The present invention relates to three novel cancer related genes, Nmi, Omi and Rim. The Nmi gene encodes a myc gene product-binding protein. The Omi gene encodes a mammalian serine protease protein comprising an amino terminal regulatory domain, which includes a signal peptidase site, a triple repeat motif, an SH3 binding domain, and a consensus Mxi2/p38 kinase phosphorylation site, and a carboxy terminus serine protease catalytic domain. The retinoblastoma-interacting myosin-like gene (Rim gene) encodes a retinoblastoma binding protein comprising two leucine zipper structures, an RB family binding motif, an E1A/CtBP binding motif, and four nuclear localization sequences. Described herein are isolated and antisense nucleic acids molecules, recombinant expression vectors, host cells and non-human transgenic animals containing an insertion or a disruption of the Nmi, Omi and Rim genes. Diagnostic, screening and therapeutic methods utilizing the compositions of the invention are also provided.

The invention relates to the Nmi, Omi and Rim genes, their products, and uses thereof.
Nmi Gene
The present invention is based, in part, on the discovery of the gene which encodes a protein which interacts with myc proteins.
Accordingly, the invention features, a recombinant, isolated, or substantially pure, preparation of an Nmi polypeptide. An Nmi polypeptide can be a full length or a fragment.
In preferred embodiments, the Nmi polypeptide has one or more of the following properties:
it is approximately 307 amino acids in length;
it has at least one activity of naturally occurring Nmi, e.g., it can bind to a Nmi ligand, e.g., a myc gene product, e.g., N-myc or C-myc;
it has a carboxy terminus which is has at least 15, more preferably 20, or 25% sequence identity with residues 79-264 of IFP 35 of SEQ ID NO:3 or at least 50, 60, 70, 80, 90, 95, 99 or 100% sequence identity with residues 102-307 of SEQ ID NO:1 on SEQ ID NO:16;
it has an amino terminus which is has at least 15, more preferably at least 20 or 22% sequence identity with the coiled coil heptad repeat of residues 104-188 of the C. elegans protein CEF59 of SEQ ID NO:2 or at least 50 60 70, 80, 90, 95, 99 or 100% sequence identity with residues 2-86 of SEQ ID NO:1 on SEQ ID NO:16;
it has at least 60, and more preferably at least 70, 80, 90, 95, 99, or 100% sequence identity with the protein encoded in SEQ ID NO:1 on SEQ ID NO:16.
In preferred embodiments, the polypeptide has biological activity, e.g., the polypeptide is either, an agonist or an antagonist, of a biological activity of a naturally occurring Nmi.
In preferred embodiments, the polypeptide is a vertebrate, e.g., a mammalian, e.g. a primate, e.g., a human, Nmi polypeptide.
In preferred embodiments, the Nmi polypeptide includes a domain, e.g., an amino terminal domain, which has at least 50, 60, 70, 80, 90, 99 or 100% sequence identity with residues 2-86 of the Nmi sequence of SEQ ID NO:1 on SEQ ID NO:16, or 50, 60, 70, 80, 90, 95, 99 or 100% sequence identity with residues 104-188 of the CEF59 sequence in SEQ ID NO:2.
In preferred embodiments, the polypeptide includes one or more of the following: a region which has at least 60, more preferably at least 70, 80, 90, or 100% sequence identity with residues 2-14 of SEQ ID NO:1 on SEQ ID NO:16 (or residues 104-116 of SEQ ID NO:2); a region which has at least 60, more preferably at least 70, 80, 90, or 100% sequence identity with residues 27-44 of SEQ ID NO:1 on SEQ ID NO:16 (or residues 129-146 of SEQ ID NO:2); a region which has at least 60, more preferably at least 70, 80, 90, or 100% sequence identity with residues 51-59 of SEQ ID NO:1 on SEQ ID NO:16 (or residues 153-161 of SEQ ID NO:2); or a region which has at least 60, more preferably at least 70, 80, 90, or 100% sequence identity with residues 76-86 of SEQ ID NO:1 on SEQ ID NO:16 (or residues 178-188 of SEQ ID NO:2).
In preferred embodiments, the Nmi polypeptide includes a coiled coil region, preferably in the carboxy terminus or half of the polypeptide, which has at least 50, 60, 70, 80, 90, 95, 99 or 100% sequence identity with the residues 102-288 of the Nmi sequence of SEQ ID NO:1 on SEQ ID NO:16 or 50, 60, 70, 80, 90, 95, 99 or 100% sequence identity with residues 79-264 of the IFP35 sequence in SEQ ID NO:3, or a segment of one of these regions sufficient to mediate binding to a Nmi ligand.
In preferred embodiments, the polypeptide includes one or more of the following: a region which has at least 60, more preferably at least 70, 80, 90, or 100% sequence identity with residues 103-126 of SEQ ID NO:1 on SEQ ID NO:16 (or residues 79-102 of SEQ ID NO:3); a region which has at least 60, more preferably at least 70, 80, 90, or 100% sequence identity with residues 133-142 of SEQ ID NO:1 on SEQ ID NO:16 (or residues 109-118 of SEQ ID NO:3); a region which has at least 60, more preferably at least 70, 80, 90, or 100% sequence identity with residues 149-168 of SEQ ID NO:1on SEQ ID NO:16 (or residues 125-144 of SEQ ID NO:3); a region which has at least 60, more preferably at least 70, 80, 90, or 100% sequence identity with residues 201-218 of SEQ ID NO:1 on SEQ ID NO:16 (or residues 177-194 of SEQ ID NO:3); a region which has at least 60, more, preferably at least 70, 80, 90, or 100% sequence identity with residues 233-250 of SEQ ID NO:1on SEQ ID NO:16 (or residues 209-226 of SEQ ID NO:3); a region which has at least 60, more preferably at least 70, 80, 90, or 100% sequence identity with residues 255-259 of SEQ ID NO:1on SEQ ID NO:16 (or residues 231-235 of SEQ ID NO:3); a region which has at least 60, more preferably at least 70, 80, 90, or 100% sequence identity with residues 270-275 of SEQ ID NO:1 on SEQ ID NO:16 (or residues 246-251 of SEQ ID NO:3); or a region which has at least 60, more preferably at least 70, 80, 90, or 100 % sequence identity with residues 281-288 of SEQ ID NO:1on SEQ ID NO:16 (or residues 257-264 of SEQ ID NO:3).
In a preferred embodiment, the Nmi polypeptide differs in amino acid sequence at up to 1, 2, 3, 5, or 10 residues, from the sequence in SEQ ID NO:1 on SEQ ID NO:16. In other preferred embodiments, the Nmi polypeptide differs in amino acid sequence at up to 1, 2, 3, 5, or 10% of the residues from a sequence in SEQ ID NO:1 on SEQ ID NO:16. In preferred embodiments, the differences are such that the Nmi polypeptide exhibits an Nmi biological activity. In other preferred embodiments the differences are such that the Nmi polypeptide does not have Nmi biological activity. In preferred embodiments,one or more, or all of the differences are conservative amino acid changes. In other preferred embodiments one or more, or all of the differences are other than conservative amino acid changes.
In preferred embodiments, the Nmi polypeptide includes a Nmi sequence described herein as well as other N-terminal and/or C-terminal amino acid sequence.
In yet other preferred embodiments, the Nmi polypeptide is a recombinant fusion protein having a first Nmi portion and a second polypeptide portion, e.g., a second polypeptide portion having an amino acid sequence unrelated to Nmi. The second polypeptide portion can be, e.g., any of glutathione-S-transferase, a DNA binding domain, or a polymerase activating domain. In preferred embodiment the fusion protein can be used in a two-hybrid assay.
In a preferred embodiment, there is at least 70, 80, 90, 95, 99, or 100% sequence identity between the Nmi polypeptide and the portions of SEQ ID NO:1 on SEQ ID NO:16 which share 100 sequence identity with the aligned SEQ ID NOS 2 and 3 of FIG. 2.
In another aspect, the invention features an Nmi polypeptide which is a fragment of a full length Nmi polypeptide, e.g., a fragment of a naturally occurring Nmi polypeptide, e.g., the polypeptide encoded in SEQ ID NO:1 on SEQ ID NO:16.
In preferred embodiments: the fragment is at least 5, 10, 20, 50, 100, or 150 amino acids in length; the fragment is equal to or less than 200, 150, 100, 50 amino acid residues in length; the fragment has a biological activity of a naturally occurring Nmi; the fragment is either, an agonist or an antagonist, of a biological activity of a naturally occurring Nmi; the fragment can inhibit, e.g., competitively or non competitively inhibit, the binding of Nmi to an Nmi-ligand, e.g., a myc protein.
In preferred embodiments, the fragment it has at least 60, and more preferably at least 70, 80, 90, 95, 99, or, 100% sequence identity with the corresponding amino acid sequence of SEQ ID NO:1 on SEQ ID NO:16.
In preferred embodiments, the fragment is a fragment of a vertebrate, e.g., a mammalian, e.g. a primate, e.g., a human, Nmi polypeptide.
In preferred embodiments, the Nmi fragment includes a domain, e.g., an amino terminal domain, which has at least 50, 60, 70, 80, 90, 95, 99 or 100% sequence identity with residues 2-86 of the Nmi sequence of SEQ ID NO:1 on SEQ ID NO:16, or 50, 60, 70, 80, 90, 95, 99 or 100% sequence identity with residues 104-188 of the CEF59 sequence in SEQ ID NO:2.
In preferred embodiments, the Nmi fragment includes one, two, three, or more amino terminal homology domains.The amino terminal homology domains are as follows: residues 2-14 of SEQ ID NO:1 on SEQ ID NO:16 (or residues 104-116 of SEQ ID NO:2); residues 27-44 of SEQ ID NO:1 on SEQ ID NO:16 (or residues 129-146 of SEQ ID NO:2); residues 51-59 of SEQ ID NO:1 on SEQ ID NO:16 (or residues 153-161 of SEQ ID NO:2); residues 76-86 of SEQ ID NO:1 on SEQ ID NO:16 (or residues 178-188 of SEQ ID NO:2). In preferred embodiments, the fragment includes a region which has at least 50, 60, 70, 80, 90, 95, 99 or 100% sequence identity with an amino terminal homology domain. In preferred embodiments, the fragment (which term includes terminal and internal deletions) lacks at least one amino terminal homology domain found in naturally occurring Nmi.
In preferred embodiments, the Nmi polypeptide includes a coiled coil region, preferably in the carboxy terminus or half of the polypeptide, which has at least 50, 60, 70, 80, 90, 95, 99 or 100% sequence identity with the residues 102-288 of the Nmi sequence of SEQ ID NO:1 or 50, 60, 70, 80, 90, 95, 99 or 100% sequence identity with residues 79-264 of the IFP35 sequence in SEQ ID NO:3, or a segment of one of these regions sufficient to mediate binding to a Nmi ligand.
In preferred embodiments, the Nmi fragment includes one, two, three, or more carboxy terminal homology domains. The carboxy terminal homology domains are as follows: residues 103-126 of SEQ ID NO:1 on SEQ ID NO:16 (or residues 79-102 of SEQ ID NO:3); residues 133-142 of SEQ ID NO:1 on SEQ ID NO:16 (or residues 109-118 of SEQ ID NO:3); residues 149-168 of SEQ ID NO:1 on SEQ ID NO:1 or residues 125-144 of SEQ ID NO:3); residues 201-218 of SEQ ID NO:1 on SEQ ID NO:16 (or residues 177-194 of SEQ ID NO:3); residues 233-250 of SEQ ID NO:1 on SEQ ID NO:1 (or residues 209-226 of SEQ ID NO:3); residues 255-259 of SEQ ID NO:1 (or residues 231-235 of SEQ ID NO:3); residues 270-275 of SEQ ID NO:1. on SEQ ID NO:16 (or residues 246-251 of SEQ ID NO:3); residues 281-288 of SEQ ID NO:1 on SEQ ID NO:16 (or residues 257-264 of SEQ ID NO:3).
In preferred embodiments, the fragment includes a region which has at least 50, 60, 70, 80, 90, 95, 99 or 100% sequence identity with a carboxy terminal homology domain. In preferred embodiments, the fragment (which term includes terminal and internal deletions) lacks at least one carboxy terminal homology domain found in naturally occurring Nmi.
In preferred embodiments, the fragment can inhibit an interaction, e.g., binding, between Nmi and an Nmi-ligand. In preferred embodiments, the fragment does not inhibit an interaction, e.g., binding, between Nmi and an Nmi-ligand.
In a preferred embodiment, the fragment differs in amino acid sequence at up to 1, 2, 3, 5, or 10 residues, from the corresponding residues in SEQ ID NO:1 on SEQ ID NO:16. In other preferred embodiments, the fragment differs in amino acid sequence at up to 1, 2, 3, 5, or 10% of the residues from the corresponding residues in SEQ ID NO:1 on SEQ ID NO:16. In preferred embodiments, the differences are such that the fragment exhibits an Nmi biological activity. In other preferred embodiments the differences are such that the fragment does not have Nmi biological activity. In preferred embodiments,one or more, or all of the differences are conservative amino acid changes. In other preferred embodiments one or more, or all of the differences are other than conservative amino acid changes.
