tissues such as skeletal joints or other mechanical disturbances caused by hereditary diseases, It is useful for repairing defects or damage to cartilage tissue that is the result of degenerative weakness such as causing arthritis. The restoring method is also useful for remodeling cartilage matrices, as in periodontal surgery as well as plastic or reconstructive surgery. The method will also be possible by the above described recovery procedure, for example accompanied by surgical recovery of meniscus, ligament or cartilage. It also prevents the onset or exacerbation of degenerative diseases, if adequately applied to post-traumatic extraction.
In one embodiment of the present invention, the method of the present invention provides a therapeutically sufficient amount of signaline to treat abnormal connective tissue to generate cartilage repair response in connective tissue by stimulating differentiation and / or proliferation of chondrocytes inserted into the tissue. Treating with a therapeutic agent. Influx of chondrocytes by treatment with a signaline therapeutic agent can subsequently lead to the synthesis of a new cartilage matrix by the treated cells. Connective tissues such as cartilage of the joints, intraarticular cartilage (meniscus), rib cartilage (which connects the ribs with the sternum), ligaments and tendons are particularly treatable with reconstruction and / or regenerative therapies using the present method. As used herein, regenerative therapies include prophylactic treatment of tissues in which the degeneration is in its early or urgent state, as well as treatment of deteriorated conditions that develop at the point where tissue damage is evident. The method can also be used to prevent the spread of lime action into the fibrous tissue by maintaining the continuous production of new cartilage.
In one embodiment, the methods of the present invention can be used to treat cartilage of the movable joint, such as the metacarpal joint of the knee, ankle, elbow, hip, wrist, finger or toe, or the temporal jaw joint. The treatment may be directed to the meniscus of the joint, articular cartilage of the joint, or both. More illustratively, the methods of the present invention may be used to treat degenerative knees, which may be the result of trauma (such as point wounds or excessive aging) or bone joints. For example, injecting a signaling agent into the joint with an arthroscopic needle can be used to treat abnormal cartilage. In some cases, the injected reagent may be in a hydrogel or other slow release materials described above to enable more extended and normal contact of the reagent.
The present invention further contemplates the use of such methods in the art of cartilage transplantation and artificial device therapy. To date, the growth of new cartilage from autologous or genetically different allogeneic cartilage has not been very successful. For example, because cartilage and fibrocartilage properties vary between joints, meniscal cartilage, ligaments and tendons, between the same ligaments or both ends of the tendons, and between other tissues, such as deep and surface tissues. A problem arises. The banding arrangement of the tissues reflects a gradual change in mechanical properties and fails when an implanted tissue that does not differentiate under the conditions loses the ability to respond properly. For example, when meniscal cartilage is used to repair the cruciate ligament, the tissues experience metaplasia in pure fibrous tissue. By promoting cartilage formation, the method can be used to specifically overcome this problem by allowing the transplanted cells to better adapt to the new environment and to effectively line up with hypertrophic cartilage cells in the early developmental stages of the tissue. As a result, as provided by the present method, the action of cartilage formation in the implanted tissue and the mechanical force for effectively remodeling the tissue can interact to make an improved transplant more suitable for the new action into which it is inserted.
In a similar manner, the methods of the present invention can be used to enhance the regeneration of artificial cartilage devices and their implantation. The need for improved therapies includes collagen-glycosaminoglycan templates (Stone et al. (1990) Clin Orthop Relat Red 252: 129), isolated chondrocytes (Grande et al. (1989) J Orthop Res 7: 208 And Takigawa et al. (1987) Bone Miner 2: 449), and cartilage forming cells attached to natural or synthetic polymers (Walitani et al. (1989) J Bone Jt Surg 71B: 74; Vacanti et al. (1991) Plast Reconstre Surg 88: 753); von Schroeder et al. (1991) J Biomed Mater Res 21:11; and Vacanti et al. US Pat. No. 5,041,138 to motivate a study aimed at making new cartilage. For example, chondrocytes are decomposed into polyglycolic acid, polylactic acid, agarose gel, or non-irritating monomers by prolonged hydrolysis of the polymer backbone. It can be grown in culture on biodegradable, biocompatible highly porous backbones formed from polymers such as other polymers. The mattress is designed to allow proper nutrition and gas exchange into the cells until infusion occurs. The cells can be cultured in vitro until the appropriate cell volume and density is developed into the cells to be transplanted. One advantage of the mattresses is that they are cast or molded into the desired shape on the basis of each, so that the end product is squeezed into the parent's ear and nose (by embodiment), or allows for manipulation when implanted into the joint. Is that it can be used.
In one embodiment of the method, the implant is contacted with a signaline therapeutic during incubation to induce and / or maintain differentiated chondrocytes in culture to further stimulate the generation of cartilage matrix in the implant. This method allows the cultured cells to maintain the morphological typical of cartilage forming cells (ie, hypertrophic) and to continue the production of the population and cartilage tissue of the matrix.
In another embodiment, the implanted device is treated with a signaline therapy to actively remodel the articulated device and make it more suitable for its intended function. As described above for tissue implants, artificial implantation involves the same defects that are not induced in settings comparable to the actual mechanical environment in which the matrix is implanted. The activity of chondrocytes of the matrix by the method can be such that the implant has characteristics similar to the tissue to be replaced.
In yet another embodiment, the method is used to enhance the attachment of an artificial device. By way of example, the method may be used for implantation of a dental prosthesis, wherein treatment of the enveloping connective tissue not only inhibits the formation of fibrous tissue in proximity to the artificial device, but also stimulates the formation of a striatal ligament for the prosthesis. do.
In yet another embodiment, the method can be used for the production of a skeleton (skeletal formation) at a site in an animal in which said skeletal tissue is defective. TGF-β, in particular BMPs, relates in particular to the creation of skeletal mattresses by skeletal cells, as well as to hypertrophic chondrocytes that are ultimately substituted with skeletal cells. As a result, the administration of a signaline therapeutic agent is a method for treating skeletal loss in a subject, for example, to control skeletal growth and maturation, as well as to prevent and / or counteract osteoporosis and other skeletal diseases. Can be used as part of For example, preparations that include a signaline agonist may, for example, mimic or enhance the activity of BMPs to mitigate or enhance the activity of BMPs, as long as they facilitate the production of cartilaginous tissue precursors that form a “model” for skeletalization. Can be used to induce anger. Therapeutic compositions of the signaline agonists can be supplemented with other osteoinductive factors, if necessary, such as skeletal growth factors (eg, activin as well as TGF-β factors such as skeletal morphogenic factors BMP-2 and BMP-4). And include or be administered with a skeletal absorption inhibitor, such as estrogen, bisphophonate, sodium fluoride, calcitonin, or tamoxifen, or related compounds.
For certain cell-types, especially in epithelial and hematopoietic cells, normal cell proliferation is driven by sensitivity to negative autocrine or paracrine growth regulators, such as those with TGFβ. Is displayed. This is usually done by differentiation of cells into post-mitotic form. However, it has been observed that a significant percentage of human cancers derived from these cell types show reduced sensitivity to growth regulators such as TGF-β. For example, some colonic, hepatic epithelial and epidermal progenitor tumors show reduced sensitivity and resistance to the growth-inhibitory effects of TGFβ as compared to their normal pairs. In this context, a salient feature of some of these transformed cell lines is the absence of detectable TGFβ receptors. Treatment of such tumors with signaline therapies offers the opportunity to mimic the effective action of TGFβ-mediated inhibition.
To further illustrate the use of the method, the therapeutic application of the signalling therapeutics may be used for the treatment of glioma. Gliomas are due to intracranial tumors of 40-50% during life. Despite the increasing use of radiotherapy, chemotherapy and sometimes immunotherapy following malignant glioma surgery, mortality and morbidity have not substantially improved. However, for a significant number of gliomas, there is increasing experimental and clinical evidence that the loss of TGFβ sensitivity is an important factor in the failure of growth regulation. Where the cause of reduced sensitivity is due to loss of receptors or loss of upstream of other TGFβ signaling proteins of signalling, treatment with signalling therapeutics can be used to effectively inhibit cell proliferation.
The signaline therapies of the present invention may also be used, for example, by fibrosis such as rheumatoid arthritis, insulin dependent diabetes mellitus, glomerulonephritis, cirrhosis, and scleroderma, particularly proliferative diseases associated with loss of TGFβ autocline or paracrine signaling. Other diseases characterized, as well as can be used for the treatment of hyperproliferative vascular diseases or asymptomatics, such as, for example, soft muscle hyperplasia (eg, such as arteriosclerosis).
For example, asymptomatic proliferation continues to limit the ability of coronary angiogenesis despite the various mechanical and pharmaceutical adjustments that have been used. An important mechanism involved in the normal regulation of vascular endothelial proliferation of soft muscle cells seems to be the induction of autograin and paracline TGFβ inhibition loops in the soft muscle cells (Scott-Burden et al. (1994) Tex Heart Inst J 21: 91- 97; Graiger et al. (1993) Cardiovasc Res 27: 2238-2247; and Grainger et al. (1993) Biochem J 294: 109-112). Loss of sensitivity to TGFβ, or alternatively, abuse of inhibitory stimuli, such as by PDGF autostimulation, may be a contributing factor to abnormal soft muscle proliferation in asymptomatic. Therefore, proliferation can be prevented or prevented by the use of gene therapy with the gene construct of the present invention, which mimics induction by TGFβdp. The signaline gene construct can be delivered by transdermal transluminal gene transfer (Mazur et al. (1994) Tex Heart Inst J 21: 104-111) using, for example, viral or lipomalmal delivery compositions. have. Representative adenovirus-mediated gene transfer techniques and compositions for the treatment of cardiac or vascular soft muscle are provided in PCT Publication No. WO94 / 11506.
TGFβ also plays an important role in local glomeruli and interstitial sites in human kidney initiation and disease. As a result, the method provides a method for treating or arresting proliferative diseases of other kidneys, including in vivo delivery of glomerulopathy and subject signaline therapeutics.
Still another object of the present invention relates to the therapeutic application of signaline therapeutics that enhance the survival of neurons and other neurons in both the central and peripheral nervous systems. The ability of the TGF-β factor to regulate neuronal differentiation during the development of the nervous system or at maturation is attributed to the ability of any signaline protein in the control, chemical or mechanically deficient cells of mature neurons to maintain normal cell function, functional capacity and aging. It indicates that it may be expected to participate in the prevention of degeneration and immaturity arising from the loss of differentiation resulting from the loss of differentiation in certain histopathological conditions, and existing histopathological conditions. In light of this understanding, the present invention provides: (i) acute, anti-acute to the nervous system, including trauma, chemical wounds, wounds and defects (such as ischemia due to shock), together with infectious / pandemic and tumor-induced wounds. Acute or chronic wounds; (ii) aging of the nervous system, such as Alzheimer's disease; (iii) chronic neurodegenerative diseases of the nervous system, including Parkinson's disease, Huntingston's chorea, muscular dystrophy, as well as spinal cord degeneration, and (iv) chronic immunological diseases or nervous systems of the nervous system, including complex sclerosis. Particular consideration is given to the application of the methods of the present invention to the treatment of neurological conditions (prevention and / or reduction of stringency) resulting from giving.
Many neurological disorders involve alteration of discrete populations of neurological elements and can be treated with therapeutic diets, including signalling therapies. For example, Alzheimer's disease involves defects in some neurotransmitters, both entering the renal cortex and remaining with the cortex. For example, nuclei in patients with Alzheimer's disease were observed to have severe loss of neurons (75%) compared to age-matched controls. Although Alzheimer's disease is the most common form of dementia, some other diseases can also make dementia. Some of these are degenerative diseases characterized by neuronal death in various parts of the central nervous system, especially the cerebral cortex. However, some forms of dementia are associated with degeneration of the white matter or thalmus, the basis of the cerebral cortex. Here, dysfunction of cognition occurs from the separation of the cortical area by degeneration of the centrifugal and afferent nerves. Huntington's disease is associated with endoscopic and cortical cholinergic neurons and GABA neurons. Pick's disease is a severe neuronal degeneration in the neocortex of the frontal and anterior transient lobes, sometimes carried out by the death of neurons in the neoplasm. Treatment of patients suffering from such degenerative symptoms, for example, not only promotes differentiation and repopulation by descendant cells in the affected area, but also modulates the differentiation and cytolytic tasks leading to neuronal loss (eg For example, to increase the survival rate of the neurons present).
In addition to degenerative-induced dementia, one or more pharmaceutical formulations of the signaline therapeutics of the present invention may be applied opportunistically to the treatment of neurodegenerative diseases with expression of tremor and unconscious movement. Parkinson's disease primarily affects subcortical structures, for example, and is characterized by the degeneration of the cortical pathways, the bald nucleus, the erythema and the motor nucleus of the vagus nerve. Ballism is associated with damage to the subthalmic nucleus and is often due to acute vascular accidents.
It is also a neurogenetic and myopathic disease that ultimately affects the body's separation of the peripheral nervous system and appears as a neuromuscular disease. In one embodiment, the method is used to treat muscular atrophy. ALS is the name given to complex diseases involving upper or lower motor neurons. Patients show notrophy of the spinal cord muscles, advanced component paralysis, primary lateral sclerosis or a combination of these symptoms. Major pathological abnormalities are characterized by the selective and developing degeneration of the lower and higher motor neurons of the spinal cord in the cerebral cortex. Therapeutic applications of the signaline therapeutics may be used alone or in combination with neurotropic factors that inhibit and / or inhibit motor neuron degeneration in ALS patients such as CNTF, BDNF, or NGF.
Signaling therapeutics can also be used to treat endogenous diseases of the peripheral nervous system, including diseases that affect the distribution of nerves in soft muscle and endocrine tissues (such as glandular tissue). For example, the method can be used to treat atrial cardiac arrhythmias or tachycardia resulting from neurodegenerative conditions that neurally distribute the heart rhabdomyomus.
In another embodiment, the method may be used for the treatment of neomyelo or aberrant proliferative mutations such as those occurring in the central nervous system. For example, the signaline therapeutics that induce differentiation of nerve cells by changing their sensitivity to TGF-β can be used to render the transformed cells post-mitotic or cytolytic. Treatment with signalin therapeutics promotes the destruction of autosecretory loops, such as the TGF-β autostimulatory loops, which are believed to be involved in neoplastic transformation of some neuronal tumors. Therefore, signaline therapies can be used, for example, in the treatment of malignant gliosis, medullocytosis, neuroectodermal tumors, and epithelial cell tumors.
Similarly, another object of the present invention includes the inhibition of T cell activation. TGFβ is known to inhibit T cell proliferation and the signaline described herein can be used in ameliorating diseases including chronic infections. In addition, TGFβ is associated with some form of tolerance (Chen et al. (1995) Nature 376: 177-180), and the present invention prior to the reception or allergy or autoimmunity of allo or xenograft. It can be used to induce T cell resistance in case of disease.
In yet another embodiment, regulation of the signaline-dependent pathway can be used to inhibit spermatogenesis. Spermatogenesis is a process involving meiosis and final differentiation of haploid cells into morphologically and functionally polarized sperm following mitotic replication of a diploid liver cell pool. The process exhibits both integrated temporal and spatial regulation as well as integrated interactions between sperm and somatic cells. It has already been published that TGFβ subfamily, especially activin-mediated signals, play an important role in coupling extracellular stimuli to the regulation of mitosis and meiosis that occur during spermatogenesis (Klaij et al. (1994) J. Endocrinol. 141: 131-141).
Similarly, the elements of the TGF family are important for the regulation of female genitalia (Wu, T.C. et al. (1994) Mol. Reprod. Dev. 38: 9-15). Thus, TGFβ inhibitors, such as the signaline antagonists produced in this assay, can be used to inhibit cystic maturation as part of the contraceptive formulation. For other purposes, induction regulation of meiosis maturation with signalin therapeutics can be used to co-occur cystic bodies for in vitro fertilization. The method can be used to provide a healthier, more viable, singler population of cysts resulting in cleavage, fertilization and development into blastocysts. In addition, the signalin therapeutics may be used to treat other diseases of the reproductive system of women that result in infertility, including polycystic syndrome.
Another object of the present invention is a transgenic non-human type expressing a heterologous signaling gene of the present invention or having one or more genomic signalling genes cleaved in at least one tissue or cell-type of an animal. To characterize animals. Accordingly, the present invention is directed to characterizing animal models for developmental disease in which animals have mis-expressed signaline traits. For example, mice can be grown to remove one or more signalin traits, or in other words to inactivate. The mouse model can consequently be used to study diseases arising from mis-expressed signaling genes, as well as to evaluate potent therapies for similar diseases.
Another object of the invention is a transgenic animal comprising the transgene of the invention and consisting of cells (of the animal) which preferably express (if any) an exogenous signalling protein in one or more cells of the animal. It is about. Signaling transgenes can encode proteins in their natural-type form or can encode not only antisense constructs, but also their homologues, including both agonists and antagonists. In a preferred embodiment, the expression of the transgene is limited to specific subsets of cells, tissues or generators using, for example, cis-acting sequences that regulate expression in a desired pattern. In the present invention, the tessellated expression of the signaline protein may be essential for many forms of alignment assays and additionally, for example, significantly alters the occurrence in small patches of normal embryonic tissue in other ways. May provide a means for investigating the effects of lack of signaline expression. To this end, tissue-specific regulatory sequences and condition regulatory sequences can be used to regulate expression of the transgene into any spatial pantone. In addition, the temporal pattern of expression can be provided by, for example, a conditional recombination system and a prokaryotic transcriptional regulatory sequence.
