Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 25 or more amino acid residues in defined sequence
Reexamination Certificate
2000-03-07
2001-05-01
McKelvey, Terry (Department: 1636)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
25 or more amino acid residues in defined sequence
C530S325000, C530S326000, C530S327000, C530S350000
Reexamination Certificate
active
06225441
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The invention is related to the area of developmental and cancer genetics. In particular it is related to the field of transcriptional regulation.
BACKGROUND OF THE INVENTION
Substantial progress in understanding the responses to tumor-derived growth factor-&bgr; (TGF-&bgr;) and related ligands has been made in the last five years (Derynck and Fang, 1997; Hoodless and Wrana, 1998; Kretzschmar and Massague, 1998). The receptors for these ligands have been cloned and shown to be serine/threonine kinases which are activated by binding to ligand. The major substrates for these kinases, besides the receptors themselves, appear to be Smad proteins. The founding member of the Smad family is the product of the Drosophila gene Mad, identified by its requirement in signaling by the TGF-&bgr; family member Dpp (Sekelsky et al., 1995). Nine homologs of Mad have since been identified in vertebrate cells and shown to transduce or inhibit signals from specific TGF-&bgr; like ligands (Heldin et al., 1997; Derynck and Fang, 1997; Hoodless and Wrana, 1998; Kretzschmar and Massague, 1998).
The phosphorylation of Smad 1, Smad2, and Smad3 stimulates their interaction with Smad4 and the transport of the resulting heteromeric complex to the nucleus (Kretzschmar et al., 1997; Lagna et al., 1996; Liu, 1997; Macias-Silva et al., 1996; Nakao et al., 1997; Nakao et al., 1997; Souchelnytskyi et al., 1997). Once in the nucleus, the Smad complex transcriptionally activates specific target genes through activation domains present at the carboxyl termini of these proteins (Liu et al., 1996). Two ways in which Smad activation could lead to transcriptional activation have been identified. First, it has been shown that human Smad3 and Smad4, but not Smad2, can bind to specific DNA sequences and activate transcription of adjacent reporters (Zawel et al., 1998). A similar sequence-specific activity is present in Drosophila Mad (Kim et al., 1997). Second, Smad2 has been shown to bind to the Xenopus forkhead activin signal transducer protein FAST-1 (xFAST-1) and to participate in a complex exhibiting sequence specific binding activity attributable to the xFAST-1 component (Chen et al., 1996; Chen et al., 1997; Liu, 1997). Although Smad4 does not directly bind to xFAST-1, Smad4 is recruited to the xFAST-1/Smad2 complex by Smad2 (Chen et al., 1997; Liu, 1997).
TGF-&bgr;-like responses are remarkably widespread in eukaryotes, and are important not only in development but also in cancer (Fynan and Reiss, 1993; Hartsough and Mulder, 1997). Further progress in understanding the varied developmental and oncogenic ramifications of these pathways in mammalian cells depends on knowledge of the relevant mammalian genes. Thus, there is a need in the art for the identification, isolation, purification, and analysis of mammalian and human genes which mediate physiological and pathological responses to TGF-&bgr; and related ligands.
SUMMARY OF THE INVENTION
It is an object of the invention to provide reagents and methods for altering TGF-&bgr; activity. These and other objects of the invention are provided by one or more of the embodiments described below.
One embodiment of the invention provides an isolated and purified hFAST-1 protein comprising the amino acid sequence shown in SEQ ID NO:2 and naturally occurring biologically active variants thereof.
Another embodiment of the invention provides a fusion protein which comprises a first protein segment and a second protein segment fused to each other by means of a peptide bond. The first protein segment consists of at least thirteen contiguous amino acids selected from the amino acid sequence shown in SEQ ID NO:2.
Still another embodiment of the invention provides an isolated and purified polypeptide which consists of at least thirteen contiguous amino acids of hFAST-1 as shown in SEQ ID NO:2.