In preferred embodiments, the fragment includes an Nmi sequence described herein as well as other N-terminal and/or C-terminal amino acid sequence.
Polypeptides of the invention include those which arise as a result of the existence of multiple genes, alternative transcription events, alternative RNA splicing events, and alternative translational and postranslational events. The polypeptide can be expressed in systems, e.g., cultured cells, which result in substantially the same postranslational modifications present when expressed Nmi is expressed in a native cell, or in systems which result in the omission of postranslational modifications present when expressed in a native cell.
The invention includes an immunogen which includes a Nmi polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for the Nmi polypeptide, e.g., a humoral response, an antibody response, or a cellular response. In preferred embodiments, the immunogen comprising an antigenic determinant, e.g., a unique determinant, from a protein represented by SEQ ID NO:1.
The present invention also includes an antibody preparation specifically reactive with an epitope of the Nmi immunogen or generally of a Nmi polypeptide, preferably an epitope which consists all or in part of residues from the amino terminal or coiled-coil domain, an epitope, which when bound to an antibody, results in the modulation of a biological activity, or carboxy or amino terminal homology domain described herein.
In another aspect, the invention features, a composition which includes a Nmi polypeptide (or a nucleic acid which encodes it) and one or more additional components, e.g., a carrier, diluent, adjuvent, or solvent. The additional component can be one which renders the composition useful for in vitro, in vivo, pharmaceutical, or veterinary use.
In another aspect, the invention provides a substantially pure nucleic acid having or comprising a nucleotide sequence which encodes a polypeptide, the amino acid sequence of which includes, or is, the sequence of a Nmi polypeptide.
In preferred embodiments,the encoded Nmi polypeptide has one or more of the following properties:
it is approximately 307 amino acids in length;
it has at least one activity of naturally occurring Nmi, e.g., it can bind to a Nmi ligand, e.g., a myc gene product, e.g., N-myc or C-myc;
it has a carboxy terminus which is has at least 15, more preferably 20, or 25% sequence identity with residues 79-264 of IFP 35 of SEQ ID NO:3 or at least 50, 60, 70, 80, 90, 95, 99 or 100% sequence identity with residues 102-307 of SEQ ID NO:1 on SEQ ID NO:16.
it has an amino terminus which is has at least 15, more preferably at least 20 or 22% sequence identity with the coiled coil heptad repeat of residues 104-188 of the c. elegans protein CEF59 of SEQ ID NO:2 or at least 50, 60, 70, 80, 90, 95, 99 or 100% sequence identity with residues 2-86 of SEQ ID NO:1 on SEQ ID NO:16;
it has at least 60, and more preferably at least 70, 80, 90, 95, 99, or 100% sequence identity with the protein encoded in SEQ ID NO:1 on SEQ ID NO:16.
In preferred embodiments, the encoded polypeptide has biological activity, e.g., the polypeptide is either, an agonist or an antagonist, of a biological activity of a naturally occurring Nmi.
In preferred embodiments, the encoded polypeptide is a vertebrate, e.g., a mammalian, e.g. a primate, e.g., a human, Nmi polypeptide.
In preferred embodiments, the encoded Nmi polypeptide includes a domain, e.g., an amino terminal domain, which has at least 50, 60, 70, 80, 95, 99 or 100% sequence identity with residues 2-86 of the Nmi sequence of SEQ ID NO:1 on SEQ ID NO:16, or 50, 60, 70, 80, 90, 95, 99 or 100% sequence identity with residues 104-188 of the CEF59 sequence in SEQ ID NO:2.
In preferred embodiments, the encoded polypeptide includes one or more of the following: a region which has at least 60, more preferably at least 70, 80, 90, or 100% sequence identity with residues 2-14 of SEQ ID NO:1 on SEQ ID NO:16 (or residues 104-116 of SEQ ID NO:2); a region which has at least 60, more preferably at least 70, 80, 90, or 100% sequence identity with residues 27-44 or SEQ ID NO:1 on SEQ ID NO:16 (or residues 129-146 of SEQ ID NO:2); a region which has at least 60, more preferably at least 70, 80, 90, or 100% sequence identity with residues 51-59 of SEQ ID NO:1 on SEQ ID NO:16 (or residues 153-161 of SEQ ID NO:2); or a region which has at least 60, more preferably at least 70, 80, 90, or 100% sequence identity with residues 76-86 of SEQ ID NO:1 on SEQ ID NO:16 (or residues 178-188 of SEQ ID NO:2).
In preferred embodiments, the encoded Nmi polypeptide includes a coiled coil region, preferably in the carboxy terminus or half of the polypeptide, which has at least 50, 60, 70, 80, 90, 95, 99 or 00%;,sequence identity with the residues 102-288 of the Nmi sequence of SEQ ID NO:1 on SEQ ID NO:16 or 50, 60, 70, 80, 90, 95, 99 or 100% sequence identity with residues 79-264 of the IFP35 sequence in SEQ ID NO:3, or a segment of one of these regions sufficient to mediate binding to a Nmi ligand.
In preferred embodiments, the encoded polypeptide includes one or more of the following: a region which has at least 60, more preferably at least 70, 80, 90, or 100% sequence identity with residues 103-126 of SEQ ID NO:1 on SEQ ID NO:16 (or residues 79-102 of SEQ ID NO:3); a region which has at least 60, more preferably at least 70, 80, 90, or 100% sequence identity with residues 133-142of: SEQ ID NO:1 on SEQ ID NO:16 (or residues 109-118 of SEQ ID NO:3); a region which has at least 60, more preferably at least 70, 80, 90, or 100% sequence identity with residues 149-168 of SEQ ID NO:1 on SEQ ID NO:16 (or residues 125-144 of SEQ ID NO:3); a region which has at least 60, more preferably at least 70, 80, 90, or 100% sequence identity with residues 201-218 of SEQ ID NO:1 on SEQ ID NO:16 (or residues 177-194 of SEQ ID NO:3); a region which has at least 60, more preferably at least 70, 80, 90, or 100% sequence identity with residues 233-250 of SEQ ID NO:1 on SEQ ID NO:16 (or residues 209-226 of SEQ ID NO:3); a region which has at least 60, more preferably at least 70, 80, 90, or 100% sequence identity with residues 255-259 of SEQ ID NO:1 on SEQ ID NO:16 (or residues 231-235 of SEQ ID NO:3); a region which has at least 60, more preferably at least 70, 80, 90, or 100% sequence identity with residues 270-275 of SEQ ID NO:1 on SEQ ID NO:16 (or residues 246-251 of SEQ ID NO:3); or a region which has at least 60, more preferably at least 70, 80, 90, or 100% sequence identity with residues 281-288 of SEQ ID NO:1 on SEQ ID NO:16 (or residues 257-264 of SEQ ID NO:3).
In a preferred embodiment, the encoded Nmi polypeptide differs in amino acid sequence at up to 1, 2, 3, 5, or 10 residues, from the sequence in SEQ ID NO:1 on SEQ ID NO:16. In other preferred embodiments, the Nmi polypeptide differs in amino acid sequence at up to 1, 2, 3, 5, or 10% of the residues from a sequence in SEQ ID NO:1 on SEQ ID NO:16. In preferred embodiments, the differences are such that the Nmi polypeptide exhibits an Nmi biological activity. In other preferred embodiments the differences are such that the Nmi polypeptide does not have Nmi biological activity. In preferred embodiments, one or more, or all of the differences are conservative amino acid changes. In other preferred embodiments, one or more, or all of the differences are other than conservative amino acid changes.
In preferred embodiments, the encoded Nmi polypeptide includes a Nmi sequence described herein as well as other N-terminal and/or C-terminal amino acid sequence.
In yet other preferred embodiments, the encoded Nmi polypeptide is a recombinant fusion protein having a first Nmi portion and a second polypeptide portion, e.g., a second polypeptide portion having an amino acid sequence unrelated to Nmi. The second polypeptide portion can be, e.g., any of glutathione-S-transferase, a DNA binding domain, or a polymerase activating domain. In preferred embodiment the fusion protein can be used in a two-hybrid assay.
In a preferred embodiment, there is at least 70, 80, 90, 95, 99 or 100% sequence identity between the encoded Nmi polypeptide and the portions of SEQ ID NO:1on SEQ ID NO:16 which share 100 sequence identity with the aligned SEQ ID NOS 2 and 3 of FIG. 2.
The encoded polypeptide can be a fragment of a full length Nmi polypeptide, e.g., a fragment of a naturally occurring Nmi polypeptide, e.g., the polypeptide encoded in SEQ ID NO:1.
In preferred embodiments: the encoded fragment is at least 5, 10, 20, 50, 100, or 150 amino acids in length; the encoded fragment is equal to or less than 200, 150, 100, 50 amino acid residues in length; the encoded fragment has a biological activity of a naturally occurring Nmi; the encoded fragment is either, an agonist or an antagonist, of a biological activity of a naturally occurring Nmi; the encoded fragment can inhibit, e.g., competitively or non competitively inhibit, the binding of Nmi to an Nmi-ligand, e.g., a myc protein.
In preferred embodiments, the encoded fragment it has at least 60, and more preferably at least 70, 80, 90, 95, 99, or 100% sequence identity with the corresponding amino acid sequence of SEQ ID NO:1 on SEQ ID NO:16.
In preferred embodiments, the encoded fragment is a fragment of a vertebrate, e.g., a mammalian, e.g. a primate, e.g., a human, Nmi polypeptide.
In preferred embodiments, the encoded Nmi fragment includes a domain, e.g., an amino terminal domain, which has at least 50, 60, 70, 80, 95, 99 or 100% sequence identity with residues 2-86 of the Nmi sequence of SEQ ID NO:1 on SEQ ID NO:16, or 50, 60, 70, 80, 90, 95, 99 or 100% sequence identity with residues 104-188 of the CEF59 sequence in SEQ ID NO:2.
In preferred embodiments, the encoded Nmi fragment includes one, two, three, or more amino terminal homology domains. The amino terminal homology domains are as follows: residues 2-1 of SEQ ID NO:1 on SEQ ID NO:16 (or residues 104-116 of SEQ ID NO:2); residues 27-44 of SEQ ID NO:1 on SEQ ID NO:16 (or residues 129-146 of SEQ ID NO:2); residues 51-59 of SEQ ID NO:1 on SEQ ID NO:16 (or residues 153-161 of SEQ ID NO:2); residues 76-86 of SEQ ID NO:1 on SEQ ID NO:16 (or residues 178-188 of SEQ ID NO:2). In preferred embodiments, the encoded fragment includes a region which has at least 50, 60, 70, 80, 90, 95, 99 or 100% sequence identity with an amino terminal homology domain. In preferred embodiments, the encoded fragment (which term includes terminal and internal deletions) lacks at least one amino terminal homology domain found in naturally occurring Nmi.
In preferred embodiments, the encoded Nmi polypeptide includes a coiled coil region, preferably in the carboxy terminus or half of the polypeptide, which has at least 50, 60, 70, 80, 90, 95, 99 or 100% sequence identity with the residues 102-288 of the Nmi sequence of SEQ ID NO:1 on SEQ ID NO:16 or 50, 60, 70, 80, 90, 95, 99 or 100% sequence identity with residues 79-264 of the IFP35 sequence in SEQ ID NO:3, or a segment of one of these regions sufficient to mediate binding to a Nmi ligand.
In preferred embodiments, the encoded Nmi fragment includes one, two, three, or more carboxy terminal homology domains. The carboxy terminal homology domains are as follows: residues 103-126 of SEQ ID NO:1 on SEQ ID NO:16 (or residues 79-102 of SEQ ID NO:3); residues 133-142 of SEQ ID NO:1 on SEQ ID NO:16 (or residues 109-118 of SEQ ID NO:3); residues 149-168of SEQ ID NO:1 on SEQ ID NO:16 (or residues 125-144 of SEQ ID NO:3); residues 201-218 of SEQ ID NO:1 on SEQ ID NO:16 (or residues 177-194 of SEQ ID NO:3); residues 233-250 of SEQ ID NO:1on SEQ ID NO:16 (or residues 209-226 of SEQ ID NO:3); residues 255-259 of SEQ ID NO:1 (or residues 231-235 of SEQ ID NO:3); residues 270-275 of SEQ ID NO:1 on SEQ ID NO:16 (or residues 246-251 of SEQ ID NO:3); residues 281-288 of SEQ ID NO:1 on SEQ ID NO:16 (or residues 257-264 of SEQ ID NO:3).
In preferred embodiments, the encoded fragment includes a region which has at least 50, 60, 70, 80, 90, 95, 99 or 100% sequence identity with a carboxy terminal homology domain. In preferred embodiments, the encoded fragment (which term includes terminal and internal deletions) lacks at least one carboxy terminal homology domain found in naturally occurring Nmi.
In preferred embodiments, the encoded fragment can inhibit an interaction, e.g., binding, between Nmi and an Nmi-ligand. In preferred embodiments, the encoded fragment does not inhibit an interaction, e.g., binding, between Nmi and an Nmi-ligand.