Genetic techniques that allow for expression of the transgene to be regulated via site-specific genetic manipulation in vivo are known in the art. For example, genetic systems that allow for controlled expression of recombinases that catalyze the genetic recombination of target sequences are useful. As used herein, the phrase “target sequence” refers to a nucleotide sequence genetically recombined by recombinase. The target sequence is flanked by the recombinase recognition sequence and is typically deleted or converted in cells expressing recombinase activity. Recombinase catalyzed recombination operations can be designed such that the target sequence causes recombination to result in the activation or inhibition of expression of one of the signal proteins of the invention. For example, deletion of a target sequence that interferes with the expression of a recombinant signaline gene, such as encoding an antagonist homolog or antisense transcript, can be designed to activate expression of the gene. Such disruption of the protein may arise from a variety of mechanisms, such as spatial separation of the signaline gene from promoter elements or stop codons therein. In addition, the transgene may be prepared such that the coding sequence of the gene is flanked by a recombinase recognition sequence and initially infected intracellularly in the 3 'to 5' direction with respect to the promoter element. In such a case, the conversion of the target sequence will be able to re-conform the gene of the invention by locating the 5 'end of the coding sequence in the direction to the promoter element causing the promoter induced transcriptional activation.
All of the transgenic animals of the present invention include the transgene of the present invention in which the transgene in a variety of cells thereof changes the shape of the "host cell" with respect to the regulation of cell growth, death and / or differentiation. Since one or more transgene constructs described herein can be used to make a transgenic organ of the present invention, the general description typically specifies the creation of a transgenic organ by referring to exogenous genetic material. This general description can be applied by one skilled in the art to insert specific transgene sequences into an organ using the methods and materials described below.
In one embodiment, the cre / loxP recombinase system of bacteriophage P1 (Lakso et al. (1992) PNAS 89: 6232-6236; Orban et al. (1992) PNAS 89: 6861-6865) or FLP of Saccharomyces cerevisiae Recombinase systems (O'Gorman et al. (1991) Science 251: 1351-1355; PCT Publication WO 92/15694) can be used to prepare in vivo site-specific genetic recombination systems. Cre recombinase catalyzes site-specific recombination of regulatory target sequences located between loxP sequences. The loxP sequence is a 34 base pair nucleotide repeat sequence to which the Cre recombinase binds and is essential for Cre recombinase mediated genetic recombination. The direction of the loxP sequence determines whether the regulatory target sequence is deleted or inverted when Cre recombinase is present (Abremski et al. (1984) J. Biol. chem. 259: 1509-1514); That is, catalyzes deletion of the target sequence when the loxP sequence appears as a direct repetition, and catalyzes the conversion of the target sequence when the loxP sequence appears as a converted repeat.
Thus, genetic recombination of the target sequence is dependent on the expression of the Cre recombinase. The expression of the recombinase can be controlled by modulatory promoter elements such as, for example, inducibility or inhibition by tissue-specific, generator-specific, externally added reagents. The regulated control will cause genetic recombination of the target sequence only in cells in which recombinase expression is mediated by promoter elements. As a result, the activated expression of the recombinant signaline protein can be regulated via the regulation of recombinase expression.
The use of the cre / loxP recombinase system to regulate the expression of recombinant signaling proteins is required for the production of transgenic animals containing transgenes encoding both the Cre recombinase and the protein of interest. Animals containing both the Cre recombinase and recombinant signalin genes can be provided through the production of "double" transgenic animals. A simple way of providing such animals is to cross two transgenic animals each containing a transgene, such as, for example, a signaline gene and a recombinase gene.
One advantage of early production of transgenic animals containing signaline transgenes in a recombinase-mediated expressible format is that the target protein may be detrimental to expression in the transgenic animal, whether agonists or antagonists. Comes from one. In such cases, a basal population in which the subject transgene is inactive in all tissues may be developed and maintained. The individual basal populations can be crossed with, for example, one or more tissues and / or animals expressing the recombinase in a desired temporal pattern. As a result, for example, a basal population in which the antagonist signalling gene is inactive may be used to study progeny from a basal body in which destruction of signalin-mediated induction in certain tissues or in some embryos is, for example, lethal. Will make it possible.
Transgenes of similar conditions may be provided using prokaryotic promoter sequences essential for stimulating prokaryotic proteins to promote expression of the signaline transgene. Representative promoters and corresponding cross-activating prokaryotic proteins are described in US Pat. No. 4,833,080.
In addition, the expression of the conditional transgene is such that, for example, a gene encoding a cross-activating protein such as recombinase or prokaryotic protein is delivered to the tissue in a manner such as cell-type specific method, and expression Can be induced by gene therapy-like methods. By this method, the signaline transgene can remain inactive at maturity until it is "on" by induction of the cross-activation.
In one embodiment, a "transgenic non-human animal" of the present invention may be prepared by introducing a transgene into the germline of a non-human animal. Various methods can be used depending on the stage of development of the embryonic target cell. Certain lines of certain animals used to practice the present invention may generally be selected for good dryness, good embryo yield, good oocyte visibility in the embryo and good genital suitability. In addition, the haplotype is a meaningful factor. For example, transgenic mice are produced and dyes such as the C57B / 6 or FVB lines are often used (Jackson Laboratory, Bar Harbor, ME). Preferred dyes are such as C57B / 6 or DBA / 1 having H-2, H-2 or H-2 haplotypes. The lines used to practice the invention may themselves be transgenic and / or may be great (ie, obtained from an animal with one or more genes partially or completely suppressed).
In one embodiment, the transgene construct may be introduced into a single stage embryo. The conjugate is the best target for micro-injection. In these mice, the male germline is about 20 micrometers in diameter allowing for reproducible infusion of 1-2 pl DNA solution. The use of a conjugate as a target for gene transfer has a major advantage in that in most cases the injected DNA will be inserted into the host gene prior to initial cleavage (Brinster et al. (1985) PNAS 82: 4438-4442). As a result, all cells of the transgenic animal will carry the inserted transgene. This will also be reflected in the effective delivery of the transgene to the descendants of the basal body, since generally 50% of the sperm cells will bear the transgene.
In general, fertilized embryos are incubated in appropriate cultures until germline emerges. At about this point, the nucleotide sequence comprising the transgene is introduced into a female or male germline as described below. In some species, such as mice, male reproductive nuclei are preferred. Most preferably, it is added to the male DNA complement of the conjugate prior to progression by the egg nucleus or the zygote female reproductive nucleus. The nucleus or female germline possibly affects the male DNA complement by replacing the protamine of the male DNA with histones, thereby facilitating the combination of female and male DNA complements that form a diploid conjugate. Emits.
As a result, it is preferred that exogenous genetic material be added to the male complement of the DNA or any other complement of the DNA before being affected by the female reproductive nucleus. For example, the exogenous genetic material is added to the early male germ nucleus as soon as possible after the male germ nucleus is formed, when the male and female germ nuclei are well separated and are all located close to the cell membrane. The endogenous genetic material may also be induced to decondense and then added to the nucleus of the sperm. Sperm containing endogenous genetic material may subsequently be added to the egg along with the transgene construct that is subsequently added as soon as possible.
The introduction of the transgenic nucleotide sequence into the embryo can be carried out using means known in the art such as, for example, microinjection, electroporation or lipofection. After influx of the transgenic nucleotide sequence into the embryo, the embryo can be incubated in vitro by varying time, retransplanted into a surrogate host, or both. In vitro incubation to maturity is within the object of the present invention. One common method is to incubate embryos for about 1-7 days, depending on the species, and eventually replant them into the surrogate host.
For the purposes of the present invention, the conjugate is essential for the formation of diploid cells that can develop into complete organs. Typically, the conjugate will consist of an egg containing a nucleus formed naturally or artificially by the fusion of two haplotype nuclei from a gamete or gametes. As a result, the germline nucleus must be one that is naturally compatible, that is, to make a living conjugate that can differentiate and develop into an organ of action. Usually, euploid conjugates are preferred. If a diploid conjugate is obtained, the number of chromosomes should not vary more than one with respect to the euploid number of the organ to which the germ is directed.
In addition to similar biological considerations, the physical ones also govern the amount of exogenous genetic material (eg, volume.) That can be added to the nucleus of the conjugate or the genetic material that forms part of the conjugate nucleus. If the dielectric material is not removed, the amount of exogenous dielectric material that can be added is limited by the amount that can be absorbed without physical destruction. Typically, the volume of exogenous genetic material inserted will not exceed about 10 picoliter. The physical effect of the addition should not be large enough to physically destroy the viability of the conjugate. The biological limitation of the number and diversity of DNA sequences is that the functions of the exogenous genetic material and the resulting conjugate, the genetic material, including the exogenous genetic material, must be able to capture and maintain the differentiation and development of the conjugate into an organ of action. It will vary depending on the particular conjugate and will be readily apparent to those skilled in the art.
The number of copies of the transgene construct added to the conjugate will depend on the total amount of exogenous genetic material added and will be an amount capable of generating genetic transformation. In theory, only one copy is required; Typically, however, many copies are used, such as 1,000-20,000 copies of the transgene construct, for example to confirm that one copy is functional. With respect to the present invention, it may sometimes be beneficial to have one or more functional copies of each exogenous DNA sequence inserted to enhance expression of the exogenous DNA sequence.
The exogenous genetic material is preferably inserted into the dielectric material of the nucleus by microinjection. Microinjection of cells and cellular structures is known and used in the art.
Replantation is performed using standard methods. Typically, the surrogate host is anesthetized and an embryo is inserted into the fallopian tube. The number of embryos transplanted into a particular host will vary from species to species, but typically is comparable to the number of naturally occurring offspring.
Gene transplant progeny of the surrogate host may be screened for the presence and / or expression of the transgene by any suitable method. Screening is often performed by Southern blot or Northern blot assays, using markers complementary to at least a portion of the transgene. Western blot assays using antibodies against proteins encoded by the transgene are used as an optional or additional method for screening for the presence of the transgenic product. Typically, DNA is prepared from tail tissue and analyzed by Southern blot assay or PCR for the transgene. In addition, tissues or cells that are believed to express the transgene at high levels may be tested for the presence and expression of the transgene using Southern assays or PCR, although any tissue or cell type may be used for the assay. do.
Optional or additional methods for assessing the presence of the transgene include, without limitation, appropriate biochemical and / or immunological assays such as enzymes, histological dyes for specific indicators or enzyme activity, flow sytometric ) Assays, and the like. In addition to assessing the effect of the transgene on the levels of various types of blood cells and other blood constructs, blood assays may also be used to detect the presence of the transgene product in the blood.
The offspring of the transgenic animal can be obtained by crossing the transgenic animal with a suitable mate, or by in vitro fertilization of eggs and / or sperm obtained from the transgenic animal. Where mating with a partner is performed, the partner may or may not be transgenic and / or knockout; If it is transgenic, it will contain the same or different transgenes or both. In addition, the pair may be a parental line. Where in vitro fertilization is used, the fertilized embryo can be transplanted into a surrogate host, incubated in vitro or both methods performed. Using one method, progeny can be assessed for the presence of the transgene using the methods described above, or other appropriate methods.
The transgenic animal produced according to the present invention will comprise exogenous genetic material. As described above, in one embodiment, the exogenous genetic material will be a DNA sequence that produces a signaline protein (agonist or antagonist) and an antisense transcript or signaline variant. In addition, in this embodiment, the sequence will be attached to a transcriptional regulatory element, such as, for example, an accelerator that preferably expresses a transgene product in a specific type of cell.
Retroviral infections can also be used to introduce transgenes into non-human animals. Generating non-human embryos can be cultured in vitro up to blastocyst. During this period, the dividers can be targeted for retroviral infection (Jaenich, R. (1976) PNAS 73: 1260-1264). Effective infection of the splits is obtained by enzymatic treatment to remove zona pellucida (Manipulating the Mouse Embryo, Hogan ed. (Cold Spring Harbor laboratory Press, Cold Spring Harbor, 1986). Viral vector systems used to control are typically replication-defective retroviruses carrying the transgene (Jahner et al. (1985) PNAS 82: 6927-6931; Van der Putten et al. (1985) PNAS 82: 6148-6152). Infection is easily and effectively obtained by culturing the dividers on a monolayer of virus-producing cells (Van der Putten, detailed above; Stewart et al. (1987) EMBO J. 6: 383-388). Virus or virus-producing cells can be injected into the blastocyst (Jahner et al. (1982) Nature 298: 623-628) Subcellular cells where the insertion forms a transgenic non-human animal Most of the bases will be mosaics for the transgene because they only occur in. In addition, the base may generally include various retroviral inserts of the transgene in other parts of the genome to be separated from offspring. It is also possible to introduce the transgene into the sperm line by intraviral retroviral infection of the embryo during pregnancy (as described by Jahner et al. (1982)).
The third type of target cell for transgene influx is embryonic liver cells (ES). ES cells are obtained from pre-transplanted embryos cultured in vitro and fused with embryos (Evans et al. (1981) Nature 292: 154-156; Bradley et al. (1984) Nature 309: 255-258; Gossler et al. (1986)). PNAS 83: 9065-9069; and Robertson et al. (1986) Natrue 322: 445-448). Transgenes can be efficiently introduced into the ES cells by DNA infection or by retrovirus-mediated migration. The infected ES cells can then be combined with an embryoid body from a non-human animal. The ES cells then colonize the embryo and contribute to the resulting sperm line of the artificial animal. For review, see Jaenisch, R. (1988) Science 240: 1468-1474.
In one embodiment, gene targeting, a method using homologous recombination to modify an animal's genome, can be used to induce changes in cultured embryonic liver cells. By targeting the subject signaline genes in ES cells, these changes can be introduced into the spermline of the animal making the virtual. The gene targeting procedure comprises fragments similar to target signaling sites and also constructs DNA targeting constructs that include intended sequence modifications (e.g., insertions, deletions, point mutations) to sequences on the signaling genome. By incorporation into cultured cells. The iron cells are then screened for correct targeting to confirm and isolate appropriately targeted.
Targeting genes in embryonic liver cells is in fact performed by the present invention as a means for disrupting signalling gene function through the use of targeted transgenic constructs designed to mimic recombination with one or more signaline genome sequences. to be. The targeting construct may be rearranged such that, for recombination with elements of the signaling gene, a positive selection indicator is inserted (or replaced) into the coding sequence of the targeted signaling gene. The inserted sequence functionally disrupts the signalin gene, while also suggesting positive selection properties. Representative signaline targeting constructs are described in more detail below.
Typically, embryonic liver cells (ES cells) used to produce knockout animals will be the same species as the knockout animals produced. As a result, for example, mouse embryonic liver cells will be commonly used for the production of knockout mice.
Embryonic liver cells are described in Doetschman et al. (1985) J. Embryol. Exp. Morphol. Produced and maintained using methods well known to those skilled in the art as described by 87: 27-45. While any ES cell line can be used, the selected line is typically chosen for the cell's ability to integrate and become part of the sperm line of the developing embryo to make the sperm line inheritance of the knockout construct. One strain commonly used for the production of ES cells is the 129J lineage. Another ES cell line is murine cell line D3 (American Type Culture Collection, catalog no.CKL 1934). Yet another preferred ES cell line is the WW6 cell line (Ioffe et al. (1995) PNAS 92: 7357-7361). Robertson's Teratocarcinomas and Embryonic Stem Cells; A Practical Approach, E.J.Robertson, published by IRL Press, Washington D.C. (1987); Current Topics in Devel. By Bradley et al. (1986). Biol. 20: 357-371; And as described in Manipulating the Mouse Embryo: A laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1986), by Hogan et al. Incubated and prepared.
Insertion of the knockout construct into the ES cell can be performed using a variety of methods well known in the art, including, for example, electroporation, microinjection and calcium phosphate treatment. The preferred insertion method is electroporation.
Each knockout construct inserted into the cell should first be straight. Therefore, if the knockout construct is inserted into a vector (described below), straightening is performed by digesting the DNA with an appropriate restriction endonuclease selected to cleave only within the vector sequence, not within the knockout construct sequence. .
For insertion, the knockout construct is added to ES cells under appropriate conditions for the chosen method of insertion, as known to those skilled in the art. Where more than one construct should be introduced into the ES cell, each knockout construct can be introduced simultaneously.
If the ES cells can be electroporated, the ES cells and knockout construct DNA are exposed to electrical pulses according to the instructions using an electroporation machine. After electroporation, the ES cells are typically allowed to recover under appropriate incubation conditions. The cells are then screened for the presence of the knockout construct.
Screening can be performed using a variety of methods. If the marker gene is an antibiotic resistance gene, for example, ES cells can be cultured in the presence of antibiotics of different lethal concentrations. Living ES cells can have a nicely integrated knockout construct. If the marker gene is different from the antibiotic resistance gene, the Southern blot of DNA on the ES cell genome can be labeled with a sequence of DNA designed to hybridize only to the marker sequence. In addition, PCR may be used. Finally, if the marker gene is a gene that encodes an enzyme whose activity can be detected (e.g., β-galactosidase), the enzyme substrate can be added to the cell under appropriate conditions, Enzymatic activity can be analyzed. Those skilled in the art will be familiar with other useful indicators and means for detecting their presence in specific cells. All such indicators are considered to be included within the object indicated by the present invention.
The knockout construct can integrate to several locations in the ES cell genome and to different locations in the genome of each ES cell due to the occurrence of random insertion. Preferred insertion sites are in positions complementary to knocked out DNA sequences, such as, for example, signaline coding sequences, transcriptional regulatory sequences, and the like. Typically, ES cells making the knockout construct less than about 1-5 percent will incorporate the knockout construct substantially within the desired location. To identify the ES cells with proper integration of the knockout construct, whole DNA can be extracted from the ES cells using standard methods. As a result, the DNA can be labeled on the Southern blot using a label or labels designed to hybridize in a pattern specific to genomic DNA digested with specific restriction enzyme (s). Alternatively, or in addition, the genomic DNA may be amplified by PCR with a label specifically designed to amplify DNA fragments of a particular size and sequence (ie, cells comprising the knockout construct in the appropriate location). Only will produce DNA fragments of the appropriate size).