Even another embodiment of the invention provides a preparation of antibodies which specifically bind to an hFAST-1 protein as shown in SEQ ID NO:2.
Yet another embodiment of the invention provides a subgenomic polynucleotide which encodes an hFAST-1 protein as shown in SEQ ID NO:2.
Still another embodiment of the invention provides a vector comprising a subgenomic polynucleotide which encodes an hFAST-1 protein as shown in SEQ ID NO:2.
Another embodiment of the invention provides a vector comprising a subgenomic polynucleotide which encodes an hFAST-1 protein as shown in SEQ ID NO:2 and which is intron-free.
Yet another embodiment of the invention provides a vector comprising a subgenomic polynucleotide which comprises the sequence shown in SEQ ID NO:1.
Even another embodiment of the invention provides a recombinant host cell which comprises a polynucleotide. The polynucleotide encodes an hFAST-1 protein as shown in SEQ ID NO:2.
Still another embodiment of the invention provides a recombinant host cell which comprises a polynucleotide. The polynucleotide encodes an hFAST-1 protein as shown in SEQ ID NO:2 and which is intron-free.
Yet another embodiment of the invention provides a recombinant host cell which comprises a polynucleotide. The polynucleotide comprises the sequence shown in SEQ ID NO:1.
A further embodiment of the invention provides a recombinant DNA construct for expressing hFAST-1 antisense nucleic acids. The recombinant DNA construct comprises a promoter and a coding sequence for hFAST-1. The coding sequence consists of at least 12 contiguous base pairs selected from SEQ ID NO:1. The coding sequence is in an inverted orientation with respect to the promoter. Upon transcription from the promoter an RNA is produced which is complementary to native mRNA encoding hFAST-1.
Another embodiment of the invention provides a method of screening test compounds for those which inhibit the action of TGF-&bgr;. A test compound is contacted with a first protein and a second protein. The first protein is all or a portion of a Smad2 protein or a naturally occurring biologically active variant thereof. The portion of the Smad2 protein is capable of binding to hFAST-1. The second protein is all or a portion of hFAST-1 or a naturally occurring biologically active variant thereof. The portion of hFAST-1 is capable of binding to the portion of the Smad2 protein. An amount selected from the group consisting of (a) the first protein bound to the second protein, (b) the second protein bound to the first protein, (c) the first protein which is not bound to the second protein, and (d) the second protein which is not bound to the first protein is determined. A test compound which decreases the amount of (a) or (b) or increases the amount of (c) or (d) is a candidate compound for inhibiting the action of TGF-&bgr;.
Even another embodiment of the invention provides a method of screening test compounds for the ability to decrease or augment TGF-&bgr; activity. A cell is contacted with a test compound. The cell comprises a first fusion protein, a second fusion protein, a reporter gene, and hSmad4 protein. The first fusion protein comprises (1) a DNA binding domain or a transcriptional activating domain and (2) all or a portion of an hFAST-1 protein. The portion of hFAST-1 consists of a contiguous sequence of amino acids selected from the amino acid sequence shown in SEQ ID NO:2. The portion of hFAST-1 is capable of binding to Smad2 protein. The second fusion protein comprises (1) a DNA binding domain or a transcriptional activating domain and (2) all or a portion of Smad2 protein, or a naturally occurring biologically active variant thereof. The portion of Smad2 is capable of binding to hFAST-1 protein. When the first fusion protein comprises a DNA binding domain, the second fusion protein comprises a transcriptional activating domain. When the first fusion protein comprises a transcriptional activating domain, the second fusion protein comprises a DNA binding domain. The interaction of the portion of the hFAST-1 protein with the portion of Smad2 protein reconstitutes a sequence-specific transcriptional activating factor. The reporter gene comprises a DNA sequence to which th
Kinzler Kenneth W.
Vogelstein Bert
Zawel Leigh
Zhou Shibin
Banner & Witcoff
McKelvey Terry
The Johns Hopkins University
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