In a preferred embodiment, the encoded fragment differs in amino acid sequence at up to 1, 2, 3, 5, or 10 residues, from the corresponding residues in SEQ ID NO:1 on SEQ ID NO:16. In other preferred embodiments, the encoded fragment differs in amino acid sequence at up to 1, 2, 3, 5, or 10% of the residues from the corresponding residues in SEQ ID NO:1 on SEQ ID NO:16. In preferred embodiments, the differences are such that the encoded fragment exhibits an Nmi biological activity. In other preferred embodiments, the differences are such that the encoded fragment does not have Nmi biological activity. In preferred embodiments, one or more, or all of the differences are conservative amino acid changes. In other preferred embodiments, one or more, or all of the differences are other than conservative amino acid changes.
In preferred embodiments, the encoded fragment includes an Nmi sequence described herein as well as other N-terminal and/or C-terminal amino acid sequence.
In preferred embodiments, the subject Nmi nucleic acid includes a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the Nmi gene sequence, e.g., to render the Nmi gene sequence suitable for use as an expression vector.
In another aspect, the invention features a purified nucleic acid which hybridizes under stringent conditions to the sense or antisense nucleic acid sequence of SEQ ID NO:1. In preferred embodiments, the purified nucleic acid is at least 10, more preferably 20, 30, 40, 50 or 100 nucleotides in length. In preferred embodiments,the purified nucleic acid: is useful as a probe or primer; has at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% homology with a sequence from SEQ ID NO:1; is at least 10, 20, 30, 50, 100, or 200 nucleotides in length; further includes a label group attached thereto. The label group can be, e.g., a radioisotope, a fluorescent compound, an enzyme, and/or an enzyme co-factor.
The invention provides a nucleic acid, e.g., RNA or DNA, encoding a polypeptide of the invention. This includes double stranded nucleic acids as well as coding and antisense single strands.
In another aspect, the invention includes: a vector including a nucleic acid which encodes a Nmi polypeptide; a host cell transfected with the vector; and a method of producing a recombinant Nmi polypeptide; including culturing the cell, e.g., in a cell culture medium, and isolating the Nmi polypeptide, e.g. from the cell or from the cell culture medium.
In another aspect, the invention features a cell or purified preparation of cells which include a Nmi transgene, or which otherwise misexpress a Nmi gene. The cell preparation can consist of human or non human cells, e.g., rodent cells, e.g., mouse or rat cells, rabbit cells, or pig cells. In preferred embodiments, the cell or cells include a Nmi transgene, e.g., a heterologous form of a Nmi gene, e.g., a gene derived from humans (in the case of a non-human cell). The Nmi transgene can be misexpressed, e.g., overexpressed or underexpressed. In other preferred embodiments, the cell or cells include a gene which misexpress an endogenous Nmi gene, e.g., a gene the expression of which is disrupted, e.g., a knockout. Such cells can serve as a model for studying disorders which are related to mutated or mis-expressed Nmi alleles or for use in drug screening.
In another aspect, the invention features a transgenic Nmi animal, e.g., a rodent, e.g., a mouse or a rat, a rabbit, or a pig. In preferred embodiments, the transgenic animal includes (and preferably express) a heterologous form of a Nmi gene, e.g., a gene derived from humans. In other preferred embodiments, the animal has an endogenous Nmi gene which is misexpressed, e.g., a knockout. Such a transgenic animal can serve as a model for studying disorders which are related to mutated or mis-expressed Nmi alleles or for use in drug screening.
For example, the invention includes a method of evaluating the effect of the expression or misexpression of a Nmi gene, on any of: a parameter related to cell growth or regulation of the cell cycle. The method includes: providing a transgenic animal having a Nmi transgene, or which otherwise misexpresses a Nmi gene; contacting the animal with an agent; and evaluating the effect of the transgene on a parameter related to cell growth or the regulation of the cell cycle (e.g., by comparing the value of the parameter for a transgenic animal with the value for a control, e.g., a wild type animal).
In another aspect, the invention provides, a method of determining if a subject mammal, e.g., a primate, e.g., a human, is at risk for a disorder related to a lesion in or the misexpression of a Nmi gene. Such disorders include, e.g., those characterized by abnormal or unwanted cell proliferation, e.g., cancer, e.g., leukemia, e.g., promyelocytic leukemia, chronic myelogenous leukemia, or lymphoblastic leukemia, lymphoma, e.g., Burkitts lymphoma, carcinoma, e.g., colorectal adenocarcinoma or lung carcinoma, colorectal cancer, melanoma, neuroblastoma, lung cancer, e.g., small cell lung cancer, or a disorder of the brain, e.g., mental retardation. The method includes detecting, in a tissue of the subject the mis-expression, or a mutation which results in misexpression, of a gene encoding a protein represented by SEQ ID NO:1 on SEQ ID NO:16. preferred embodiments: detecting the mutation or misexpression includes ascertaining the existence of at least one of: a mutation in the gene or in a region which controls expression of the gene; an alteration in the level of a messenger RNA transcript of the gene; the presence of a non-wild type splicing pattern of a messenger RNA transcript of the gene; or a non-wild type level of the protein.
In another aspect, the invention features, a method of determining if a subject mammal, e.g., a primate, e.g., a human, is at risk for a disorder, e.g., a disorder related to a lesion in, or the misexpression of, an Nmi gene. Such disorders include, e.g., those characterized by abnormal or unwanted cell proliferation, e g., cancer, e.g., leukemia, e.g., promyelocytic leukemia, chronic myelogenous leukemia, or lymphoblastic leukemia, lymphoma, e.g., Burkitts lymphoma, carcinoma, e.g., colorectal adenocarcinoma or lung carcinoma, colorectal cancer, melanoma, neuroblastoma, lung cancer, e.g., small cell lung cancer, or a disorder of the brain, e.g., mental retardation. The method includes detecting, in a tissue of the subject, the presence or absence of a mutation in a Nmi gene, or a homolog thereof. In preferred embodiments: detecting the mutation includes ascertaining the existence of at least one of: a deletion of one or more nucleotides from the gene; an insertion of one or more nucleotides into the gene, a point mutation, e.g., a substitution of one or more nucleotides of the gene, a gross chromosomal rearrangement of the gene, e.g., a translocation, inversion, or deletion.
For example, detecting the genetic lesion can include: (i) providing a probe/primer including an oligonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence from SEQ ID NO:1 or naturally occurring mutants thereof or 5xe2x80x2 or 3xe2x80x2 flanking sequences naturally associated with a Nmi gene; (ii) contacting the probe/primer with nucleic acid of the tissue; and detecting, e.g., by hybridization, e.g., in situ hybridization, of the probe/primer to the nucleic acid, the presence or absence of the genetic lesion.
In another aspect, the invention provides, a method of determining if a subject mammal, e.g., a primate, e.g., a human, is at risk for a disorder related to a Nmi gene. Such disorders include, e.g., those characterized by abnormal or unwanted cell proliferation, e.g., cancer, e.g., cancer, e.g., leukemia, e.g., promyelocytic leukemia, chronic myelogenous leukemia, or lymphoblastic leukemia, lymphoma, e.g., Burkitts lymphoma, carcinoma, e.g., colorectal adenocarcinoma or lung carcinoma, colorectal cancer, melanoma, neuroblastoma, lung cancer, e.g., small cell lung cancer, or a disorder of the brain, e.g., mental retardation. The method includes detecting, in a tissue of the subject, a non-wild type level of a Nmi RNA or polypeptide.
Diagnostic methods disclosed herein can be performed prenatally, on infants, children, or adults.
The invention includes methods of determining the stage of a cancer, or the degree of resistance to a chemotherapy agent. Advanced stage, or resistance to chemotherapy, is correlated with amplification or overexpression of Nmi.
Thus, in another aspect, the invention provides, a method of determining if a subject mammal, e.g., a primate, e.g., a human, at risk for a disorder related to a Nmi gene, is resistant to chemotherapy. Such disorders include, e.g., those characterized by abnormal or unwanted cell proliferation, e.g., cancer, e.g., cancer, e.g., leukemia, e.g., promyelocytic leukemia, chronic myelogenous leukemia, or lymphoblastic leukemia, lymphoma, e.g., Burkitts lymphoma, carcinoma, e.g., colorectal adenocarcinoma or lung carcinoma, colorectal cancer, melanoma, neuroblastoma, lung cancer, e.g., small cell lung cancer, or a disorder of the brain. The method includes detecting, in a tissue of the subject, amplification of the Nmi gene or an a non-wild type, e.g., elevated, level of a Nmi RNA or polypeptide. Amplification of the Nmi gene or elevated (e.g., as compared to normal or control tissue) levels of Nmi mRNA or polypeptide is indicative of resistance to chemotherapy. In preferred embodiments: the subject has been administered a chemotherapeutic agent prior to detection of Nmi gene, RNA, or polypeptide levels; the subject has been administered a chemotherapeutic agent prior to detection of Nmi gene, RNA, or polypeptide levels and is administered a second different treatment modality, e.g., a different chemotherapeutic agent, or a non-chemotherapeutic treatment modality, after the detection of Nmi gene, RNA, or polypeptide levels; the subject is administered a non-chemotherapeutic treatment modality, after the detection of Nmi gene, RNA, or polypeptide levels.
In another aspect, the invention features, a method of evaluating a compound for the ability to interact with an Nmi polypeptide. The interaction can be any interaction between the compound and Nmi, e.g., binding of the compound with an Nmi polypeptide, alteration of the three dimensional structure of the compound or the Nmi polypeptide, or covalent or non-covalent modification of the compound or the Nmi polypeptide. The method includes: contacting the compound with the Nmi polypeptide; and evaluating the ability of the compound and the Nmi polypeptide to interact. This method can be performed in vitro, e.g., in a cell free system, or in vivo, e.g., in a two-hybrid interaction trap assay. This method can be used to identify naturally occurring or synthetic molecules which interact with Nmi polypeptides, and is useful for evaluating compounds for the ability to inhibit or mimic an Nmi.
In preferred embodiments, the compound is: a ligand of an Nmi polypeptide; a myc polypeptide, e.g., an N-myc, C-myc, Max, or Mxil protein; a protein having a basic-leucine zipper region, e.g., a fos protein; a daughterless protein; a transcription factor, e.g., a zip transcription factor; a polypeptide which includes any of a bHLH-Zip region, a bHLH region, a zip region; a polypeptide which binds DNA or RNA, e.g., a polypeptide which binds DNA or RNA is a sequence specific manner; a polypeptide which does not include a zinc-finger region. In particularly preferred embodiments the compound is a fragment or an analog of one of the above recited polypeptides.
The method is particularly useful for identifying compounds, e.g., fragments or analogs of Nmi ligands, which bind Nmi and which, e.g., are agonists or antagonists of a Nmi ligand.
In another aspect, the invention features, a method of evaluating a fragment or analog of the Nmi protein for a biological activity or for the ability to interact with a compound. The interaction can be any interaction between the compound and the fragment or analog, e.g., binding of the compound with the Nmi fragment or analog, alteration of the three dimensional structure of the compound or the Nmi fragment or analog, or covalent or non-covalent modification of the compound or the Nmi fragment or analog. The method includes: contacting the fragment or analog of the Nmi protein with the compound; and evaluating the ability of the compound and the Nmi fragment or analog to interact. This method can be performed in vitro, e.g., in a cell free system, or in vivo, e.g., in a two-hybrid interaction trap assay.
In preferred embodiments, the compound is: a ligand of an Nmi polypeptide; a myc polypeptide, e.g., an N-myc, C-myc, Max, Mxil, or protein; ; a protein having a basic-leucine zipper region, e.g., a fos protein; a daughterless protein; a transcription factor, e.g., a zip transcription factor; a polypeptide which includes any of a bHLH-Zip region, a bHLH region, a zip region, a polypeptide which binds DNA or RNA, e.g., a polypeptide which binds DNA or RNA is a sequence specific manner; a polypeptide which does not include a zinc-finger region. In particularly preferred embodiments the compound is a fragment or an analog, e.g., an Nmi-interacting fragment or analog, of one of the above recited polypeptides.
The method is particularly useful for identifying fragments or analogs of Nmi which have biological activity or which can bind a candidate compound. This method is useful for identifying fragments or analogs of Nmi which are agonists or antagonists of Nmi.
In yet another aspect, the invention features a method for evaluating a first compound, for the ability to modulate an interaction of an Nmi polypeptide with a second compound, e.g., a myc protein. The first compound can be, e.g., an inhibitor candidate, e.g., a fragment or analog of an Nmi-ligand e.g., a fragment of a myc protein. The method includes the steps of:
(i) providing a reaction mixture which includes the first compound (e.g., an inhibitor candidate), an Nmi polypeptide, and the second compound (e.g., a myc polypeptide), preferably under conditions wherein in the absence of the first compound, the Nmi polypeptide and the second compound, interact; and
(ii) detecting an interaction between the Nmi polypeptide and the second compound, e.g., detecting the formation (or dissolution) of a complex which includes the second compound, and the Nmi polypeptide. A change, e.g., a decrease or increase in an interaction, e.g., in the formation of the complex in the presence of the first compound (relative to what is seen in the absence of the first compound), being indicative of a modulation.
The interaction can be any interaction between the second compound and Nmi, e.g., binding of the second compound with an Nmi polypeptide, alteration of the three dimensional structure of the compound or the Nmi polypeptide, or covalent or non-covalent modification of the second compound or the Nmi polypeptide.