After appropriate ES cells containing the knockout construct in the appropriate locations have been identified, the cells can be inserted into the embryo. Insertion can be performed in a variety of ways known to those skilled in the art, but the preferred method is microinjection. For microinjection, about 10-30 cells are collected into micropipets and injected into embryos, which is an appropriate stage of development that allows the integration of external ES cells containing knockout constructs into developmental embryos. For example, transformed ES cells can be microinjected into the blastocyst.
The appropriate developmental stage for embryos used for the insertion of ES cells is very species-dependent, but about 3.5 days for mice. The embryo is obtained by perfusing the uterus of a pregnant female. Suitable methods for doing this are known to those skilled in the art and are described, for example, by Bradley et al. (Described above).
While the correct developmental stage of any embryo is suitable for use, the preferred embryo is male. In mice, preferred embryos also have a gene encoding for a hair color different from the hair color encoded by the ES cell gene. In this method, progeny can be easily screened for the presence of knockout solids by observing mosaic fur color (indicating that the ES cells are inserted into developing embryos). As a result, for example, if the ES cell line has a gene for white hair, the selected embryo has a gene for black or brown hair.
The number of ES cells introduced into the embryo, the embryo can be implanted into the womb of fertility wool during fertility. While any wool can be used, the wool is typically chosen for its ability to be well pregnant and produce, and to care for infants. The wool is typically prepared by mating with a vasectomy male of the same species. The fertility timing of wool is important for successful transplantation, which is species dependent. For mice, this period is about 2-3 days.
Descendants born of wool can be screened first for mosaic hair color where the hair color selection method (described above) is used. Additionally, or alternatively, DNA obtained from descendant tail tissue can be screened for the presence of knockout constructs using Southern blots and / or PCR as described above. The descendants that appear as mosaics can result in cross-crossing with each other if they are thought to have knockout constructs in their sperm lines to produce homozygous knockout animals. Homozygotes can be identified by Southern blot of equal amounts of genomic DNA obtained from mice known to be heterozygous and natural mice, as well as mice of the product of these crosses.
Other means of identifying and characterizing the knockout progeny are also useful. For example, northern blots can be used to label mRNA for the presence or absence of transcripts encoding the knocked out gene, indicator gene, or both. In addition, Western blots can be used to test the expression level of knocked out signaling genes in various tissues of the offspring by labeling the western blots with an antibody against an indicator gene product in which the gene is expressed or an antibody against a particular signaling protein. have. Finally, in situ assays and / or Fluorescence activated cell sorting (FACS) assays of various cells obtained from descendants can be performed using antibodies suitable for observing the presence or absence of the knockout constituent citron product.
Still other methods of making knock-out or degraded transgenic animals are also generally known. For example, Manipulating the Mouse Embryo. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Recombinase dependent knockout can also be achieved by homologous recombination inserting the target sequence such that tissue specific and / or temporary regulation of the inactivation of the signaling gene can be regulated by the recombinase sequence (described below). Can be generated.
Animals containing one or more knockout constructs and / or one or more transgene expression constructs are prepared in several ways. A preferred method of preparation is to produce a series of mammals each containing one of the desired transgene traits. The animals are mated together through heterocrossing, heterogeneous breeding and selection to finally produce one animal containing all the desired knockout constructs and / or expression constructs, where the animals are in other words knockout construct (s). ) And / or covalently (genetically the same) for the native type except for the presence of the transgene (s).
Hybridization and posthybridization are typically performed by crossing off offspring and sibling or parental lines, depending on the goals of each particular step in the breeding process. In some cases, it is necessary to produce a large number of offspring to produce a single offspring containing the transgene at each knockout construct and / or in the appropriate chromosomal position. For example, genes encoding signals and other TGFβ-like genes (eg, skeletal morphogenic proteins, activin, nodal, etc.), other tumor suppressor genes (eg, p53, DCC, p21 ^cip1 , P27 ^kip1 , Rb And / or E2F), or developmental genes (eg, hedgehog, dosulin, neurotropic factors). As a result, in order to produce mice with both signaling and other knocked out genes, two choices are necessary. First, double knockouts can be made by injecting a single ES cell with both signaline and other gene knockout constructs, and screened for transformed cells incorporating both constructs into the same chromosomes within the same ES cell. have.
Also, as a more preferred embodiment, two knockout animals may be produced, one containing a signaline knockout construct and one containing another gene knockout construct. The animals can eventually breed together, successfully interbreed and screen until a descendant containing all knockout constructs is obtained on the same chromosome.
Representative transgenic hybrids that can be made with any of the signaline transgenic animals of the present invention are those in which another tumor suppressor gene is functionally disrupted, causing one tumor gene to be overexpressed or lose negative control (functionally overexpressed). Descendants mating with a second transgenic animal. For example, the signal ^{cleavage agent} of the present invention is another gene that is cleaved in at least one site for tumor suppressor genes such as, for example, p53, DCC, p16 ^ink4 , p21 ^cip1 , p27 ^kip1 , Rb and / or E2F. Can be crossbred with the transplanted animal (same species). In another embodiment, the signal degrading agents of the present invention overexpress at least one tumor gene, for example ras, myc, cdc25A or B, Bcl-2, Bcl-6, a transforming growth factor (eg , TGFα, TGFβ, etc.), neu, int-3, polyoma virus intermediate T antigen, SV40 giant T antigen, one or both of papillomaviral E6 and E7 proteins, CDK4, or cyclin ( cyclin) can be crossbred with a transgenic animal whose expression and / or bioactivity is released for at least one tumor gene such as D1.
In yet another embodiment, the second transgenic animal is a degradation or overexpression of differentiation factors such as TGFβ (eg, BMP, etc.), Hedgehog, dosulin, neurotropic factors, or the like, or neurotropic factor receptors, The signal generated may be altered by functional degradation or overexpression of receptors or signal transduction proteins involved in inducing differentiation such as modulated TGFβ receptors (eg, activin receptors), WT-1, and the like.
As can be appreciated from the following, the variety of F1 × F1 hybrids that can be produced arises both from the control and / or pattern of defects provided by the transgenic construct, as well as from the effects of the transgene itself. For example, the hybrids can be made between a homozygous or heterozygous signaline transgenic animal and also a second transgenic animal which may be homozygous or heterozygous. Signaling defects of the transgenic animals of the present invention used in hybrid-crossing may be tissue-specific, developmentally specific or ubiquitous, as are the transgenic defects of the crossed second transgenic animal. For example, under the control of transcriptional regulatory sequences, the transgene can be regulated in a tissue-specific or ubiquitous manner. Similarly, such regulatory elements may provide for constitutive or inducible expression. As an example, the signal degrading agents described in the appended examples can be crossbred with a transgenic animal comprising an activated ras tumor gene induced by a Whey acidic protein (WAP) promoter. While the signalin deficiency is generalized (eg, depending on the level of mosiasism), recombinant expression of the ras tumor gene will in principle be limited to the resulting hybrid mammary epithelium. The animal can be used, for example, as a breast cancer model. In addition, as a replacement for the WAP-ras transgene, the signalin cleavage agent may be crossed with a transgenic animal that expresses a tumor gene under transcriptional control of a tyrosinase promoter / enhancer element. For example, the crossed transgenic animals can include tumor genes such as ras, cyclin D1 or CDK4 R24C variants that are regulated up and down the transcription of tyrosinase promoters.
Other representative examples of genetic xenotyping with the signaline transgenic animals of the present invention include:
Hybrid with ζ-globin / v-Ha-ras transgene: The transgenic expresses v-Ha-ras under the zeta-globin promoter, which is described in Leder et al. (1990) PNAS 87: 9178-9182. Developed and characterized, and commercially available by the Charles River Laboratory. The transgenic lineage enables the development of dermal papilloma and impression cell malignancies under skin treatment with phorbol esters (growth promoters).
Hybrid with MMTV / c-myc Transgene: The transgene expresses c-myc under the MMTV (mouse mammary tumor virus) promoter, Stewart et al. (1984) Cell 38: 627-637; (1987) Cell 49: 465-475), and was characterized and commercially available by the Charles River Laboratory. The transgenic lineage develops spontaneous mammary adenocarcinomas and other tumors.
Hybrid with Eμ-myc Transgene: The transgene expresses c-myc under Eμ enhancer promoters (especially immunoglobulin promoters are expressed in lymphoid cells). The transgene develops spontaneous B-cell lymphoma (Adams et al. (1985) Nature 318: 533-538).
Hybrid with mTR transgene: Mouse gene encoding RNA component of telomerase rebonucleoprotein was cloned (Blasio et al. (1995) Science 269: 1267-1270). Transgenic mice that overexpress MTR or degraded for MTR expression can be crossed with the signaline transgenic animals of the invention. The genetic hybrid provides valuable information and disease models. For example, the animals can be used to determine the effect of signaline-deficiency on tumor development (tumor may appear earlier or develop rapidly into the most malignant and invasive stage). Signalin-deficiency can affect the type of tumor or their localization, and therefore they can constitute a new animal model for malignancy in certain humans. The animals can also constitute an excellent animal model for assaying chemotherapeutic diets because they allow direct comparisons between various signalin ⁺ and signalin ^- tumor traits.
Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited to the following Examples.
Example 1
RT-PCR Replication of Signaling cDNA
This example describes a method used to obtain cDNA copies that encode elements of the signaling family of signal transduction molecules. Primers were contacted with BamHI or EcoRI linkers, 5 'and 3' were generated, respectively, and used to amplify fragments of Xenopus signalin cDNA. The sequence of the upstream primer used in this study is CGGGATCCTIGA (T / C) GGI (A / C) GI (T / C) TICA (A / G) (A / G) T and the downstream used in this study Primer sequence was CGGAATTCTA (A / G) TG (A / G) TAIGG (A / G) TT (T / G / A) AT (A / G) CA. The cDNA template used in this study was derived from Xenopus embryos in steps 2, 11 and 40. PCR was performed under the following conditions: 93 ° C., 3 minutes; 42 ° C., 1.5 minutes; 74 ° C., 1 cycle; Thereafter, 93 ° C., 1 minute; 42 ° C., 1.5 minutes; 72 ° C., 4 cycles per minute; After 93 ° C., 1 min; 55 ° C. 1.5 minutes; 72 ° C., 30 cycles per minute; And finally 72 ° C., one cycle of 5 minutes. The PCR fragments were subreplicated with pBluescript KSII.
The PCR fragments were sequenced and used as markers to screen the Xenopus cystic cDNA library. Several copies were isolated from the cyst library and subreplicated with pBluescript KSII and then sequenced on both strands.
Example 2
Xenopus signaline proteins transduce individual subgroups of the TGFβ subfamily.
(1) Experimental procedure
Preparation of Synthetic mRNA for Microinjection
To prepare synthetic mRNAs encoding signaline proteins, pSP64T-derived plasmids containing whole signaline cDNA were linearized with XbaI and described in (Krieg and Melton, 1987 Methods in Enzymology 155, 397-415). As transcribed in vitro. The copy is called pSP64TNE-Xe Signaling 1 (also known as pSP64TNE-545-1) and pSP64TNE-Xe Signaling 2 (also known as pSP64TNE-545-4). In addition, the truncated type I BMP receptor (tBR) (Graff et al. 1994, Cell 79, 169-179) and the truncated type II activin receptor (tAR) (Hemmati-Brivanlou and Melton, 1992, Nature 359, 609-). 614 is described. Embryos were not injected (control) or injected with 2 ng of Xe signaling 1 or Xe signaling 2 mRNA. Smaller amounts of mRNA also include mesoderm during infusion, for example 60 pg of Xe signalin 2 contains mesoderm markers (not shown).
Developmental method
Embryos were obtained, microinjected, cultured and animal caps were cut as described above (Thomsen and Melton, 1993 Cell 74,433-441; Graff et al. 1994 Cell 79, 169-179). Histological sections were cut from paraffin containing samples and stained with geimsa for photography (Graff et al. 1994, detailed). All developmental stages are according to Nieuwkoop and Faber (1967 Normal Table of Xenopus laevis (Daudin) (Amsterdam, North Holland Publishing Company) .The mesoderm containing protein is buffered with 0.5 MMR and 0.5% bovine serum albumin. Activin was donated by Dr. Mather at Genentech, and BMP-4 was provided by Dr. Celeste of the Genetics Institute.
RNA analysis by RT-PCR
Proteinase K digestion, RNA extraction and RT-PCR assays have already been disclosed (Graff et al. 1994 Cell 79, 169-179; Wilson and Melton, 1994 Current Biology 4, 676-686). The intensity of the radiation band amplified by RT-PCR reflects the richness of the mRNA (Graff et al., 1994 Cell 79, 169-179; Wilson and Melton, 1994 Current Biology 4, 676-686), which indicates the amount of cDNA template Can be demonstrated during the experiment by further confirming that the strength of the mand is consistent with the abundance of the mRNA. In each experiment (Experiments 4, 7A-C and 8), the PCR amplified product of each line represents the fraction of RNA (approximately 1 / 50th) isolated from the pool of animal caps.
Conditions for PCR detection of RNA transcripts and sequences of most primers are already disclosed for brachyury, goosecoid, muscle actin, NCAM, EF1α and globin (Graff et al. 1994 Cell 79,169). -179; Hemmati-Brivanlou and Melton 1992 Nature 359, 609-614; Wilson, PA and Melton, DA 1994 Current Biology 4, 676-686). Primer sequences not already disclosed are listed below 5 'to 3' and both primer groups were used for 25 cycles.
Xe Signaling 1 Upstream: ACA GCA GCA TTT TTG TTC AG
Downstream: GAG ACC GAG GAG ATG GGA TT
Xe Signalin 2 Upstream: TCC CCT TCA GTC CGC TGC
Downstream: CCA ACA AGG TGC TTT TCG
Cyst Injection and Protein Fractionation
Stage VI cysts were isolated and injected with 30 ng of Xe signaline mRNA and incubated in culture containing ³⁵ S-amino acids that label the newly translated protein as previously disclosed (Smith, L. et al. 1991 Cell 67 , 79-87; Kessler and Melton, 1995 Development 121 2155-216). Briefly, the cysts were manually separated and defolliculated with collagenase. As a result, the cysts were injected with 30 ng of signaline-encoded mRNA. After injection, the cyst sieve were incubated in culture medium containing ³⁵ S- cysteine and ³⁵ S- methionine labeling solidifying the translated protein rupge new. Cultures containing secret proteins were isolated. 20 cysts were treated with 400 μl of 4 ° C. buffer 94A + [0.25M Sucrose, 20 mM Hepes pH 7.4, 50 mM KCl, 0.5 mM MgCl ₂ , 1 mM K-EGTA pH 7.4, 1 mM PMSF, 1 μg / ml Homogenized in [eptin] and the fraction is shown as a whole in FIG. 6. Membrane and cytosolic fractions were separated by centrifugation at 1000 × g for 5 minutes at 4 ° C. at low speed and centrifugation at 4 ° C. for 45 minutes (Evans and Kay, 1991 Methods in Cell Biology). 36, 133-148). Nuclei were isolated by manual antagonism (Evans and Kay, 1991 Methods in Cell Biology 36, 133-148). One cyst fraction of each compartment was analyzed by 10% SDS-PAGE in the presence of dithiothreitol, a reducing agent. Cultures containing the secret protein were isolated (Smith, L. et al. 1991 Cell 67, 79-87; Kessler and Melton, 1995 Development 121, 2155-216).
(ii) Xe signaling is a gene family
Altered polymerase chain reaction (PCR) primers were used to screen the Xenopus cystic body library, four different Xe signaline cDNAs were cloned (FIG. 6), two of which were characterized herein. The sequences of Xe signaline 1 and Xe signaline 2 are shown in FIG. 6. Xe signaline 1 is 76% identical to Mad, and Xe signaline 2 is 62% identical. This high degree of sequence conservation indicates that the Xe signaline is a vertebrate homologue of the Drosophila Mad gene. Vertebrate Xe signaling is also similar to three Mad-related C. elegans (CEM-1, CEM-2 and CEM-3), also called C. elegans Mad, identified by the C. elegans genome sequencing project ( Sekelsky et al. 1995 Genetics 139, 1347-1358; Savage et al. 1996 Proc. Nat. Acad. Sci. 93, 790-794). Xe signalin 2 contains an optionally conjugated exon that appears to be found at the same position in CEM-3 (Sekelsky et al., 1995 Genetics 139, 1347-1358). Until recently, in cloning of cDNAs or genes in frogs, mice, and humans, 6 different Xe signals were identified, which appear to be divided into four groups that closely match the sequences identified in invertebrates (JG and DAM unpublished). Observation). Open reading frames predict proteins with molecular weights between 50,000 and 55,000 Daltons that do not contain any homologs to any signal sequence, cross-membrane domain, or other known protein sequence motif.
(iii) Signalin induces mesoderm formation.
Xenopus laevis animal pole explants typically become ectoderm (ciliary cortex), but can be converted to mesoderm of the spine or abdomen depending on the TGF-β phase and ligand used as inducers. Activin, Vg1, TGF-β and nodal all induce spinal mesoderm (Rosa et al. 1998 Science 239, 783-785; Thomsen et al. 1990 Cell 63, 485-493; Green et al. 1990 Development 108, 173-183 Dale et al., 1993 EMBO J. 12, 4471-4480; Thomsen and melton, 1993 Cell 74, 433-441; Jones et al. 1995 Development 121, 3651-3662), whereas BMP- and BMP- induce abdominal mesoderm. (Loster et al. 1991 Mechanisms of Development 33, 191-200; Dale et al. 1992 Development 115, 573-585; Jones et al. 1992 Development 115, 639-647; Hemmati-Brivanlou and Thomsen, 1995 Developmental Genetics 17, 78-89). The two types of ectoderm, the spine and the abdomen, are easily distinguished by morphology, tissue and molecular markers. To test whether direct expression of Xe signalin induces mesoderm (sending TGFβ-like signals), synthetic mRNA encoding Xe signaline protein was injected into the animal poles of the fertilized egg, and the animal cap was removed. After culture, the cells were assayed for mesoderm induction (FIG. 1). Xe signalin 1 is expressed in the form of the ventral mesoderm, which is an animal pole explant, as shown by the liquid-filled material containing the mesenchymal and mesoderm (Fig. 3). Animal caps injected with Xe signalin 1 do not express spinal mesoderm markers, guscoids, muscle actin or neural markers, NCAM, but express globin, a definitive marker of abdominal mesoderm (FIG. 4). Unexpectedly, the formation of abdominal mesoderm by Xe signalin 1 is manifested without the expression of early markers on mesoderm, such as the Brachiari (FIG. 4). This deficiency of Xe Brachiari expression is observed at all early time points. In all cases, the data indicate that Xe signalin 1 induces the abdomen, the same type of mesoderm observed when the animal cap is induced by BMP-2 or BMP-4 (Koster et al. Mechanisms of Development 33, 191). -200, 1991; Dale et al. 1992 Development 115, 573-585; Jones et al. 1992 Development 115, 639-647; Hemmati-Brivanlou and Thomsen, 1995 Developmental Genetics 17, 78-89).
In contrast, when Xe signalin 2 is expressed in animal poles, the tissue is expanded in a manner to the mesoderm of the spine (FIG. 2), and histological analysis shows the presence of muscle and spinal cord (FIG. 3). This is confirmed by immunohistochemistry with muscle specific monoclonal antibodies, 12/101 and spinal specific antibodies, Tor70.1 (not shown). Molecular assays show that mesoderm induced by Xe signalin 2 does not express abdominal marker globin but expresses spinal marker, guscoid and muscle actin (FIG. 4). Therefore, Xe signalin 2 such as activin, Vg1, TGF-β and nodal induce spinal mesoderm. As a result, Xe signalin 1 and 2 produce two distinct and easily distinguished biological responses; Xe signalin 1 produces ventral mesoderm and Xe signalin 2 produces spinal mesoderm.
To further assert that the pronounced responses shown with Xe signaling 1 and Xe signaling 2 are qualitative differences and not concentration dependent differences, we examined the two Xe signaling at concentrations ranging from 15 pg to 2 ng ( 7A-C). Xe signalin 2 induces mesoderm over a broad concentration range of ˜125 pg to 2 ng (7A) and may induce mesoderm formation at an amount of 60 pg (not shown). In FIG. 7A, RNA was analyzed by RT-PCR for the presence of the indicated transcript. Xe signalin 2 was expressed in a 2-fold dilution series from 2 ng to 15.6 pg. Xe signaline 2 induces expression of other molecular markers starting at about 125 pg of RNA in a concentration-dependent manner. Higher concentrations of Xe signalin 2 induce the expression of most spine mesoderm marker guscoid. At lower Xe signalin 2 concentrations, no guscoids are expressed, but Xwnt-8, a ventral-lateral marker, is expressed. Meaning, no concentration of Xe signalin 2 resulted in the expression of globin, an abdominal marker. These results reproduce the concentration effect obtained by varying the amount of activin and Vg1, the TGF-β molecule that induces spinal mesoderm (Green et al. 1990 Development 108, 173-183; Green et al. 1992 Cell 71, 731-739; Wilson; And Melton, 1994 Current Biology 4, 676-686; Kessler and Melton 1995 Development 121, 2155-216).