In preferred embodiments, the one or more of the first compound (e.g., an inhibitor candidate), the Nmi polypeptide, and the second compound (e.g., a myc polypeptide), is a purified preparation.
In preferred embodiments, the second compound can be: a ligand of an Nmi polypeptide; a myc polypeptide, e.g., an N-myc, C-myc, Max, Mxii, or protein; a protein having a basic-leucine zipper region, e.g., a fos protein; a daughterless protein; a transcription factor, e.g., a zip transcription factor; a polypeptide which includes any of a bHLH-Zip region, a zip region; a bHLH region, a polypeptide which binds DNA or RNA, e.g., a polypeptide which binds DNA or RNA is a sequence specific manner; a polypeptide which does not include a zinc-finger region. In particularly preferred embodiments the compound is a fragment or an analog, e.g., an Nmi-interacting fragment or analog, of one of the above recited polypeptides.
The method can be performed in a cell-free system, e.g., a cell lysate or a reconstituted protein mixture. The Nmi polypeptide, and the second compound can be expressed in a cell, and the cell contacted with the first compound, e.g. in an interaction trap assay (e.g., a two-hybrid assay).
In yet another aspect, the invention features a two-phase method (e.g., a method having an in vitro, e.g., a cell free system, and an in vivo phase) for evaluating a compound, for the ability to modulate an interaction of an Nmi polypeptide, with a second compound.
The method includes steps in vitro evaluation method described herein, e.g., (i) and (ii) of the method described immediately above performed in vitro, and further includes: (iii) determining if the compound modulates the interaction in vitro, e.g., in a cell free system, and if so; (iv) administering the compound to a cell or animal; and optionally,(v) evaluating the in vivo effect of the compound on an interaction of an Nmi polypeptide, with a second compound, e.g., by the effect on cell growth or the regulation of the cell cycle, or by the effect on the expression of a reporter gene.
In another aspect, the invention features a method for evaluating a compound, e.g., for the ability to modulate, e.g., to inhibit or promote, a Nmi polypeptide-mediated phenomenon, e.g., an aspect of intracellular signaling, the cell cycle, or cell proliferation, or to evaluate test compounds for use as therapeutic agents. The method includes: contacting the test compound with a cell. or a cell free system, which includes a reporter gene functionally linked to a Nmi; polypeptide regulatory sequence, and detecting the modulation of the expression of the reporter gene, modulation of the expression of the reporter gene being correlated to efficacy of the compound.
In another aspect, the invention features a two-phase method (e.g., a method having a primary in vitro and a secondary in vivo phase) for evaluating a treatment. The method can be used to evaluate a treatment for the ability to modulate, e.g., to inhibit or promote, a Nmi polypeptide-mediated phenomenon, e. g., progression through the cell cycle or cell proliferation, or to evaluate test compounds for use as therapeutic agents. The method includes: (i) an in vitro phase in which the test compound is contacted with a cell, or a cell free system, which includes a reporter gene functionally linked to a Nmi polypeptide regulatory sequence, and detecting the modulation of the expression of the reporter gene and (ii) if the test compound modulates the expression, administering the test compound to an animal, and evaluating the in vivo effects of the compound on a parameter related to an Nmi-related parameter, e.g., progression through the cell cycle or cell proliferation, intracellular signaling,
In another aspect, the invention features, a method of evaluating a compound for the ability to bind a nucleic acid encoding a Nmi polypeptide regulatory sequence. The method includes: contacting the compound with the nucleic acid; and evaluating ability of the compound to form a complex with the nucleic acid.
In another aspect, the invention features a method of making a fragment or analog of an Nmi polypeptide, e.g., a fragment or analog having a biological activity of a naturally occurring Nmi polypeptide. The method includes: altering the sequence, e.g., by substitution or deletion of one or more residues, of an Nmi polypeptide, e.g., altering the sequence of a non-conserved region, or a domain or region referred to herein, and, optionally testing the altered polypeptide for the desired activity.
In another aspect, the invention features, a method of evaluating a treatment for the ability to modulate the growth or regulation of the cell cycle of a cell which has a greater than wild type level of Nmi activity. The method is useful in assessing the usefulness of a particular treatment for disorders which are mediated by Nmi activity. The method includes: administering the treatment to a cell which over expresses Nmi, e,g., a cell which has been genetically engineered to over express the Nmi gene, e.g., a cell which has one or more additional copies of an Nmi encoding sequence, or an Nmi encoding sequence coupled to a control region which results in over expression (as compared to wild type for that cell); and determining if there is an effect on cell growth. The cell can be e.g., a microbial cell, e.g., a yeast cell, or an animal cell e.g., a mammalian cell, e.g., a rodent, e.g., rat or mouse, or a human cell. The cell can, e.g., be a cultured cell or a cell derived from a transgenic animal.
In another aspect, the invention features a method of treating a mammal, e.g., a human, at risk for a disorder, e.g., a disorder characterized by aberrant or unwanted level of Nmi activity. Such disorders include, e.g., those characterized by abnormal or unwanted cell proliferation, e. g., cancer, e.g., leukemia, e.g., promyelocytic leukemia, chronic myelogenous leukemia, or lymphoblastic leukemia, lymphoma, e.g., Burkitts lymphoma, carcinoma, e.g., colorectal adenocarcinoma or lung carcinoma, colorectal cancer, melanoma, neuroblastoma, or lung cancer, e.g., small cell lung cancer. The method includes reducing the activity of Nmi, e.g., by administering to the mammal a therapeutically effective amount of any of:
a Nmi polypeptide encoding nucleic acid, wherein the encoded polypeptide is an antagonist of Nmi, e.g., it inhibits the interaction of Nmi with an Nmi ligand;
a Nmi polypeptide, wherein the polypeptide is an antagonist of Nmi, e.g., it inhibits the interaction of Nmi with an Nmi ligand;
an anti-Nmi antibody, e.g., an intrabody, which, e.g., inhibits the interaction of Nmi with an Nmi ligand; or
an antisense molecule, or a nucleic acid which encodes an antisense molecule, which inhibits the expression or activity of Nmi.
In preferred embodiments,the method includes further reducing the activity of a Nmi ligand, e.g., a myc gene product, e.g., by administering to the mammal a therapeutically effective amount of any of:
a Nmi ligand encoding nucleic acid, wherein the encoded polypeptide is an antagonist of the Nmi ligand, e.g., it inhibits the interaction of Nmi with a Nmi ligand;
a Nmi ligand fragment or analog, wherein the fragment of analog is an antagonist of the ligand, e.g., it inhibits the interaction of Nmi with an Nmi ligand;
an anti-Nmi ligand antibody, e,g., an intrabody, which, e.g., inhibits the interaction of Nmi with a Nmi ligand; or
an antisense molecule, or a nucleic acid which encodes an antisense molecule, which inhibits the expression or activity of a Nmi ligand.
In another aspect, the invention features a method of treating a mammal, e.g., a human, at risk for a disorder, e.g., a disorder characterized by an insufficient level of Nmi activity. Such disorders include disorders of a tissue in which Nmi is not normally expressed, e.g., the brain. Such disorders include, e.g., mental retardation, and disorders characterized by unwanted cell proliferation, e.g., cancers of the brain or peripheral nervous system, and melanoma. The method includes increasing the activity of Nmi, e.g., by administering to the mammal a therapeutically effective amount of any of:
a Nmi polypeptide encoding nucleic acid; or
a Nmi polypeptide.
In another aspect, the invention features a method of treating a mammal, e.g., a human, at risk for a disorder, e.g., a disorder characterized by aberrant or unwanted level of Nmi activity. The method includes administering to the mammal a treatment, e.g., a therapeutically effective amount a Nmi polypeptide.
In another aspect, the invention features, a human cell, e.g., a hematopoietic stem cell, transformed with a nucleic acid which encodes a Nmi polypeptide, or transformed with a nucleic acid which encodes an Nmi antisense molecule.
Nmi polypeptides and nucleic acids are useful for: identifying cells which express a Nmi gene; the production of peptides or antisense molecules which can modulate the cell cycle or cell proliferation, in vivo or in vitro; for the generation of anti-Nmi antibodies, which are useful for identifying cells which express Nmi or for evaluating levels of Nmi expression; for producing Nmi binding molecules; and for therapeutic and diagnostic applications.
Omi Gene
The present invention is based, in part, on the discovery of the gene which encodes the mammalian serine protease, Omi. Accordingly, the present invention features a purified or isolated preparation or a recombinant preparation of Omi, or an Omi polypeptide. An Omi polypeptide can be a full length sequence or a fragment of the full length of Omi.
In a preferred embodiment, Omi has at least about 60% to about 70%, more preferably at least about 80%, even more preferably at least about 90% to about 95%, and most preferably at least about 99% sequence identity with human Omi, e.g., the human Omi of SEQ ID NO:5. Omi can be identical to a human Omi sequence, e.g., that of SEQ ID NO:5. In another embodiment, Omi is encoded by a nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule of the nucleic acid sequence shown in SEQ ID NO:4. In addition, Omi can have substantially the same electrophoretic mobility as human Omi. Omi has a predicted molecular weight of about 57 kDa. Yet another preferred embodiment of the invention features an Omi which is reactive with an Omi-specific antibody, e.g., an antibody which binds to the epitope recognized by a monoclonal antibody, or a polyclonal antibody. Antibodies against Omi can be made by methods exemplified herein.
In another preferred embodiment, Omi is expressed by a recombinant cell, e.g., a bacterial cell, a cultured cell (e.g., a cultured eukaryotic cell) or a cell of a non-human transgenic organism, e.g., a transgenic animal or transgenic plant. Cultured cells can include CHO cells or SF8 cells. Expression of Omi in a transgenic animal can be general or can be under the control of a tissue specific promoter. Preferably, one or more sequences which encode Omi or a fragment thereof are expressed in a preferred cell-type by a tissue specific promoter. Exemplary sequences encoding fragments of Omi include, e.g., a sequence encoding the amino terminal regulatory domain of Omi, e.g., a signal peptidase site, a domain that includes one of more of consensus sequence PRAXXTXXTP (SEQ ID NO:6), where X can be any amino acid residue (triple repeat), a sequence encoding an SH3 binding domain, a sequence encoding a consensus phosphorylation site, e.g., a consensus phosphorylation site for Mxi2 kinase, and/or a carboxy terminal serine protease catalytic domain.
In a preferred embodiment, the recombinant Omi differs from Omi isolated from tissue in one or more of the following: its pattern of glycosylation, myristilation, phosphorylation, or other posttranslational modifications.
In a preferred embodiment, the recombinant Omi preparation is free of other placental proteins, pancreatic proteins, tumor proteins, or other human proteins.
In a preferred embodiment, the recombinant Omi preparation contains at least 1, 10, or 100 xcexcg of Omi, or an Omi polypeptide.
In a preferred embodiment, the recombinant Omi preparation contains at least 1, 10, or 100 mg of Omi, or an Omi polypeptide.
In a preferred embodiment, the Omi polypeptide has one or more of the following biological activities: 1) it is phosphorylated by the Mxi2 kinase; 2) it interacts with, e.g., binds to, the Mxi2 kinase; 3) it interacts with, e.g., binds to, a target, e.g., a serine protease inhibitor; 4) it proteolytically cleaves a substrate, e.g., a protein; 5) it is a serine protease; 6) it is a member of the MAP kinase cell signalling pathway; 7) it is involved in mammalian pathologies, e.g., ischemia of the kidney, the heart, or the forebrain; inflammatory response; septic shock; and 8) it modulates a cellular response to stress.
In other preferred embodiments: the Omi polypeptide includes an amino acid sequence with at least 60%, 80%, 90%, 95%, 98%, or 99% sequence identity to an amino acid sequence from SEQ ID NO:5; the Omi polypeptide includes an amino acid sequence essentially the same as the amino acid sequence in SEQ ID NO:5; the Omi polypeptide is at least 5, 10, 20, 50, 100, or 150, 200, 250 amino acids in length; the Omi polypeptide includes at least 5, preferably at least 10, more preferably at least 20, most preferably at least 50, 100, or 150, 200, 250 contiguous amino acids from SEQ ID NO:5; the Omi polypeptide is either, an agonist or an antagonist, of a biological activity of naturally occurring Omi; the Omi polypeptide is a vertebrate, e.g., a mammalian, e.g. a primate, e.g., a human, Omi polypeptide.
In preferred embodiments, the Omi polypeptide is encoded by the nucleic acid in SEQ ID NO:4, or by a nucleic acid having at least about 50%, more preferably at least about 60% to about 70%, and most preferably at least about 75% sequence identity with the nucleic acid from SEQ ID NO:4.
In preferred embodiments, the Omi polypeptide includes an amino terminal regulatory domain (RD) which includes a putative signal peptidase cleavage site, a triple repeat sequence of consensus sequence PRAXXTXXTP (SEQ ID NO:6), where X can be any amino acid residue), an SH3 domain (PPPASPR, SEQ ID NO:7), a potential consensus Mxi2/p38 kinase phosphorylation site (SPRS, SEQ ID NO:8), and a carboxy terminal serine protease catalytic domain.
In preferred embodiments, the Omi polypeptide includes at least one putative signal peptidase cleavage site. Generally, the domain is about 2 residues in length, and preferably, has about 50, 60, 70, 80, 90, or 95% sequence identity with the protein .sequence shown in SEQ ID NO:5 (between amino acid residues 26 and 27).
In preferred embodiments, the Omi polypeptide includes a consensus amino acid sequence P-R-A-X-X-T-X-X-T-P (SEQ ID NO:6), where X can be any amino acid. In preferred embodiments, the Omi polypeptide includes at least one consensus sequence, more preferably 2 consensus sequences, and most preferably about 3 consensus sequences, e.g., a triple repeat sequence. Generally, the consensus sequence is about 10 residues in length, and preferably, has about 70, 80, 90, or 95% sequence identity with the protein sequence shown in SEQ ID NO:5 (amino acid residues 117-126, 139-148 and 150-159).
In preferred embodiments, the Omi polypeptide includes at least one SH3 binding domain (e.g., a domain having the consensus sequence PPPASPR, SEQ ID NO:7). Generally, the domain is about 7 residues in length, and preferably, has about 70, 80, 90, or 95% sequence identity with the protein sequence shown in SEQ ID NO:5 (amino acid residues 209-215). In other embodiments, the SH3 binding domain overlaps with one or both the RD and the putative Mxi2/p38 kinase consensus phosphorylation site.
In preferred embodiments, the Omi polypeptide includes at least one potential consensus phosphorylation site for Mxi2/p38 kinase (e.g., a domain having the consenus sequence SPRS, SEQ ID NO:8). Generally, the phosphorylation site is about 4 residues in length, and preferably, has about 70, 80, 90, or 95% sequence identity with the protein sequence shown in SEQ ID NO:5 (amino acid residues 213-216).
In preferred embodiments, the Omi polypeptide includes a serine protease catalytic domain similar to L56 and HtrA serine protease catalytic domains. Generally, the serine protease catalytic domain is about 312 residues, and preferably has about 50, 60, 70, 80, 90, or 95% sequence identity with the protein sequence shown in SEQ ID NO:5 (amino acid residues 217-529).
In a preferred embodiment, the Omi polypeptide differs in amino acid sequence at up to 1, 2, 3, 5, or 10 residues, from a sequence in SEQ ID NO:5. In other preferred embodiments, the Omi polypeptide differs in amino acid sequence at up to 1, 2, 3, 5, or 10% of the residues from a sequence in SEQ ID NO:5. Preferably, the differences are such that: the Omi polypeptide exhibits an Omi biological activity, e.g., the Omi polypeptide retains a biological activity of a naturally occurring Omi. A position differs if it is a different amino acid, is deleted, or is an insertion, as compared to the sequence in SEQ ID NO:5.
In preferred embodiments, the Omi polypeptide includes an Omi sequence described herein as well as other N-terminal, and/or a C-terminal amino acid sequence.
In preferred embodiments, the Omi polypeptide includes all or a fragment of an amino acid sequence from SEQ ID NO:5, fused, in reading frame, to additional amino acid residues, preferably to residues encoded by genomic DNA 5xe2x80x2 to the genomic DNA which encodes a sequence from SEQ ID NO:5.
In yet other preferred embodiments, the Omi polypeptide is a recombinant fusion protein having a first Omi portion and a second polypeptide portion, e.g., a second polypeptide portion having an amino acid sequence unrelated to Omi. The second polypeptide portion can be, e.g., any of glutathione-S-transferase, a DNA binding domain, or a polymerase activating domain. In a preferred embodiment, the fusion protein can be used in a two-hybrid assay.
For example, a first Omi portion, e.g., an Omi portion containing a serine protease catalytic domain, e.g., amino acids 209 to end encoded by the last exon, can be fused to a DNA binding domain. In a two hybrid assay, the first Omi portion is co-expressed in a cell with a second polypeptide portion containing a transcription activation domain fused to an expression library, e.g., a HeLa cervical carcinoma expression library.
In a preferred embodiment, the Omi polypeptide includes: amino acid residues 1-116, or a sequence that has 80, 90, 95, 99% sequence identity with 1-116, of SEQ ID NO:5; amino acid residues 117-126, or a sequence that has 80, 90, 95, 99% sequence identity with 117-126, of SEQ ID NO:5; amino acid residues 139-148, or a sequence that has 80, 90, 95, 99% sequence identity with 139-148, of SEQ ID NO:5; amino acid residues 150-159, or a sequence has that 80, 90, 95, 99% sequence identity with 150-159, of SEQ ID NO:5; amino acid residues 209-215, or a sequence that has 80, 90, 95, 99% sequence identity with 209-215, of SEQ ID NO:5; amino acid residues 213-216, or a sequence that has 80, 90, 95, 99% sequence identity with amino acids 213-216, of SEQ ID NO:5; and/or amino acid residues 217-529, or a sequence has 80, 90, 95, 99% sequence identity with amino acids 217-529, of SEQ ID NO:5.
In preferred embodiments, the Omi polypeptide has an antagonistic activity or agonistic activity, and is capable of: modulating a cellular response to stress.
In a preferred embodiment, the Omi polypeptide is a fragment of a naturally occurring Omi which inhibits a cellular response to stress.
In another aspect, the invention features an Omi polypeptide which is a fragment of a full length Omi polypeptide, e.g., a fragment of a naturally occurring Omi polypeptide, e.g., the polypeptide encoded by the nucleic acid of SEQ ID NO:4.
In preferred embodiments: the fragment is at least 5, 10, 20, 50, 100, or 150 amino acids in length; the fragment is equal to or less than 200, 150, 100, 50 amino acid residues in length; the fragment has a biological activity of a naturally occurring Omi; the fragment is either, an agonist or an antagonist, of a biological activity of a naturally occurring Omi; the fragment can inhibit, e.g., competitively or non competitively inhibit, the binding of Omi to an Omi-interacting protein, e.g., an Mxi2 protein.
In preferred embodiments, the fragment it has at least 60, and more preferably at least 70, 80, 90, 95, 99, or 100% sequence identity with the corresponding amino acid sequence of SEQ ID NO:5.
In preferred embodiments, the fragment is a fragment of a vertebrate, e.g., a mammalian, e.g. a primate, e.g., a human, Omi polypeptide.
In preferred embodiments, the Omi fragment includes an amino terminal regulatory domain, which has at least 50, 60, 70, 80, 90, 95, 99 or 100% sequence identity with residues 1-216 of the Omi sequence of SEQ ID NO:5.
In preferred embodiments, the Omi fragment includes a signal peptidase site, which has at least 50, 60, 70, 80, 90, 95, 99 or 100% sequence identity with residues 26-27 of the Omi sequence of SEQ ID NO:5.
In preferred embodiments, the Omi fragment includes includes a consensus amino acid sequence P-R-A-X-X-T-X-X-T-P (SEQ ID NO:6), where X can be any amino acid. In preferred embodiments, the Omi fragment includes at least one consensus sequence, more preferably 2 consensus sequences, and most preferably about 3 consensus sequences (e.g., a triple repeat sequence) having at least 50, 60, 70, 80, 90, 95, 99 or 100% sequence identity with residues 117-126, 139-148 and 150-159 of the Omi sequence of SEQ ID NO:5.
In preferred embodiments, the Omi fragment includes an SH3 binding domain, which has at least 50, 60, 70, 80, 90, 95, 99 or 100% sequence identity with residues 209-215 of the Omi sequence of SEQ ID NO:5.
In preferred embodiments, the Omi fragment includes a potential consensus Mxi2/p38 kinase phosphorylation site, which has at least 50, 60, 70, 80, 90, 95, 99 or 100% sequence identity with residues 213-216 of the Omi sequence of SEQ ID NO:5.
In preferred embodiments, the Omi fragment includes a carboxy terminal serine protease catalytic domain, which has at least 50, 60, 70, 80, 90, 95, 99 or 100% sequence identity with residues 217-529 of the Omi sequence of SEQ ID NO:5.
In preferred embodiments, the fragment can inhibit an interaction, e.g., binding, between Omi and an Omi-interacting protein.
In a preferred embodiment, the fragment differs in amino acid sequence at up to 1, 2, 3, 5, or 10 residues, from the corresponding residues in SEQ ID NO:5. In other preferred embodiments, the fragment differs in amino acid sequence at up to 1, 2, 3, 5, or 10% of the residues from the corresponding residues in SEQ ID NO:5. In preferred embodiments, the differences are such that the fragment exhibits an Omi biological activity. In other preferred embodiments, the differences are such that the fragment does not have Omi biological activity. In preferred embodiments, one or more, or all of the differences are conservative amino acid changes. In other preferred embodiments, one or more, or all of the differences are other than conservative amino acid changes.
In a preferred embodiment, the Omi polypeptide, or fragment thereof, differs in amino acid sequence from the amino acid sequence encoded by the EST fragments shown in Table 1 herein.
By xe2x80x9cdiffersxe2x80x9d is meant an amino acid sequence other than the amino acid sequence encoded by those EST fragments shown in Table 1, e.g., it differs from the amino acid sequence encoded by those EST fragments shown in Table 1 by at least one amino acid residue, e.g., the Omi polypeptide is at least one amino acid residue shorter, one amino acid residue longer, differs in sequence at least at one position, has a different N terminus, or has a different C terminus, as compared with the amino acid sequence encoded by those EST fragments shown in Table 1.
Polypeptides of the invention include those which arise as a result of the existence of multiple genes, alternative transcription events, alternative RNA splicing events, and alternative translational and postranslational events. The Omi polypeptide can be expressed in systems, e.g., cultured cells, which result in substantially the same postranslational modifications present when expressed Omi is expressed in a native cell, or in systems which result in the omission of postranslational modifications present when expressed in a native cell.
The invention includes an immunogen which includes an Omi polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for the Omi polypeptide, e.g., a humoral response, an antibody response, or a cellular response. In preferred embodiments, the immunogen comprises an antigenic determinant, e.g., a unique determinant, from a protein represented by SEQ ID NO:5.
The present invention also includes an antibody preparation specifically reactive with an epitope of the Omi immunogen or generally of an Omi polypeptide, preferably an epitope which consists all or in part of residues from the the amino acid sequence of SEQ ID NO:5, or an epitope, which when bound to an antibody, results in the modulation of a biological activity.
In preferred embodiments,the Omi-like polypeptide, as expressed in the cells in which it is normally expressed or in other eukaryotic cells, has a molecular weight of about 57 kDa as estimated from the nucleic acid sequence SEQ ID NO:1.
In another embodiment, the Omi polypeptide comprises amino acid residues 1-529 of FIG. 4a (SEQ ID NO:5).
In a preferred embodiment, the recombinant Omi polypeptide has one or more of the following characteristics:
(i) it is approximately 529 amino acids in length;
(ii) it has the ability to cleave a substrate, e.g., a protein;
(iii) it binds to Mxi2 in vivo and in vitro;
(iv) it does not bind to p38;
(v) it has a molecular weight, amino acid composition or other physical characteristic of Omi of SEQ ID NO:5;
(vi) it has an overall sequence similarity of at least 50%, preferably at least 60%, more preferably at least 70, 80, 90, or 95%, with an Omi polypeptide of SEQ ID NO:5;
(vii) it is found in all human tissues;
(viii) it is most abundantly found in the placenta and the pancreas;
(ix) it is found in tumor cell lines, e.g., promyelocytic leukemia HL-60 cells, chronic myelogenous leukemia K-562, Burkitt""s lymphoma Raji,and human colorectal carcinoma SW480 cell lines;
(x) it is translated from at least two species of mRNA, one abundant transcript is about 2.1 kb, and another less abundant transcript of about 4.5 kb;
(xi) it is encoded by a gene localized on the human chromosome 2p12;
(xii) it has at least one signal peptidase site which is preferably about 70%, 80%, 90% or 95% identical to amino acid residues 26-27 of SEQ ID NO:5;
(xiii) it has a domain that includes a consensus amino acid sequence P-R-A-X-X-T-X-X-T-P (SEQ ID NO:6), more preferably 2, and most preferably about 3 consensus sequences (e.g., a triple repeat sequence), each of which is preferably about 70%, 80%, 90%, or 95% identical to amino acid residues 117-126, 139-148 and 150-159 of SEQ ID NO:5;
(xiv) it has at least one SH3 binding domain which is preferably about 70%, 80%, 90% or 95% identical to amino acid residues 209-215 of SEQ ID NO:5;
(xv) it has a putative consensus phosphorylation site for Mxi2/p38 kinase kinase which is preferably about 70%, 80%, 90% or 95% identical to amino acid residues 213-216 of SEQ ID NO:5; and
(xvi) it has a carboxy terminal serine protease catalytic domain containing at least one site of serine protease activity which is preferably about 70%, 80%, 90% or 95% identical to amino acid residues 181-529 of SEQ ID NO:5.
Also included in the invention is a composition which includes an Omi polypeptide (or a nucleic acid which encodes it) and one or more additional components, e.g., a carrier, diluent, or solvent. The additional component can be one which renders the composition useful for in vitro and in vivo pharmaceutical or veterinary use.
In another aspect, the invention provides an isolated or substantially pure nucleic acid having or comprising a nucleotide sequence which encodes a polypeptide, the amino acid of which includes, or is, the sequence of an Omi polypeptide.