The result obtained with Xe signaline 1 is in contrast to that produced by Xe signaline 2 (FIG. 7B). In no amount Xe signalin 1 induces any of spinal markers, guscoids, actin or NCAM, while Xe signalin 1 induces expression of globins resembling BMP-2 and BMP-4. In addition, Xe signaling 1 appears to be much less potent than Xe signaling 2, which requires nanogram amounts of mRNA to make mesoderm. The two mimic effects observed with ligands as BMP are less potent than activin or Vg1 (Thomsen et al. 1990 Cell 63, 485-493, Thomsen and Melton, 1993 Cell 74, 433-441, Hemmati-Brivanlou and Thomsen, 1995 Developmental Genetics 17, 78-89).
Co-injection of mRNAs encoding Xe signaling 1 and 2 results in the formation of abdominal and spinal mesoderm. In FIG. 7C, animal caps expressing either Xe signaling 1 (2ng), Xe signaling 2 (2ng) or Xe signaling 2 (M1 + M2, 2ng each) were incubated up to tadpole stage 38. , Total RNA was collected. Xe signalin 1 induces expression of abdominal marker globin, Xe signalin 2 induces expression of spinal marker actin and the combination results in expression of both markers.
Taken together, the data suggest that Xe signalin 1 induces ventral ectoderm that complements the effects of BMP-2 and BMP-4, while Xe signalin 2 mimics the effects of spinal induction ligands such as activin and Vg1. Induces spinal mesoderm. As a result, the Xe signal protein has qualitatively differentiated activity in developmental mesoderm induction.
(iv) phosphorylation of signaline proteins
The Xenopus signalin coding sequence is subcloned into an expression vector to include a myc epitope fused to the backbone of the signalin coding sequence. The fusion protein is continuously expressed in COS cells. Briefly, the rkradua COS cells are labeled with γ- [ ³² P] -ATP, after incubation, homogenized and immunoprecipitated with an antibody against the myc-tag. ³² P-labeled proteins were detected in the precipitate by SDS-PAGE and radiographs. Importantly, the myc-tagged protein was also shown to be active by the animal cap assay described above.
(v) Signalin acts downstream of the TGF-β receptor.
To identify the location of the Xe signaline in the TGF-β signaling cascade, a truncated receptor was used that acts as the dominant negative receptor. By using the dominant negative form of the receptor, it is expected that the signal does not act downstream of the receptor, whereas the signal acting on the upstream of the receptor blocked by the truncated receptor is not affected (Herskowitz, 1987). Nature 329, 219-222; Amaya et al. 1995 Cell 66, 257-270; Hemmati-Brivanlou and melton, 1992 Nature 359, 609-614; Graff et al. 1994 Cell 79, 169-179; Suzuki et al. 1994 Proc. Natl. Acad. Sci. 91, 10255-10259; Umbhauer et al. 1995 Nature 376, 58-62). Xe signalin 1 appears to be located in the BMP-specific pathway, and the cornered BMP receptor is formed so that the endoplasmic reticulum, mesenchymal and mesothelial are not weakened when Xe signalin 1 co-expresses with the dominant negative BMP receptor As evidenced by the fact that it does not affect Xe signaline 1-dependent form or histological induction of mesoderm of the abdomen (FIG. 9). In contrast to the effects of this deficiency on morphology and histology, the edible BMP receptor blocks the Xe signalin 1-dependent induction of globin (9B). Formation of the endoplasmic reticulum, mesenchymal is an early and potent direct effect of the expression of Xe signaline 1 (and BMP-signaling), whereas the expression of globin is a late effect requiring virtually many steps. The latter stage can be modified without blocking the Xe signaling 1 function per second. Blocking of globin expression can be explained by BMP receptors that inhibit endogenous BMP-signaling present in animal caps (Graff et al. 1994 Cell 79, 169-179; Suzuki et al., 1994 Proc. Natl. Acad. Sci. 91 , 10255-10259; Hawley et al. 1995 Genes and Development 9, 2923-2935; Sasai et al. 1995 Nature 376, 333-336; Schmidt et al. 1995 Developmental Biology 169, 37-50; Wilson and Hemmati-Brivanlou, 1995 Nature 376, 331- 333). If ectopic expression of Xe signalin 1 requires endogenous BMP activity to induce globin, the truncated BMP receptor will eliminate globin expression by blocking endogenous BMP signaling. As a supplement to this view, coexpression of BMP-4 and Xe signalin 1 mRNAs in amounts that have no effect on their own results in the induction of globin (not shown).
Another way to determine if Xe signalin 1 is downstream of the receptor is to test whether Xe signalin 1 can reverse the transgenic effects of the cornered dominant negative receptor. The cornered BMP receptors that block BMP-signaling result in weak induction of neural tissue as shown by induction of N-CAM (FIG. 9) (Sasai et al. 1995 Nature 376, 333-336; Hawley et al. 1995 Genes and Development 9, 2923-2935). Similarly, the truncated activin receptors that block all tested TGF-β signals, including BMP, induce neural tissues more strongly than the truncated BMP receptors (FIG. 9) (Hemmati-Brivanlou and Melton, 1992). , Nature 359, 609-614; Schulte-Merker et al. 1994, EMBO Journal 13, 3533-3541; Kessler and Melton, 1995 Development 121, 2155-216, Hemmati-Brivanlou and Thomsen, 1995 Developmental Genetics 17, 78-89). Xe signalin 1 completely reverses the induction of N-CAM by one of the truncated receptors, meaning that Xe signalin 1 acts downstream of the receptor. This reversal of N-CAM expression does not appear when BMP-4 is coexpressed with the truncated BMP receptor (Sasai et al. 1995 Nature 376, 333-336).
Since Xe signalin 2 appears to act within the activin / Vg1-like spinal pathway, it is important to determine if the dominant negative activin receptor blocks xe signaline 2 function. The cornered activin receptors block activin and Vg1 functions and block the formation of all vertebrate mesoderm (Hemmati-Brivanlou and Melton, 1992 Nature 359, 609-614; Schulte-Merker et al. 1994 EMBO Journal 13, 3533 -3541; Kessler and Melton, 1995 Development 121, 2155-216). Microinjection of the truncated activin receptor leads to expression of NCAM that determines whether the dominant negative activin receptor is active (FIG. 9D) (Hemmati-Brivanlou and Melton, 1992 Nature 359, 609-614). Co-expression of the dominant negative activin receptor with Xe signalin 2 does not block (not shown) genotypic expansion induced by Xe signalin 2. In addition, the dominant negative activin receptor has no effect on mesoderm formed by Xe signalin 2, as indicated by the lack of influence on molecular marker brachial and muscle actin. These results support that Xe signaline acts downstream of the receptors.
(vi) Xe signalin is expressed singly during embryonic development
Since each Xe signalin induces one but not both of the ventral or spinal mesoderm, their placement or other activation may explain how the embryonic mesoderm is initially established and patterned. The spatial distribution of the Xe signalin transcripts within the various regions that generate embryos by reverse transcription-PCR (RT-PCR) was determined. Xe signaline RNA is matured because the cDNA was recovered from the cyst library. The RNAs are in the blastocyst, and both Xe signalin 1 and 2 mRNAs are present at approximately the same level in all blastocyst regions. Similarly, during the early blastocyst phase, Xe Signalin 1 and Xe Signalin 2 mRNAs appear to be distributed equally within the abdominal and spinal margin regions (FIG. 8). The time course of Xe signaline 1 and Xe signaline 2 expression shows that the RNAs are present at nearly constant levels from the 2-cell phase to the tadpol phase (not shown). Spatial and temporal agreement during the formation of the spinal-abdominal mesoderm pattern indicates that TGF-β signaling activates other Xe signaling proteins on the other side of the embryo.
To test whether mesoderm induction by TGF-β phase and ligand affects the transcription of the Xe signaline gene, we added BMP-4 or activin protein to the ectoderm explants and when mesoderm were induced Xe signaline mRNA levels were analyzed at intervals of up to 40 minutes. As expected, both BMP-4 and activin induce mesoderm tested at 160 min by expression of Brachyri RNA herein (FIG. 8). The levels of Xe Signaling 1 and Xe Signaling 2 mRNA are not affected at all 4 time points (FIG. 8), indicating that transcription of Xe Signaling 1 and Xe Signaling 2 does not change significantly by mesoderm induction. . Taken together, the data indicate the presence of a constant amount and nearly uniform amount of Xe signaling 1 and Xe signaling 2 mRNA in early development.
(vii) the location of signaline proteins relative to the cytosol and nucleus
To determine the intracellular location of the Xe signaling protein, we injected stage VI cysts with 30 ng of Xe signaling mRNA and incubated in culture containing ³⁵ S-amino acids. Cysts were fractionated and total, secret, membrane related, nuclear, or cytosolic proteins were analyzed by SDS-PAGE. Figure 10 shows the results obtained with Xe signal Rin 2, the same result is Xe signal Rin 1 or Xe signal Rin 2 Xe signal having a synthesized mRNA that encodes one was obtained Lin 1 cystic body, the cyst sieve are ³⁵ S Incubated with containing amino acids. Newly synthesized proteins were examined from cyst body cultures (containing secret proteins), manually isolated nuclei, and biochemically fractionated membranes and cytoplasm. Gel fractionation of the newly synthesized protein (FIG. 10) shows that the Xe signaline protein is present in both the nucleus and cytoplasm, but not in the membrane fraction hidden in solution. Close examination of the nucleus and cytoplasmic lines reveals that the nucleus Xe signaling protein is slightly larger. This reproducible effect indicates that proteins in the nucleus can be post-translationally modified. To rule out the possibility that the nucleus or cytoplasmic position of Xe signaline is due to overexpression, Xe signaline was expressed at low concentrations and their intracellular location was determined by Western blot. When the Xe signalin is expressed at the detection limit of the antibody (20-100 times smaller than the mRNA used in Figure 10), the protein is still found in both the cytosol and the nucleus.
The results presented herein indicate that Xe signalin is a component of the vertebrate TGF-β signaling pathway. Expression of each Xe signaline protein mimics the effect of specific subgroups of TGF-β signaling on mesoderm induction in Xenopus by producing spinal or ventral mesoderm. In addition, experiments showing that the truncated receptors do not block Xe signaling signaling coupled with top tests genetically indicative of the need for signaling in DPP-sensitized cells have shown that Xe signaling has been described as TGF. It supports the notion of downstream of ligands and receptors in the -β signal transduction cascade.
Immunohistochemical studies conducted with biochemical fractions in the Drosophila Mad protein (submitted Newfeld et al. 1996) and Xenopus cysts showing that the Xe signaline is an intracellular protein are the same as the above review. The data shown in Figures 9A-C indicate that there is a difference between the nuclear and cytoplasmic forms of the Xenopus Xe signaline protein. Given the precedent of other signal transduction cascades, it is possible for ligand-dependent changes to result in the cross-location of Xe signaling proteins from one compartment to another (Verma et al. 1995 Genes and Development 9, 2723-2735). Since the Xe signaline is part of the signaling cascade initiated by the receptor serine-threonine kinase, it is possible that a size difference between the nucleus and the cytosolic unit is caused by phosphorylation. In fact, preliminary experiments indicate that the xE signaline is a phosphate protein.
Bending The Xe signalin 1 appears to carry a BMP group of signals to the ventral mesoderm, while the Xe signalin 2 carries an activin / Vg1 / NodaVTGF-β signal that forms spinal mesoderm. In turn, the Xe signaling acts as an integration point in the signaling pathway. Within Xenopus there are at least two different mature Xe signaling (Xe signaling 3, 4), which are still functionally related to TGF-β signaling.
In order to understand the mesoderm induction of Xenopusso, the results presented in the present invention do not show any difference in the distribution of parental or conjugated Xe signaling mRNA, and as a prediction, their corresponding proteins are uniformly distributed along the future body axis. In other words, all cells in the residual region of the early embryo can theoretically respond to spinal or ventral mesoderm induced signals by having Xe signaling 1 and Xe signaling 2 mRNAs. As a result, the BMP signal is easy to activate Xe signaling 1 on the ventral side of the embryo, while the spinal-induced signal (possibly Vg1 or activin) activates Xe signaling 2 on the future spine.
It is an unexpected finding that the formation of abdominal mesoderm by Xe signalin 1 occurs without the appearance of Brachyery (FIG. 4). Xe signalin 1 can directly activate differentiation to the ventral mesoderm and does not require expression of Xbra. In fact, while Xbra is being considered as a general marker for embryonic mesoderm, there is no experiment showing whether all mesodermal formation requires Xbra expression. In an embodiment, gene neuroD disrupts neuronal formation in Xenopus and can clearly avoid the early inhibitory effects of direct conversion of animal cap cells to neurons (Lee et al. 1995 Science 268, 836-844). .
All infusions reported herein were performed with mRNA encoding the natural-type but not mutant or structurally active forms of the Xe signaline protein. Several mechanisms can be proposed to explain why the injection of native-type Xe signaling mRNA already present in the embryo leads to mesoderm formation. Clearly, the injection of Xe signaline mRNA leads to the generation of active Xe signaline protein, which can be caused by many mechanisms. Animal cap cells have endogenous BMP and activin mRNA and are optionally exposed to low levels of BMP and activin signaling pathways, even at levels not sufficient to induce mesoderm (Hemmati-Brivanlou and Melton, 1992 Nature 359, 609 Graff et al., 1994 Cell 79, 169-179; Hawley et al., 1995 Genes and Development 9, 2923-2935; Sasai et al., 1995 Nature 376, 333-336; Schmidt et al. 1995 Developmental Biology 169, 37-50; Wilson; And Hemmati-Brivanlou, 1995 Nature 376, 331-333).
Ectopic expression of Xe signaling in combination with this structural pathway increases the level of signaling (BMP for Xe signaling 1 and activin / Vg1 / nodal) for Xe signaling 2 leading to mesodermal induction. Another possibility is that Xe signaling is under negative control, and providing excess Xe signaling protein will overrule this control. Similar to the results obtained with Xe signaling, mRNA insertion of glycogen synthase kinase-3 or several components of the Wnt signaling pathway, such as each, results in the activity of the Wnt signal (He et al., Et al. 1995 Nature 374, 617-622; Pierce and Kimelman, 1995 Development 121, 755-765; Sokol et al. 1995 Development 121, 1637-1647).
As described above, Xe signaling appears to be the point at which information is integrated in that each Xe signal delivers input from the TGF-β phase and a subgroup of ligands. This makes another sense in that Xe signaling can relate to integrating information, ie measuring the amount of signal a cell receives. When Xenopus blastocyst cells are exposed to various concentrations of activin, a variety of spinal mesoderm are produced (Green et al., 1990 Development 108, 173-183; Green et al., 1992 Cell 71, 731-739; Wilson and melton, 1994 Current Biology 4, 676-686. For example, high concentrations produce chocks, and low concentrations make muscles. Similarly, varying amounts of Xe signaling 2 reflect speculative varying amounts of Xe signaling 2 activity leading to the expression of markers of various types of mesoderm (FIGS. 7A-C). Therefore, it is possible that Xe signaline is a counting device used in cells to measure the concentration of ligand. For example, post-translational modifications such as phosphorylation can regulate the nuclear: cytoplasmic ratio of Xe signaline. In addition, the activity of each Xe signalin can be determined, in other words, by the number of phosphorylated residues that reflect the concentration of the ligand. Determining which of these biochemical mechanisms modulate Xe signaling activity may help to understand how morphogenesis signals regulate cell fate during development.
Example 3
RT-PCR Cloning of Human Signaling cDNA
Using human PCR primers as described in Examples 1 and 2, several human signaling copies were isolated. In summary, using the denatured PCR primers obtained in Examples 1 and 2, human cDNA samples were amplified by the following PCR conditions: tag polymerase in 9 μl of 25 mM MgCl / 124 μl standard buffer; Temperature cycling, 3 minutes at 95 ° C., then 25 seconds at 95 ° C., 15 seconds at 42 ° C., then 10 seconds at 72 ° C., 25 seconds at 95 ° C., 10 seconds at 55 ° C., 10 seconds at 72 ° C. and 73 ° C. 4 cycles for 10 seconds. The resulting cDNA was sequenced by standard methods.
Example 4
Diverse Expression of Signaling Gene Products in Human Tissues
Using denaturing PCR primers for the signaline family, human cDNA samples were amplified from various tissues using the conditions as described for cloning in Example 2 above. Strong and dominant bands at the correct size for the signaline transcript fragments were amplified with 31 cycles obtained from kidney, liver, lung, mammary gland, pancreas, visa, testis and thymus. This indicates that at least one signaling element is expressed in each of said mature tissues.
By "A" -track sequencing (eg, only reading the A terminus), the data obtained are ubiquitously expressed in the signaline gene product as a whole, while some signalins vary in expression in the above-mentioned tissues. Represents sea bream. The relative amounts of the signaline transcripts (known materials) are shown in Table 1 below:
Human signal type Agencyhu-1hu-2hu-3hu-4hu-5hu-6hu-7 kidney2OneOne-OneOne- Visa-One-OneOne2- liver--5One--- Pancreas--5---One
Note that Hu-signalin 3 predominates in the two digestive organs, liver and pancreas. On the other hand, at least 4-5 different forms (known as data) are expressed in kidneys and visas. The data suggest how the TGF signaling pathway can be degraded in a tissue specific manner. Finally, the A-track data still indicates that other signaling transcripts are still present, eg, the seven sequences for the human signaling family provided herein do not include the entire family.
Example 5
Identification of Human Signaling from Expressed Sequence Tag (EST) Sequences
Using the program BLAST (Basic Local Alignment Search Tool; National Center for Biotechnology Information), some replicated signal sequences were compared to a standard database and sequences with similarity to the replicated signal sequence were examined. In particular, many human EST sequences (for review, see Boguski (1995) Trends Biochemical Science 20: 295-296) have been identified as similar to some of the replicated signaling. Using the guidance of the sub-classification of the invention of the cloned signaline, we were able to combine parts of the EST sequence, correcting for sequencing errors (especially frameshift errors), and some human signaline copies. More complete coding sequences could be derived.
In particular, the N-terminal fragment of human cDNA was harvested from a specific EST sequence and included the signaline motif of hu-signalin 1, a human cloned sequence. The 170 residue fragment represented by SEQ ID NO: 12 (nucleotide) and SEQ ID NO: 25 (amino acid) is an element of an α- subfamily that is substantially homologous to other elements of the α- subfamily outside the signaline motif.
In a similar manner, the 121 residue C-terminal portion of the human signal replica was collected from the EST sequence based on the sequence for the Xenopus signal replica. Analysis of the nucleotide (SEQ ID NO: 13) and amino acid (SEQ ID NO: 26) sequences of the fragments is very similar to xe-signalin 2, thus clearly indicating that it is part of the transcript for the γ- subfamily.
Example 6
Since the previous data of this application, many full-length human signaling (or named DOT, dpc-4 and MAD-like proteins) have been disclosed in the literature. Representative signalins include Genebank Application Numbers U76622, U59913, U59911, U65019, U65019, U68018, U68019, 1438077, U59913 and U59912, among others. Without exception, each copy comprises a signaline motif represented by Formula SEQ ID NO: 27 (or referred to herein as a v domain) and a χ domain represented by Formula SEQ ID NO: 29.
Standing column list
(1) General Information
(i) Applicant:
(A) Name: Onto Jenny, Inc.
(B) Street: Moulton Street 45
(C) Poetry: Cambridge
(D) Note: Massachusetts
(E) Country: United States
(F) Zip code: 02138
(A) Name: President & Fellows of Harvard College
(B) Street: Quincy Street 17
(C) city: Boston
Note: Metachusetts
(E) Country: United States
(F) Zip code: 02138
(ii) Title of the Invention: TGFβ Signaling Proteins, Genes and Uses Associated therewith
(iii) SEQ ID NO: 26
(iv) Payee Address:
(A) Recipients: Lahib & Cockfield, LLP
(B) Street: State Street 60
(C) city: Boston
(D) Note: Massachusetts
(E) Country: United States
(F) Zip code: 02109-1875
(v) computer readable form:
(A) Media Type: Floppy Diskette
(B) Computer: IBM PC Compatible
(C) operating system: PC-DOS / MS-DOS
(D) Software: ASCII (Document)
(vi) Current application data:
(A) Application number:
(B) filing date:
(vii) Preliminary Application Data:
(A) Application number: 08 / 580,031
(B) Application date: December 20, 1995
(viii) Patent Attorney Information:
(A) Name: Vincent, Machu P.
(B) registration number: 36,709
(C) Case Number: ONI-190PC
(ix) Communications Information:
(A) Telephone: (617) 227-7400
(B) Fax: (617) 227-5941
(2) Information about SEQ ID NO: 1
(i) Sequence features:
(A) Length: 1769 base pairs
(B) Type: nucleic acid
(C) Number of chains: 1 and 2 strands
(D) topology: linear
(ii) Type of molecule: cDNA
(ix) Characteristic part:
(A) Name / abbreviation: CDS
(B) Location: 161..1552