In preferred embodiments, the encoded Omi polypeptide has one or more of the following properties:
(i) it is approximately 529 amino acids in length;
(ii) it has the ability to cleave a substrate, e.g., a protein;
(iii) it binds to Mxi2 in vivo and in vitro;
(iv) it does not bind to p38;
(v) it has a molecular weight, amino acid composition or other physical characteristic of Omi of SEQ ID NO:5;
(vi) it has an overall sequence similarity of at least 50%, preferably at least 60%, more preferably at least 70, 80, 90, or 95%, with an Omi polypeptide of SEQ ID NO:5;
(vii) it is found in all human tissues;
(viii) it is most abundantly found in the placenta and the pancreas;
(ix) it is found in tumor cell lines, e.g., promyelocytic leukemia HL-60 cells, chronic myelogenous leukemia K-562, Burkitt""s lymphoma Raji,and human colorectal carcinoma SW480 cell lines;
(x) it is translated from at least two species of mRNA, one abundant transcript is about 2.1 kb, and another less abundant transcript of about 4.5 kb;
(xi) it is encoded by a gene localized on the human chromosome 2p12;
(xii) it has at least one signal peptidase site which is preferably about 70%, 80%, 90% or 95% identical to amino acid residues 26-27 of SEQ ID NO:5;
(xiii) it has a domain that includes a consensus amino acid sequence P-R-A-X-X-T-X-X-T-P (SEQ ID NO:6), more preferably 2, and most preferably about 3 consensus sequences (e.g., a triple repeat sequence), each of which is preferably about 70%, 80%, 90%, or 95% identical to amino acid residues 117-126, 139-148 and 150-159 of SEQ ID NO:5;
(xiv) it has at least one SH3 binding domain which is preferably about 70%, 80%, 90% or 95% identical to amino acid residues 209-215 of SEQ ID NO:5;
(xv) it has a putative consensus phosphorylation site for Mxi2/p38 kinase kinase which is preferably about 70%, 80%, 90% or 95% identical to amino acid residues 213-216 of SEQ ID NO:5; and
(xvi) it has a carboxy terminal serine protease catalytic domain containing at least one site of serine protease activity which is preferably about 70%, 80%, 90% or 95% identical to amino acid residues 181-529 of SEQ ID NO:5.
In preferred embodiments, the encoded polypeptide has a biological activity, e.g., the polypeptide is either, an agonist or an antagonist, of a biological activity of a naturally occurring Omi.
In preferred embodiments,the encoded polypeptide is a vertebrate, e.g., a mammalian, e.g. a primate, e.g., a human, Omi polypeptide.
In preferred embodiments, the encoded Omi polypeptide includes a signal peptidase site which has at least 60, more preferably at least 70, 80, 90, or 100% sequence identity with at nucleotides 325-326 of SEQ ID NO:4 (or residues 26-27 of SEQ ID NO:5).
In preferred embodiments, the encoded Omi polypeptide includes a consensus amino acid sequence P-R-A-X-X-T-X-X-T-P (SEQ ID NO:6), where X can be any amino acid. In preferred embodiments, the Omi polypeptide includes at least one consensus sequence, more preferably 2 consensus sequences, and most preferably about 3 consensus sequences. Preferably, each consensus amino acid sequence is at least 60, more preferably at least 70, 80, 90, or 100% sequence identity with nucleotides 596-624, 661-691 and 695-724 of SEQ ID NO:4 (or residues I 17-126, 139-148 and 150-159 of SEQ ID NO:5).
In preferred embodiments, the encoded Omi polypeptide includes an SH3 binding domain which has at least 60, more preferably at least 70, 80, 90, or 100% sequence identity with nucleotides 872-891 of SEQ ID NO:4 (or residues 209-215 of SEQ ID NO:5).
In preferred embodiments, the encoded Omi polypeptide includes a potential consensus Mxi2/p38 kinase phosphorylation site which has at least 60, more preferably at least 70, 80, 90, or 100% sequence identity with nucleotides 885-891 of SEQ ID NO:4 (or residues 213-216 of SEQ ID NO:5).
In preferred embodiments, the encoded Omi polypeptide includes a carboxy terminal serine protease catalytic domain, which has at least 50, 60, 70, 80, 90, 95, 99 or 100% sequence identity with residues 217-529 of the Omi sequence of SEQ ID NO:5.
In a preferred embodiment, the encoded Omi polypeptide differs in amino acid sequence at up to 1, 2, 3, 5, or 10 residues, from the sequence in SEQ ID NO:5. In other preferred embodiments, the Omi polypeptide differs in amino acid sequence at up to 1, 2, 3, 5, or 10% of the residues from a sequence in SEQ ID NO:5. In preferred embodiments,the differences are such that the Omi polypeptide exhibits an Omi biological activity. In other preferred embodiments the differences are such that the Omi polypeptide does not have Omi biological activity. In preferred embodiments,one or more, or all of the differences are conservative amino acid changes. In other preferred embodiments one or more, or all of the differences are other than conservative amino acid changes.
In preferred embodiments, the encoded Omi polypeptide includes an Omi sequence described herein as well as other N-terminal and/or C-terminal amino acid sequence.
In yet other preferred embodiments, the encoded Omi polypeptide is a recombinant fusion protein having a first Omi portion and a second polypeptide portion, e.g., a second polypeptide portion having an amino acid sequence unrelated to Omi. The second polypeptide portion can be, e.g., any of glutathione-S-transferase, a DNA binding domain, or a polymerase activating domain. In preferred embodiment, the fusion protein can be used in a two-hybrid assay.
In preferred embodiments, there is at least 70, 80, 90, 95, 99, or 100% sequence identity between the encoded Omi polypeptide and the amino acid sequence of SEQ ID NO:5.
The encoded polypeptide can be a fragment of a full length Omi polypeptide, e.g., a fragment of a naturally occurring Omi polypeptide, e.g., the polypeptide encoded in SEQ ID NO:5.
In preferred embodiments, the encoded fragment is at least 5, 10, 20, 50, 100, or 150 amino acids in length; the encoded fragment is equal to or less than 200, 150, 100, 50 amino acid residues in length; the encoded fragment has a biological activity of a naturally occurring Omi; the encoded fragment is either, an agonist or an antagonist, of a biological activity of a naturally occurring Omi; the encoded fragment can inhibit, e.g., competitively or non competitively inhibit, the binding of Omi to an Omi interacting protein, e.g., Mxi2.
In preferred embodiments,the encoded fragment it has at least 60, and more preferably at least 70, 80, 90, 95, 99, or 100% sequence identity with the corresponding amino acid sequence of SEQ ID NO:5.
In preferred embodiments, the encoded fragment is a fragment of a vertebrate, e.g., a mammalian, e.g. a primate, e.g., a human, Omi polypeptide.
In preferred embodiments, the encoded Omi fragment includes a domain, e.g., an amino terminal domain, which has at least 50, 60, 70, 80, 90, 95, 99 or 100% sequence identity with residues 1-216 of the Omi sequence of SEQ ID NO:5.
In preferred embodiments, the encoded Omi fragment includes an amino terminal regulatory domain. The amino terminal regulatory domain is as follows: residues 1-117 of SEQ ID NO:5; residues 26-27 of SEQ ID NO:5; residues 117-126 of SEQ ID NO:5; residues 139-148 of SEQ ID NO:5; residues 150-159 of SEQ ID NO:5; residues 160-208 of SEQ ID NO:5; residues 209-215 of SEQ ID NO:5; and residues 213-216 of SEQ ID NO:5. In preferred embodiments,the encoded fragment includes a region which has at least 50, 60, 70, 80, 90, 95, 99 or 100% sequence identity with an amino terminal regulatory domain. In preferred embodiments,the encoded fragment (which term includes terminal and internal deletions) lacks at least one amino terminal regulatory domain found in naturally occurring Omi.
In preferred embodiments, the encoded Omi fragment includes a carboxy terminal serine protease catalytic domain. The carboxy terminal serine protease catalytic domain is as follows: residues 217-529 of SEQ ID NO:5.
In preferred embodiments,the encoded fragment includes a region which has at least 50, 60, 70, 80, 90, 95, 99 or 100% sequence identity with a carboxy terminal serine protease catalytic domain. In preferred embodiments, the encoded fragment (which term includes terminal and internal deletions) lacks at least one carboxy terminal serine protease catalytic domain found in naturally occurring Omi.
In preferred embodiments, the encoded fragment can inhibit an interaction, e.g., binding, between Omi and an Omi interacting protein. In preferred embodiments,the encoded fragment does not inhibit an interaction, e.g., binding, between Omi and an Omi interacting protein.
In a preferred embodiment, the encoded fragment differs in amino acid sequence at up to 1, 2, 3, 5, or 10 residues, from the corresponding residues in SEQ ID NO:5. In other preferred embodiments, the encoded fragment differs in amino acid sequence at up to 1, 2, 3, 5, or 10% of the residues from the corresponding residues in SEQ ID NO:5. In preferred embodiments,the differences are such that the encoded fragment exhibits an Omi biological activity. In other preferred embodiments the differences are such that the encoded fragment does not have Omi biological activity. In preferred embodiments,one or more, or all of the differences are conservative amino acid changes. In other preferred embodiments one or more, or all of the differences are other than conservative amino acid changes.
In preferred embodiments, the encoded fragment includes an Omi sequence described herein as well as other N-terminal and/or C-terminal amino acid sequence.
In a preferred embodiment, the nucleic acid encodes a polypeptide or fragment thereof which differs by at least one amino acid residue from the amino acid sequence encoded by those EST fragments shown in Table 1.
In a preferred embodiment, the nucleic acid differs by at least one nucleotide from the nucleotide sequence in those EST fragments shown in Table 1.
By xe2x80x9cdiffersxe2x80x9d is meant a nucleic acid which has a nucleotide sequence other than a nucleotide sequence which encodes the amino acid sequence encoded by those EST fragments shown in Table 1, e.g., it differs from the sequence encoded by those EST fragments shown in Table 1 by at least one nucleotide, e.g., the Omi encoding nucleic acid is at least one nucleotide shorter, one nucleotide longer, differs in sequence at least one position, has a different 5xe2x80x2 terminus, or has a different 3xe2x80x2 terminus, as compared with a sequence encoded by those EST fragments shown in Table 1.
In preferred embodiments, the subject Omi nucleic acid includes a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the Omi gene sequence, e.g., to render the Omi gene sequence suitable for use as an expression vector.
A preferred embodiment of the invention features a nucleic acid molecule having a nucleotide sequence at least about 85% sequence identity to a nucleotide sequence of SEQ ID NO:4. In other preferred embodiments, the Omi polypeptide is encoded by a nucleic acid molecule having a nucleotide sequence with at least about 90% to about 95%, and more preferably about 98% to about 99% sequence identity to the nucleotide sequence from SEQ ID NO:4. In another preferred embodiment, the Omi polypeptide is encoded by the nucleic acid molecule of SEQ ID NO:4.
In yet a further preferred embodiment, the nucleic acid which encodes an Omi polypeptide of the invention, hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 12 consecutive nucleotides of SEQ ID NO:4. In preferred embodiments, the purified nucleic acid is at least 10, more preferably 20, 30, 40, 50 or 100 nucleotides in length.
The invention also provides a probe or primer which includes or comprises a substantially purified oligonucleotide. The oligonucleotide includes a region of nucleotide sequence which hybridizes under stringent conditions to at least 10 consecutive nucleotides of sense or antisense sequence from SEQ ID NO:4, or naturally occurring mutants thereof. In preferred embodiments,the purified nucleic acid: is useful as a probe or primer; has at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% homology with a sequence from SEQ ID NO:4; is at least 10, 20, 30, 50, 100, or 200 nucleotides in length. In preferred embodiments, the probe or primer further includes a label group attached thereto. The label group can be, e.g., a radioisotope, a fluorescent compound, an enzyme, and/or an enzyme co-factor.
The invention involves nucleic acids, e.g., RNA or DNA, encoding an Omi polypeptide of the invention. This includes double stranded nucleic acids as well as coding and antisense single strands.
In another aspect, the invention features a cell or purified preparation of cells which include an Omi transgene, or which otherwise misexpress an Omi gene. The cell preparation can consist of human or non human cells, e.g., rodent cells, e.g., mouse or rat cells, rabbit cells, or pig cells. In preferred embodiments, the cell or cells include an Omi transgene, e.g., a heterologous form of an Omi gene, e.g., a gene derived from humans (in the case of a non-human cell). The Omi transgene can be misexpressed, e.g., overexpressed or underexpressed. In other preferred embodiments, the cell or cells include a gene which misexpress an endogenous Omi gene, e.g., a gene the expression of which is disrupted, e.g., a knockout. Such cells can serve as a model for studying disorders which are related to mutated or mis-expressed Omi alleles or for use in drug screening.
In another aspect, the invention features a transgenic Omi animal, e.g., a rodent, e.g., a mouse or a rat, a rabbit, a pig, a goat, or a cow. In preferred embodiments, the transgenic animal includes (and preferably express) a heterologous form of an Omi gene, e.g., a gene derived from humans. In a further embodiment, the Omi transgene includes a tissue specific promoter, e.g., a kidney-specific promotor, a cardiac-specific promotor, a neuronal specific promotor, e.g., neuron specific enolase. In other preferred embodiments, the animal has an endogenous Omi gene which is misexpressed, e.g., a knockout. Such a transgenic animal can serve as a model for studying disorders which are related to mutated or mis-expressed Omi alleles or for use in drug screening.