(2) Information about SEQ ID NO: 2:
(i) Sequence features:
(A) Length: 1708 base pairs
(B) Type: nucleic acid
(C) Number of chains: 1 and 2 strands
(D) topology: linear
(ii) Type of molecule: cDNA
(ix) Characteristic part:
(A) Name / abbreviation: CDS
(B) Location: 51..1451

(2) Information about SEQ ID NO: 3:
(i) Sequence features:
(A) Length: 2594 base pairs
(B) Type: nucleic acid
(C) Number of chains: 1 and 2 strands
(D) topology: linear
(ii) Type of molecule: cDNA
(ix) Characteristic part:
(A) Name / abbreviation: CDS
(B) Location: 259..1656

(2) Information about SEQ ID NO: 4:
(i) Sequence features:
(A) Length: 2879 base pairs
(B) Type: nucleic acid
(C) Number of chains: 1 and 2 strands
(D) topology: linear
(ii) Type of molecule: cDNA
(ix) Characteristic part:
(A) Name / abbreviation: CDS
(B) Location: 258..2042

(2) Information about SEQ ID NO: 5:
(i) Sequence features:
(A) Length: 1642 base pairs
(B) Type: nucleic acid
(C) Number of chains: 1 and 2 strands
(D) topology: linear
(ii) Type of molecule: cDNA
(ix) Characteristic part:
(A) Name / abbreviation: CDS
(B) Location: 84..1478

(2) Information about SEQ ID NO: 6:
(i) Sequence features:
(A) Length: 132 base pairs
(B) Type: nucleic acid
(C) Number of chains: 1 and 2 strands
(D) topology: linear
(ii) Type of molecule: cDNA
(ix) Characteristic part:
(A) Name / abbreviation: CDS
(B) Location: 1..132
(2) Information about SEQ ID NO: 7:
(i) Sequence features:
(A) Length: 132 base pairs
(B) Type: nucleic acid
(C) Number of chains: 1 and 2 strands
(D) topology: linear
(ii) Type of molecule: cDNA
(ix) Characteristic part:
(A) Name / abbreviation: CDS
(B) Location: 1..132
(2) Information about SEQ ID NO: 8:
(i) Sequence features:
(A) Length: 129 base pairs
(B) Type: nucleic acid
(C) Number of chains: 1 and 2 strands
(D) topology: linear
(ii) Type of molecule: cDNA
(ix) Characteristic part:
(A) Name / abbreviation: CDS
(B) Location: 1..129
(2) Information about SEQ ID NO: 9:
(i) Sequence features:
(A) Length: 132 base pairs
(B) Type: nucleic acid
(C) Number of chains: 1 and 2 strands
(D) topology: linear
(ii) Type of molecule: cDNA
(ix) Characteristic part:
(A) Name / abbreviation: CDS
(B) Location: 1..132
(2) Information about SEQ ID NO: 10:
(i) Sequence features:
(A) Length: 132 base pairs
(B) Type: nucleic acid
(C) Number of chains: 1 and 2 strands
(D) topology: linear
(ii) Type of molecule: cDNA
(ix) Characteristic part:
(A) Name / abbreviation: CDS
(B) Location: 1..132
(2) Information about SEQ ID NO: 11:
(i) Sequence features:
(A) Length: 132 base pairs
(B) Type: nucleic acid
(C) Number of chains: 1 and 2 strands
(D) topology: linear
(ii) Type of molecule: cDNA
(ix) Characteristic part:
(A) Name / abbreviation: CDS
(B) Location: 1..132
(2) Information about SEQ ID NO: 12:
(i) Sequence features:
(A) Length: 519 base pairs
(B) Type: nucleic acid
(C) Number of chains: 1 and 2 strands
(D) topology: linear
(ii) Type of molecule: cDNA
(ix) Characteristic part:
(A) Name / abbreviation: CDS
(B) Location: 16..519
(2) Information about SEQ ID NO: 13:
(i) Sequence features:
(A) Length: 363 base pairs
(B) Type: nucleic acid
(C) Number of chains: 1 and 2 strands
(D) topology: linear
(ii) Type of molecule: cDNA
(ix) Characteristic part:
(A) Name / abbreviation: CDS
(B) Location: 1..363
(2) Information about SEQ ID NO: 14:
(i) Sequence features:
(A) Length: 464 amino acids
(B) type: amino acid
(D) topology: linear
(ii) Type of molecule: protein
(2) Information about SEQ ID NO: 15:
(i) Sequence features:
(A) Length: 467 amino acids
(B) type: amino acid
(D) topology: linear
(ii) Type of molecule: protein
(2) Information about SEQ ID NO: 16:
(i) Sequence features:
(A) Length: 466 amino acids
(B) type: amino acid
(D) topology: linear
(ii) Type of molecule: protein