In another aspect, the invention features, a method of modulating, e.g., increasing or decreasing, a stress response in a subject animal or cell. The method includes modulating the activity and/or expression of Omi gene or Omi polypeptide by contacting said cell with an Omi agent. The Omi agent can be an agonist, e.g., an Omi polypeptide or nucleic acid encoding an Omi polypeptide, or an antagonist of Omi activity. Most preferably, the method decreases the stress response in the subject animal or cell, e.g., by using an antagonist of Omi activity. Preferred Omi antagonists include, e.g., a drug, e.g., a protease inhibitor; an antisense oligonucleotide; or an antibody against Omi. The method can be performed in vivo, or in vitro. In in vivo methods the Omi agent is administered to the subject. The administration can be directed to the site where a decrease in cellular stress response is desired, e.g., by topical application or by injection, or administered in a systemic fashion.
In preferred embodiments, the Omi agent is exogenous (e.g., administered to a subject) or is recombinant.
The administration of the Omi agent can be repeated.
In preferred embodiment, the cell is a mammalian cell or a human cell. Exemplary cells include e.g., immune cells; tumor cells, e.g., leukemic or carcinoma cells; or heart cells.
In another aspect, the invention features, a method of rendering a tumor cell more susceptible to chemotherapeutic agents. The method includes modulating, e.g., decreasing, activity and/or expression of an Omi gene or polypeptide by contacting said tumor cell with an Omi agent. The Omi agent can be an agonist, e.g., an Omi polypeptide or nucleic acid encoding an Omi polypeptide, or an antagonist of Omi activity. Most preferably, the Omi agent renders the tumor more suceptible to chemotherapeutic agents by using an antagonist of Omi activity. Preferred antagonists include, e.g., a drug, e.g., a protease inhibitor; an antisense oligonucleotide; or an antibody against Omi. The method can be performed in vivo, or in vitro. In in vivo methods, the Omi agent is administered to the subject. The administration can be directed to the site where an increased suceptibility to hemotherapeutic agents is desired, e.g., by topical application or by injection, or administered in a systemic fashion.
In preferred embodiments, the tumor cell is a leukemic cell, a Burkitt""s lymphoma cell, a carcinoma, e.g., colorectal adenocarcinoma or lung carcinoma, colorectal cancer, melanoma, neuroblastoma, or lung cancer, e.g., small cell lung cancer, In preferred embodiments, the Omi agent is exogenous (e.g., administered to a subject) or is recombinant.
The administration of the Omi agent can be repeated.
In another aspect, the invention features, a method of modulating an inflammatory response, e.g., a response to septic shock; a response to ischemia, e.g., very early ischemic injury or post-ischemic injury of an organ, e.g., a kidney, heart, forebrain, e.g., global forebrain ischemia. The method includes modulating, e.g., decreasing, the activity and/or expression of an Omi gene or polypeptide by contacting said tumor cell with an Omi agent. The Omi agent can be an agonist or an antagonist of Omi activity. Most preferably, the method decreases an inflammatory response by, e.g., using an antagonist to Omi activity, e.g., a drug, e.g., a protease inhibitor; an antisense oligonucleotide; or an antibody against Omi. The method can be performed in vivo, or in vitro. In in vivo, methods the Omi agent is administered to the subject. The administration can be directed to the site where a decrease in an inflammatory response is desired, e.g., by topical application or by injection, or administered in a systemic fashion.
In preferred embodiments, the Omi agent is exogenous (e.g., administered to a subject) or is recombinant.
The administration of Omi can be repeated.
In another aspect, the invention provides, a method of treating or preventing in a subject an Omi-related disorder. The method includes: administering to the subject an effective amount of Omi agent, effective to treat or prevent the Omi-related disorder in the subject. The Omi agent can be an agonist, e.g., an Omi polypeptide or nucleic acid encoding an Omi polypeptide, or an antagonist of Omi activity. The administration can be directed to the site where treatment or prevention is desired, e.g., by topical application or by injection, or administered in a systemic fashion.
In preferred embodiments, an Omi-related disorder includes, e.g., a disorder associated with the misexpression of Omi; a disorder associated with aberrant stress response; a cancer e.g., a leukemia, e.g., promyelocytic leukemia, chronic myelogenous leukemia, Burkitt""s lymphoma, or carcinoma, e.g., colorectal adenocarcinoma or lung carcinoma, colorectal cancer, melanoma, neuroblastoma, or lung cancer, e.g., small cell lung cancer; a disorder associated with a genetic lesion at chromosome 2, region p12; a disorder associated with abnormal proteolysis of proteins; a disorder associated with septic shock; or an inflammatory condition, e.g., ischemia, e.g., ischemia of the heart, the kidney, or the forebrain.
In preferred embodiments, the subject is a mammal, e.g., human or non-human.
In preferred embodiments, the Omi agent is exogenous (e.g., administered to a subject) or is recombinant.
The administration of the Omi agent can be repeated.
In another aspect, the invention provides, a method of determining if a subject is at risk for a disorder related to a lesion in or the misexpression of a gene which encodes an Omi described herein.
Such Omi-related disorders include, e.g., a disorder associated with the misexpression of Omi; a disorder associated with aberrant stress response; a cancer, e.g., a leukemia, e.g., promyelocytic leukemia, chronic myelogenous leukemia, Burkitt""s lymphoma, or carcinoma, e.g., colorectal adenocarcinoma or lung carcinoma, colorectal cancer, melanoma, neuroblastoma, or lung cancer, e.g., small cell lung cancer; a disorder associated with a genetic lesion at chromosome 2, region p12; a disorder associated with abnormal proteolysis of proteins; a disorder associated with septic shock; or an inflammatory condition, e.g., ischemia, e.g., ischemia of the heart, the kidney, or the forebrain.
In preferred embodiments, the molecule is exogenous (e.g., administered to a subject) or is recombinant.
The administration of Omi can be repeated.
The method includes one or more of the following:
detecting, in a tissue of the subject, the presence or absence of a mutation which affects the expression of the Omi gene, or other gene which encodes a subunit of Omi, e.g., detecting the presence or absence of a mutation in a region which controls the expression of the gene, e.g., a mutation in the 5xe2x80x2 control region;
detecting, in a tissue of the subject, the presence or absence of a mutation which alters the structure of the Omi gene;
detecting, in a tissue of the subject, the misexpression of the Omi gene, at the mRNA level, e.g., detecting a non-wild type level of an Omi mRNA;
detecting, in a tissue of the subject, the misexpression of the Omi gene, at the protein level, e.g., detecting a non-wild type level of an Omi polypeptide.
In preferred embodiments,the method includes: ascertaining the existence of at least one of: a deletion of one or more nucleotides from the Omi gene; an insertion of one or more nucleotides into the gene, a point mutation, e.g., a substitution of one or more nucleotides of the gene, a gross chromosomal rearrangement of the gene, e.g., a translocation, inversion, or deletion.
For example, detecting the genetic lesion can include: (i) providing a probe/primer including an oligonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence from SEQ ID NO:4, or naturally occurring mutants thereof or 5xe2x80x2 or 3xe2x80x2 flanking sequences naturally associated with the Omi gene; (ii) exposing the probe/primer to nucleic acid of the tissue; and detecting, by hybridization, e.g., in situ hybridization, of the probe/primer to the nucleic acid, the presence or absence of the genetic lesion.
In preferred embodiments, detecting the misexpression includes ascertaining the existence of at least one of: an alteration in the level of a messenger RNA transcript of the Omi gene; the presence of a non-wild type splicing pattern of a messenger RNA transcript of the Omi gene; or a non-wild type level of Omi.
Methods of the invention can be used prenatally or to determine if a subject""s offspring will be at risk for a disorder.
In preferred embodiments,the method includes determining the structure of an Omi gene, an abnormal structure being indicative of risk for the disorder.
In preferred embodiments,the method includes contacting a sample form the subject with an antibody to the Omi protein or a nucleic acid which hybridizes specifically with the Omi gene.
In another aspect, the invention features, a method of evaluating a compound for the ability to interact with, e.g., bind to, a subject Omi polypeptide, e.g., Omi or a fragment thereof, e.g., a regulatory amino terminal domain, a triple repeat motif, an SH3 binding domain, a consensus Mxi2/p38 kinase phosphorylation site, and/or a carboxy terminal serine protease catalytic domain of Omi. The method includes: contacting the compound with the subject Omi polypeptide; and evaluating ability of the compound to interact with, e.g., to bind or form a complex with the subject Omi polypeptide. This method can be performed in vitro, e.g., in a cell free system, or in vivo, e.g., in a two-hybrid interaction trap assay. This method can be used to identify naturally occurring molecules which interact with subject Omi polypeptide. It can also be used to find natural or synthetic inhibitors of subject Omi polypeptide.
In another aspect, the invention features, a method of evaluating a compound, e.g., a polypeptide, e.g., a naturally occurring ligand of, or a naturally occuring substrate which binds to, a subject Omi polypeptide, e.g., Omi or a fragment thereof, e.g., a regulatory amino terminal domain, a signal peptidase site, a triple repeat motif, an SH3 binding domain, a consensus Mxi2/p38 kinase phosphorylation site, and/or a carboxy terminal serine protease catalytic domain, of Omi, for the ability to bind a subject Omi polypeptide. The method includes: contacting the compound with the subject Omi polypeptide; and evaluating the ability of the compound to interact with, e.g., to bind or form a complex with the subject Omi polypeptide, e.g., the ability of the compound to inhibit a subject Omi polypeptide/ligand interaction. This method can be performed in vitro, e.g., in a cell free system, or in vivo, e.g., in a two-hybrid interaction trap assay. This method can be used to identify compounds, e.g., fragments or analogs of a subject Omi polypeptide, which are agonists or antagonists of a subject Omi polypeptide.
In another aspect, the invention features, a method of evaluating a first compound, e.g., a subject Omi polypeptide, e.g., Omi or a fragment thereof, e.g., a regulatory amino terminal domain, a triple repeat motif, a signal peptidase site, an SH3 binding domain, a consensus Mxi2/p38 kinase phosphorylation site, and/or a carboxy terminal serine protease catalytic domain, of Omi, for the ability to bind a second compound, e.g., a second polypeptide, e.g., a naturally occurring ligand of an Omi polypeptide, or substrate which binds to a subject Omi polypeptide. The method includes: contacting the first compound with the second compound; and evaluating the ability of the first compound to form a complex with the second compound. This method can be performed in vitro, e.g., in a cell free system, or in vivo, e.g., in a two-hybrid interaction trap assay. This method can be used to identify compounds, e.g., fragments or analogs of a subject Omi polypeptide, which are agonists or antagonists of a subject Omi polypeptide.
In yet another aspect, the invention features a method for evaluating a compound, e.g., for the ability to modulate an interaction, e.g., the ability to inhibit an interaction of a subject Omi polypeptide, e.g., Omi or a fragment thereof, e.g., a regulatory amino terminal domain, a signal peptidase site, a triple repeat motif, an SH3 binding domain, a consensus Mxi2/p38 kinase phosphorylation site, and/or a carboxy terminal serine protease catalytic domain, of Omi, with a second polypeptide, e.g., a polypeptide, e.g., a natural ligand of a subject Omi polypeptide or a substrate which binds to a subject Omi polypeptide, or a fragment thereof. The method includes the steps of (i) combining the second polypeptide (or preferably a purified preparation thereof), a subject Omi polypeptide, (or preferably a purified preparation thereof), and a compound, e.g., under conditions wherein in the absence of the compound, the second polypeptide, and the subject Omi polypeptide, are able to interact, e.g., to bind or form a complex; and (ii) detecting the interaction, e.g., detecting the formation (or dissolution) of a complex which includes the second polypeptide, and the subject Omi polypeptide. A change, e.g., a decrease or increase, in the formation of the complex in the presence of a compound (relative to what is seen in the absence of the compound) is indicative of a modulation, e.g., an inhibition or promotion, of the interaction between the second polypeptide, and the subject Omi polypeptide. In preferred embodiments: the second polypeptide, and the subject Omi polypeptide, are combined in a cell-free system and contacted with the compound; the cell-free system is selected from a group consisting of a cell lysate and a reconstituted protein mixture; the subject Omi polypeptide, and the second polypeptide are simultaneously expressed in a cell, and the cell is contacted with the compound, e.g. in an interaction trap assay (e.g., a two-hybrid assay).
In yet another aspect, the invention features a two-phase method (e.g., a method having an in vitro, e.g., in a cell free system, and an in vivo phase) for evaluating a compound, e.g., for the ability to modulate, e.g., to inhibit or promote, an interaction of a subject Omi polypeptide subject Omi polypeptide, e.g., Omi or a fragment thereof, e.g., a regulatory amino terminal domain, a signal peptidase site, a triple repeat motif, an SH3 binding domain, a consensus Mxi2/p38 kinase phosphorylation site, and/or a carboxy terminal serine protease catalytic domain, of Omi, with a second compound, e.g., a second polypeptide, e.g., a naturally occurring ligand of an Omi polypeptide, or a substrate which binds to a subject Omi polypeptide, or a fragment thereof. The method includes steps (i) and (ii) of the method described immediately above performed in vitro, and further includes: (iii) determining if the compound modulates the interaction in vitro, e.g., in a cell free system, and if so; (iv) administering the compound to a cell or animal; and (v) evaluating the in vivo effect of the compound on an interaction, e.g., inhibition, of a subject Omi polypeptide, with a second polypeptide.