(2) Information about SEQ ID NO: 17:
(i) Sequence features:
(A) Length: 595 amino acids
(B) type: amino acid
(D) topology: linear
(ii) Type of molecule: protein

(2) Information about SEQ ID NO: 18:
(i) Sequence features:
(A) Length: 465 amino acids
(B) type: amino acid
(D) topology: linear
(ii) Type of molecule: protein

(2) Information about SEQ ID NO: 19:
(i) Sequence features:
(A) Length: 44 amino acids
(B) type: amino acid
(D) topology: linear
(ii) Type of molecule: protein
(2) Information about SEQ ID NO: 20:
(i) Sequence features:
(A) Length: 44 amino acids
(B) type: amino acid
(D) topology: linear
(ii) Type of molecule: protein
(2) Information about SEQ ID NO: 21:
(i) Sequence features:
(A) Length: 43 amino acids
(B) type: amino acid
(D) topology: linear
(ii) Type of molecule: protein
(2) Information about SEQ ID NO: 22:
(i) Sequence features:
(A) Length: 44 amino acids
(B) type: amino acid
(D) topology: linear
(ii) Type of molecule: protein
(2) Information about SEQ ID NO: 23:
(i) Sequence features:
(A) Length: 44 amino acids
(B) type: amino acid
(D) topology: linear
(ii) Type of molecule: protein
(2) Information about SEQ ID NO: 24:
(i) Sequence features:
(A) Length: 44 amino acids
(B) type: amino acid
(D) topology: linear
(ii) Type of molecule: protein
(2) Information about SEQ ID NO: 25:
(i) Sequence features:
(A) Length: 168 amino acids
(B) type: amino acid
(D) topology: linear
(ii) Type of molecule: protein
(2) Information about SEQ ID NO: 26:
(i) Sequence features:
(A) Length: 121 amino acids
(B) type: amino acid
(D) topology: linear
(ii) Type of molecule: protein

权利要求:
Claims (93)
[1" claim-type="Currently amended] Isolated or Recombinant Signaling Polypeptides of Vertebrates.
[2" claim-type="Currently amended] The polypeptide of claim 1, wherein the vertebrate is an amphibian.
[3" claim-type="Currently amended] The polypeptide of claim 1, wherein the vertebrate is a mammal.
[4" claim-type="Currently amended] The polypeptide of claim 3, wherein said mammal is a seal.
[5" claim-type="Currently amended] The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence comprising a signaling motif represented by the general formula SEQ ID NO: 28.
[6" claim-type="Currently amended] The polypeptide of claim 1, wherein the polypeptide stimulates intracellular signal transduction pathways mediated by TGFβ receptors.
[7" claim-type="Currently amended] The polypeptide of claim 1, wherein the polypeptide acts against an intracellular signal transduction pathway mediated by a TGFβ receptor.
[8" claim-type="Currently amended] The polypeptide of claim 5, wherein the polypeptide comprises an amino acid sequence represented by one of SEQ ID NOs: 14-26.
[9" claim-type="Currently amended] The polypeptide of claim 1, wherein the polypeptide has a molecular weight in the range of 45-70 Kd.
[10" claim-type="Currently amended] A signal amino acid sequence that is at least 70% similar to the amino acid sequence represented by one or more of SEQ ID NOs: 14-26, and specifically modulating the signal transduction activity of the receptor for transforming growth factor β (TGFβ) An isolated and / or recombinant signaline polypeptide characterized by.
[11" claim-type="Currently amended] The polypeptide of claim 10, wherein said polypeptide is at least 80% similar.
[12" claim-type="Currently amended] The polypeptide of claim 10, wherein the polypeptide has a molecular weight in the range of 45-70 Kd.
[13" claim-type="Currently amended] The polypeptide of claim 10, wherein the polypeptide has at least 25 amino acid residues in length.
[14" claim-type="Currently amended] The polypeptide of claim 10, wherein the polypeptide stimulates intracellular signal transduction pathways mediated by TGFβ receptors.
[15" claim-type="Currently amended] The polypeptide of claim 10, wherein said polypeptide acts against an intracellular signal transduction pathway mediated by a TGFβ receptor.
[16" claim-type="Currently amended] The polypeptide of claim 10, wherein said TGFβ receptor is not a receptor for dpp sub-family proteins.
[17" claim-type="Currently amended] The polypeptide of claim 10, wherein the signaline amino acid sequence comprises a signaline motif represented by Formula SEQ ID NO: 28.
[18" claim-type="Currently amended] 18. The polypeptide of claim 17, wherein the signaline motif coincides with a signaline motif represented by one of SEQ ID NOs: 14-26.
[19" claim-type="Currently amended] The polypeptide of claim 10, wherein the signaline amino acid sequence comprises a ν domain represented by Formula SEQ ID NO: 27.
[20" claim-type="Currently amended] The polypeptide of claim 19, wherein the v domain coincides with a v domain represented by one of SEQ ID NOs: 14-26.
[21" claim-type="Currently amended] The polypeptide of claim 10, wherein the signaline amino acid sequence comprises a χ domain represented by the general formula SEQ ID: 29.
[22" claim-type="Currently amended] The polypeptide of claim 21, wherein the signaline amino acid sequence comprises a χ domain represented by one of SEQ ID NOs: 14-26.
[23" claim-type="Currently amended] Purified or recombinant signal polypeptide comprising a signal motif.
[24" claim-type="Currently amended] 24. The signaline polypeptide of claim 23, wherein the polypeptide regulates intracellular signal transduction pathways mediated by TGFβ receptors.
[25" claim-type="Currently amended] 24. The signalling polypeptide of claim 23, wherein the signalling motif is represented by Formula SEQ ID NO: 28.
[26" claim-type="Currently amended] The signal polypeptide of claim 23, wherein the signal motif corresponds to a signal motif represented by one of SEQ ID NOs: 14-26.
[27" claim-type="Currently amended] 26. The signaline polypeptide of claim 25, wherein the polypeptide comprises an amino acid sequence represented by the following sequence:
LDGRLQVSHRKGLPHVIYCRVWRWPDLQSHHELKPXXXCEXPFXSKQKXV.
[28" claim-type="Currently amended] The signal polypeptide according to claim 23, wherein the polypeptide comprises an amino acid sequence represented by the following sequence:
LDGRLQVAGRKGFPHVIYARLWXWPDLHKNELKHVKFCQXAFDLKYDXV.
[29" claim-type="Currently amended] The signal polypeptide according to claim 23, wherein the polypeptide comprises an amino acid sequence represented by the following sequence:
LDGRLQVXHRKGLPHVIYCRLWRWPDLHSHHELKAIENCEYAFNLKKDEV.
[30" claim-type="Currently amended] The signaline polypeptide of claim 23, wherein the polypeptide comprises at least one fragment of a polypeptide sequence that matches amino acids 225-300 of SEQ ID NO: 14 or 230-301 of SEQ ID NO: 16.
[31" claim-type="Currently amended] 24. The signaline polypeptide of claim 23, wherein the polypeptide comprises at least one fragment of a polypeptide sequence that matches amino acids 186-304 of SEQ ID NO: 15.
[32" claim-type="Currently amended] 24. The signaline polypeptide of claim 23, wherein the polypeptide comprises at least one fragment of a polypeptide sequence that matches amino acids 170-332 of SEQ ID NO: 17.
[33" claim-type="Currently amended] The signal polypeptide of claim 23, wherein the polypeptide comprises a signalin ν domain represented by Formula SEQ ID NO: 27.
[34" claim-type="Currently amended] The signaline polypeptide of claim 33, wherein the v domain coincides with a v domain represented by one of SEQ ID NOs: 14-26.
[35" claim-type="Currently amended] The signal polypeptide of claim 23, wherein the polypeptide further comprises a signaline χ domain represented by Formula SEQ ID NO: 29.
[36" claim-type="Currently amended] 36. The signaline polypeptide of claim 35, wherein the χ domain is identical to the χ domain represented by one of SEQ ID NOs: 14-26.
[37" claim-type="Currently amended] 24. The signal chain polypeptide of claim 23, wherein the polypeptide is a fusion protein further comprising a second polypeptide sequence having an amino acid sequence independent of the signal chain polypeptide sequence, in addition to the signal motif.
[38" claim-type="Currently amended] The fusion protein of claim 37, wherein the fusion protein comprises a polypeptide that acts as a second polypeptide sequence, as a detectable label for detecting the presence of the fusion protein, or as a matrix-binding domain for immobilizing the fusion protein. Signaling polypeptide, characterized in that.
[39" claim-type="Currently amended] A nucleic acid encoding a signaline polypeptide represented by one of SEQ ID NOs: 14-26.
[40" claim-type="Currently amended] Purified or recombinant signal polypeptide encoded by a nucleic acid hybridized under strict conditions to one or more nucleotide sequences represented by SEQ ID NO: 1-13.
[41" claim-type="Currently amended] An isolated nucleic acid encoding a polypeptide comprising a signaline motif as a polypeptide, characterized in that it specifically modulates the signal transduction activity of a receptor for transforming growth factor β (TGFβ).
[42" claim-type="Currently amended] The nucleic acid according to claim 41, wherein the signaline motif is represented by the general formula SEQ ID NO: 28.
[43" claim-type="Currently amended] The nucleic acid of claim 42, wherein the signaline motif matches a signaline motif represented by one of SEQ ID NOs: 14-26.
[44" claim-type="Currently amended] The nucleic acid of claim 42, wherein the polypeptide comprises an amino acid sequence represented by the following sequence:
LDGRLQVSHRKGLPHVIYCRVWRWPDLQSHHELKPXECCEXPFXSKQKXV.
[45" claim-type="Currently amended] The nucleic acid of claim 42, wherein the polypeptide comprises an amino acid sequence represented by the following sequence:
LDGRLQVAGRKGFPHVIYARLWXWPDLHKNELKHVKFCQXAFDLKYDXV.
[46" claim-type="Currently amended] The nucleic acid of claim 42, wherein the polypeptide comprises an amino acid sequence represented by the following sequence:
LDGRLQVXHRKGLPHVIYCRLWRWPDLHSHHELKAIENCEYAFNLKKDEV.
[47" claim-type="Currently amended] The nucleic acid of claim 42, wherein the polypeptide comprises at least one fragment of an amino acid sequence represented by amino acids 225-300 of SEQ ID NO: 14 or 230-301 of SEQ ID NO: 16.
[48" claim-type="Currently amended] The nucleic acid of claim 42, wherein the polypeptide comprises at least one fragment of an amino acid sequence that matches amino acids 186-303 of SEQ ID NO: 15.
[49" claim-type="Currently amended] The nucleic acid of claim 42, wherein the polypeptide comprises at least one fragment of an amino acid sequence that matches amino acids 170-332 of SEQ ID NO: 17.
[50" claim-type="Currently amended] The nucleic acid of claim 42, wherein the polypeptide comprises a signaline ν domain represented by Formula SEQ ID NO: 31.
[51" claim-type="Currently amended] 51. The nucleic acid according to claim 50 wherein said v domain coincides with a v domain represented by one of SEQ ID NOs: 14-26.
[52" claim-type="Currently amended] The nucleic acid of claim 42, wherein the polypeptide further comprises a signaline χ domain represented by Formula SEQ ID NO: 29.
[53" claim-type="Currently amended] The nucleic acid of claim 52 wherein the χ domain is identical to the χ domain represented by one of SEQ ID NOs: 14-26.
[54" claim-type="Currently amended] 43. The nucleic acid of claim 42 wherein the polypeptide is a fusion protein further comprising a second polypeptide sequence having an amino acid sequence independent of the nucleic acid sequence, in addition to the signaline motif.
[55" claim-type="Currently amended] 55. The polypeptide of claim 54, wherein the fusion protein is a second polypeptide sequence that acts as a detectable label for detecting the presence of the fusion protein or as a matrix-binding domain for immobilizing the fusion protein. Nucleic acid, characterized in that it comprises a.
[56" claim-type="Currently amended] The nucleic acid of claim 42 wherein the polypeptide stimulates intracellular signal transduction pathways mediated by TGFβ receptors.
[57" claim-type="Currently amended] The nucleic acid according to claim 42 wherein said polypeptide acts against an intracellular signal transduction pathway mediated by a TGFβ receptor.
[58" claim-type="Currently amended] The nucleic acid of claim 42, wherein said nucleic acid hybridizes to a nucleic acid label having a sequence represented by at least 60 contiguous nucleotides of one or more senses or antisenses of SEQ ID NO: 1-13 under stringent conditions.
[59" claim-type="Currently amended] 43. The nucleic acid of claim 42 further comprising a transcriptional regulatory sequence operably linked to the nucleotide sequence to make the nucleic acid suitable for use as an expression vector.
[60" claim-type="Currently amended] An expression vector capable of replicating in at least one of prokaryotic and eukaryotic cells, comprising the nucleic acid of claim 42.
[61" claim-type="Currently amended] A host cell infected with the expression vector of claim 60 and expressing said recombinant polypeptide.
[62" claim-type="Currently amended] 62. A method of making a recombinant signaline polypeptide, comprising culturing the cells of claim 61 in a cell culture to express said recombinant polypeptide and isolating said recombinant polypeptide from said cell culture.
[63" claim-type="Currently amended] A transgenic animal comprising a cell having a transgene encoding a signaling polypeptide, which is a vertebrate.
[64" claim-type="Currently amended] A transgenic animal having a cell in which the gene for signalling is degraded and which is a vertebrate.
[65" claim-type="Currently amended] (i) a genetic construct comprising a nucleic acid of claim 54 operably linked to a transcriptional regulatory sequence that causes expression of the signaline polypeptide in eukaryotic cells, and
and (ii) a gene delivery composition for delivering said gene construct to a cell and causing said cell to be infected with said gene construct.
[66" claim-type="Currently amended] 66. The recombinant infection system of claim 65, wherein said gene delivery composition is selected from the group consisting of recombinant viral particles, liposomes and multi-cation nucleic acid binders.
[67" claim-type="Currently amended] Under stringent conditions, a nucleic acid composition comprising a substantially purified oligonucleotide comprising a region of nucleotide sequence that hybridizes to at least 25 consecutive nucleotides of the sense or antisense of a vertebrate signal gene.
[68" claim-type="Currently amended] 68. The nucleic acid composition of claim 67 wherein said oligonucleotide hybridizes to at least 50 consecutive nucleotides of the sense or antisense sequence of the vertebrate signal gene under stringent conditions.
[69" claim-type="Currently amended] 68. The nucleic acid composition of claim 67 wherein the oligonucleotide further comprises a detectable label group attached thereto.
[70" claim-type="Currently amended] 68. The nucleic acid composition of claim 67 wherein the oligonucleotide has at least one non-crosslinkable bond between two adjacent nucleotide subunits.
[71" claim-type="Currently amended] A test kit for detecting in a cell sample a cell containing a signaline mRNA transcript comprising the nucleic acid composition of claim 67 for measuring the level of nucleic acid encoding a signal protein.
[72" claim-type="Currently amended] The cells are treated with an effective amount of reagent that modulates the signal transduction activity of the signaling polypeptide, thereby comparing at least one of (i) growth rate, (ii) differentiation, or (iii) cell viability of the cells, as compared to cells without said reagents. A method for regulating one or more of the growth, differentiation, or survival rate of a mammalian cell in response to signaline-mediated induction comprising the step of varying the number of cells.
[73" claim-type="Currently amended] 73. The method of claim 72, wherein said reagent is similar to the effect of a naturally-occurring signaline protein on said cell.
[74" claim-type="Currently amended] 73. The method of claim 72, wherein said reagent counteracts the effect of naturally-occurring signaline protein on said cell.
[75" claim-type="Currently amended] The method of claim 72, wherein the cells are testicular cells and the reagents regulate spermatogenesis.
[76" claim-type="Currently amended] 75. The method of claim 72, wherein said cells are osteogenic cells and said reagent modulates bone formation.
[77" claim-type="Currently amended] 73. The method of claim 72, wherein said cells are chondrogenic cells and said reagent modulates chondrogenesis.
[78" claim-type="Currently amended] 73. The method of claim 72, wherein said reagent regulates the differentiation of nerve cells.
[79" claim-type="Currently amended] Antibodies to Signaling Polypeptides.
[80" claim-type="Currently amended] 80. The antibody of claim 79, wherein the antibody is monoclonal.
[81" claim-type="Currently amended] A signaline polypeptide that specifically modulates the signal transduction activity of TGFβ receptors other than TGFβ receptors against dpp subfamily elements.
[82" claim-type="Currently amended] 83. The polypeptide of claim 81, wherein said receptor is a receptor for BMP5, BMP6, BMP7, BMP8 or 60A.
[83" claim-type="Currently amended] 84. The polypeptide of claim 81, wherein said receptor is a receptor for GDF5, GDF6, GDF7, GDF1, GDF3, Vg1 or dosulin.
[84" claim-type="Currently amended] 84. The polypeptide of claim 81, wherein said receptor is a receptor for BMP3, GDF10 or nodal.
[85" claim-type="Currently amended] 83. The polypeptide of claim 81, wherein said receptor is a receptor for Inh bA or Inh bB.
[86" claim-type="Currently amended] 84. The polypeptide of claim 81, wherein said receptor is a receptor for TGFβ1, TGFβ5, TGFβ2 or TGFβ3.
[87" claim-type="Currently amended] 84. The polypeptide of claim 81, wherein said receptor is a receptor for MIS, GDF9, inhibin or GDNF.
[88" claim-type="Currently amended] A signaline polypeptide that specifically modulates signal transduction activity of a TGFβ receptor, characterized in that it is at least 50 percent similar to SEQ ID NO: 15 or SEQ ID NO: 17.
[89" claim-type="Currently amended] In a cell sample, the presence or absence of a genetic defect characterized by at least one of (i) excessive modification or mutation of a gene encoding a signal protein, and (ii) mis-expression of said gene in the cell sample Detecting, wherein the natural-type of the gene is responsible for the proliferation or differentiation of unwanted cells characterized by the encoding of signaline proteins characterized by the ability to modulate the signal transduction activity of the TGFβ receptor. Diagnostic assay to identify a cell or cells at risk of disease manifested by.
[90" claim-type="Currently amended] 90. The assay of claim 89 wherein detecting the defect comprises:
i. Providing a diagnostic label comprising a nucleotide sequence hybridizing to a sense or antisense of the gene, or a naturally occurring mutation thereof, or a nucleic acid comprising a 5 'or 3' flanking sequence naturally associated with the gene ,
ii. Binding the nucleic acid and the label of the cell sample, and
iii. By hybridization of said cellular nucleic acid with said label, deletion of one or more nucleotides from said gene, addition of one or more nucleotides to said gene, substitution of one or more nucleotides of said gene, all of said genes Or detecting the presence of at least one of some large chromosomal rearrangements, a large change in the level of mRNA transcripts of said gene, or a non-natural conjugation pattern of mRNA transcripts of said gene.
[91" claim-type="Currently amended] 93. The assay of claim 90, wherein the hybridization of the label further comprises polymerase chain reaction (PCR) of the label and cellular nucleic acid and detecting abnormality in the amplified product.
[92" claim-type="Currently amended] 91. The assay of claim 90, wherein the hybridization of the label further comprises ligation of the label and cellular nucleic acid (LCR) and detecting abnormality in the amplified product.
[93" claim-type="Currently amended] 91. The assay of claim 90, wherein said labeling hybridizes to a nucleic acid represented by one or more of SEQ ID NOs: 1-13 under stringent conditions.

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EP0868513A1|1998-10-07|

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DE868513T1|1999-12-09|

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引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
1995-12-20|Priority to US08/580,031

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1995-12-20|Priority to US8/580,031

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1996-12-20|Application filed by 온토제니, 인코오포레이티드, 조이스 브린톤, 프레지던트 앤드 펠로우즈 오브 하바드 칼리지

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1996-12-20|Priority to PCT/US1996/020745

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2000-11-06|Publication of KR20000064501A

优先权:

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申请号 | 申请日 | 专利标题

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US08/580,031|US6428977B1|1995-12-20|1995-12-20|Signalin family of TGFβ signal transduction proteins, and uses related thereto|

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US8/580,031|1995-12-20|

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PCT/US1996/020745|WO1997022697A1|1995-12-20|1996-12-20|TGFβ SIGNAL TRANSDUCTION PROTEINS, GENES, AND USES RELATED THERETO|