In another aspect, the invention features, a method of evaluating a compound for the ability to bind a nucleic acid encoding a subject Omi polypeptide, e.g., Omi or a fragment thereof, e.g., a regulatory amino terminal domain, a signal peptidase site, a triple repeat motif, an SH3 binding domain, a consensus Mxi2/p38 kinase phosphorylation site, and/or a carboxy terminal serine protease catalytic domain, of Omi. The method includes: contacting the compound with the nucleic acid; and evaluating ability of the compound to form a complex with the nucleic acid.
In another aspect, the invention features a method of making an Omi polypeptide, e.g., a peptide having a non-wild type activity, e.g., an antagonist, agonist, or super agonist of a naturally occurring Omi polypeptide, e.g., a naturally occurring Omi polypeptide. The method includes: altering the sequence of an Omi polypeptide, e.g., altering the sequence, e.g., by substitution or deletion of one or more residues of a non-conserved region, a domain or residue disclosed herein, and testing the altered polypeptide for the desired activity.
In another aspect, the invention features a method of making a fragment or analog of an Omi polypeptide having a biological activity of a naturally occurring Omi polypeptide. The method includes: altering the sequence, e.g., by substitution or deletion of one or more residues, of an Omi polypeptide, e.g., altering the sequence of a non-conserved region, or a domain or residue described herein, and testing the altered polypeptide for the desired activity.
In another aspect, the invention features, a human cell, e.g., a tumor cell, a blood cell, e.g., a white blood cell, e.g., an immune cell, e.g., lymphocyte, transformed with nucleic acid which encodes a subject Omi polypeptide.
In another aspect, the invention includes: an Omi nucleic acid, e.g., an Omi nucleic acid inserted into a vector; a cell transformed with an Omi nucleic acid; an Omi made by culturing a cell transformed with an Omi nucleic acid; and a method of making an Omi polypeptide including culturing a cell transformed with an Omi nucleic acid.
Rim Gene
The present invention is based, in part, on the discovery of the gene which encodes a retinoblastoma-interacting Myosin-like polypeptide, Rim polypeptide. Accordingly, the present invention features a purified or isolated preparation or a recombinant preparation of Rim, or a Rim polypeptide. A Rim polypeptide can be a full length Rim or a fragment.
In a preferred embodiment, Rim has at least 60% to about 70%, more preferably at least about 80%, even more preferably at least about 90% to about 95%, and most preferably at least about 99% sequence identity with human Rim, e.g., the human Rim of SEQ ID NO:10. Rim can be identical to a human Rim sequence, e.g., that of SEQ ID NO:10. In another embodiment, Rim is encoded by a nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule of the nucleic acid sequence shown in SEQ ID NO:9. In addition, Rim can have substantially the same electrophoretic mobility as human Rim. Rim has a predicted molecular weight of about 102 kDa. Yet another preferred embodiment of the invention features a Rim which is reactive with a Rim-specific antibody, e.g., an antibody which binds to the epitope recognized by a monoclonal antibody, or a polyclonal antibody. Antibodies against Rim can be made by methods exemplified herein.
In another preferred embodiment, Rim is expressed by a recombinant cell, e.g., a bacterial cell, a cultured cell (e.g., a cultured eukaryotic cell) or a cell of a non-human transgenic organism, e.g., a transgenic plant or animal. Cultured cells can include CHO cells or SF8 cells. Expression of Rim in a transgenic animal can be general or can be under the control of a tissue specific promoter. Preferably, one or more sequences which encode Rim or a fragment thereof are expressed in a preferred cell-type by a tissue specific promoter. Exemplary sequences encoding fragments of Rim include, e.g., two potential coiled-coil leucine zipper structures, an RB family binding motif, an E1A/CtBP binding motif, and four putative nuclear localization sequences.
In a preferred embodiment, the recombinant Rim differs from Rim isolated from tissue in one or more of the following: its pattern of glycosylation, myristilation, phosphorylation, or other posttranslational modifications.
In a preferred embodiment, the recombinant Rim preparation is free of other keratinocyte proteins, lymphocyte proteins, pancreatic proteins, blood cell proteins, or other human proteins.
In a preferred embodiment, the recombinant Rim preparation contains at least 1, 10, or 100 xcexcg of Rim, or a Rim polypeptide.
In a preferred embodiment, the recombinant Rim preparation contains at least 1, 10, or 100 mg of Rim, or a Rim polypeptide.
In a preferred embodiment, the Rim polypeptide has one or more of the following biological acitivities: 1) it binds to a retinoblastoma (RB) protein in vitro and in vivo; 2) it modifies the activity of the RB; 3) it regulates the cell cycle; and/or 4) it mediates oncogenic transformation of a cell.
In other preferred embodiments: the Rim polypeptide includes an amino acid sequence with at least 60%, 80%, 90%, 95%, 98%, or 99% sequence identity to an amino acid sequence from SEQ ID NO:10; the Rim polypeptide includes an amino acid sequence essentially the same as the amino acid sequence in SEQ ID NO:10; the Rim polypeptide is at least 5, 50, 100, 200, 500, 600 or 750 amino acids in length; the Rim polypeptide includes at least 5, preferably at least 10, more preferably at least 20, most preferably at least 5, 50, 100, 200, 500, 600 or 750 contiguous amino acids from SEQ ID NO:10; the Rim polypeptide is either, an agonist or an antagonist, of a biological activity of naturally occurring Rim; the Rim polypeptide is a vertebrate, e.g., a mammalian, e.g. a primate, e.g., a human, Rim polypeptide.
In preferred embodiments: the Rim polypeptide is encoded by the nucleic acid in SEQ ID NO:9, or by a nucleic acid having at least about 50%, more preferably at least about 60% to about 70%, and most preferably at least about 75% sequence identity with the nucleic acid from SEQ ID NO:9.
In preferred embodiments, the Rim polypeptide includes two or more potential coiled-coil leucine zipper motifs, an RB family binding motif, an E1A/CtBP binding motif, and four putative nuclear localization sequences.
In preferred embodiments, the Rim polypeptide includes a leucine zipper motif. In other preferred embodiments, the Rim polypeptide includes two leucine zipper motifs. Generally, the leucine zipper motif is about 21 residues in length, and preferably, has about 70, 80, 90, or 95% sequence identity with the protein sequence shown in SEQ ID NO:10 (amino acid residues 120-141 and 740-761). Preferably, each leucine zipper motif folds into two independent structural domains, one at the amino terminus and the other closer to the carboxy terminus of the protein.
In preferred embodiments, the Rim polypeptide includes an RB family binding motif, e.g., a motif having the amino acid sequence LXCXE (SEQ ID NO:11), wherein X can be any amino acid. Generally, the consensus RB family binding motif is about 5 amino acids, and preferably has about 70, 80, 90, or 95% sequence identity with the protein sequence shown in SEQ ID NO:10 (amino acid residues 153-157). Preferably, the RB family binding motif mediates binding of Rim to an RB polypeptide.
In preferred embodiments, the Rim polypeptide includes an E1A/CtBP binding motif, e.g., a motif having the amino acid sequence PLDLS (SEQ ID NO:12). Generally, the E1A/CtBP binding motif is about 4 amino acids, and preferably has about 70, 80, 90, or 95% sequence identity with the protein sequence shown in SEQ ID NO:10 (amino acid residues 490-494). Preferably, the E1A/CtBP binding motif mediates the binding of Rim to CtBP, and/or CtBP-like moieties.
In preferred embodiments, the Rim polypeptide includes a putative nuclear localization sequence. In other preferred embodiments, it includes up to four nuclear localization sequence. Generally, the putative nuclear localization sequences are about 3 amino acids, and preferably has about 70, 80, 90, or 95% sequence identity with the protein sequence shown in SEQ ID NO:10 (amino acid residues 355-358, 446-449, 877-880 and 878-881). Preferably, the putative nuclear localization sequences mediate nuclear localization of this protein.
In yet another embodiment, the invention features a Rim polypeptide that does not include or has an inactivation in at least one domain, e.g., the amino terminal domain, which serves as an antagonist to one or more Rim biological activities.
In a preferred embodiment, the Rim polypeptide differs in amino acid sequence at up to 1, 2, 3, 5, or 10 residues, from a sequence in SEQ ID NO:10. In other preferred embodiments, the Rim polypeptide differs in amino acid sequence at up to 1, 2, 3, 5, or 10% of the residues from a sequence in SEQ ID NO:10. Preferably, the differences are such that: the Rim polypeptide exhibits a Rim biological activity, e.g., the Rim polypeptide retains a biological activity of a naturally occurring Rim. A position differs if it is a different amino acid, is deleted, or is an insertion, as compared to the sequence of SEQ ID NO:10.
In preferred embodiments,the Rim polypeptide includes a Rim sequence described herein as well as other N-terminal, and/or a C-terminal amino acid sequence.
In preferred embodiments, the Rim polypeptide includes all or a fragment of an amino acid sequence from SEQ ID NO:10, fused, in reading frame, to additional amino acid residues, preferably to residues encoded by genomic DNA 5xe2x80x2 to the genomic DNA which encodes a sequence from SEQ ID NO:10.
In yet other preferred embodiments, the Rim polypeptide is a recombinant fusion protein having a first Rim portion and a second polypeptide portion, e.g., a second polypeptide portion having an amino acid sequence unrelated to Rim. The second polypeptide portion can be, e.g., any of glutathione-S-transferase, a DNA binding domain, or a polymerase activating domain. In a preferred embodiment the fusion protein can be used in a two-hybrid assay.
For example, a Rim portion, e.g., a Rim portion containing a coiled-coil leucine zipper domain, e.g., amino acid residues 120-141 and 740-761 of SEQ ID NO:10, can be fused to a DNA binding domain. Alternatively, a Rim portion can be an RB family binding motif, e.g., amino acids 153-157 of SEQ ID NO:10; or an E1A/CtBP binding motif, e.g., amino acids 490-494 of SEQ ID NO:10. In a two hybrid assay, the Rim portion is co-expressed in a cell with a second polypeptide portion containing a transcription activation domain fused to an expression library, e.g., a keratinocyte library.
In a preferred embodiment, the Rim polypeptide includes: amino acid residues 120-141, or a sequence that has 80, 90, 95, 99% sequence identity with 120-141, of SEQ ID NO:10; amino acid residues 740-761, or a sequence that has 80, 90, 95, 99% sequence identity with 740-761 of SEQ ID NO:10; amino acid residues 153-157, or a sequence that has 80, 90, 95, 99% sequence identity with 153-157, of SEQ ID NO:10; amino acid residues 490-494, or a sequence that has 80, 90, 95, 99% sequence identity with 490-494, of SEQ ID NO:10; amino acids 355-358, or a sequence that has 80, 90, 95, 99% sequence identity with 355-358, of SEQ ID NO:10; amino acids 446-449, or a sequence that has 80, 90, 95, 99% sequence identity with 446-449, of SEQ ID NO:10; amino acids 877-880, or a sequence that has 80, 90, 95, 99% sequence identity with 877-880, of SEQ ID NO:10; or amino acids 878-881, or a sequence that has 80, 90, 95, 99% sequence identity with 878-881, of SEQ ID NO:10.
In preferred embodiments, the Rim polypeptide may have an antagonistic or agonists activity, and is capable of: binding to an RB protein.
In a preferred embodiment, the Rim polypeptide is a fragment of a naturally occurring Rim which binds RB protein.
In another aspect, the invention features an Rim polypeptide which is a fragment of a full length Rim polypeptide, e.g., a fragment of a naturally occurring Rim polypeptide, e.g., the polypeptide encoded in SEQ ID NO:10.
In preferred embodiments: the fragment is at least 5, 10, 20, 50, 100, or 150 amino acids in length; the fragment is equal to or less than 200, 150, 100, 50 amino acid residues in length; the fragment has a biological activity of a naturally occurring Rim; the fragment is either, an agonist or an antagonist, of a biological activity of a naturally occurring Rim; the fragment can inhibit, e.g., competitively or non competitively inhibit, the binding of Rim to an Rim-interacting protein, e.g., an RB protein.
In preferred embodiments,the fragment it has at least 60, and more preferably at least 70, 80, 90, 95, 99, or 100% sequence identity with the corresponding amino acid sequence of SEQ ID NO:10.
In preferred embodiments,the fragment is a fragment of a vertebrate, e.g., a mammalian, e.g. a primate, e.g., a human, Rim polypeptide.
In preferred embodiments, the Rim fragment includes a domain, e.g., an RB binding motif, e.g., a motif having the sequence LXCXE (SEQ ID NO:11), wherein X can be any amino acid, which has at least 50, 60, 70, 80, 90, 95, 99 or 100% sequence identity with residues 153-157 of the Rim sequence of SEQ ID NO:10.
In preferred embodiments, the Rim fragment includes an E1A/CtBP binding motif, which has at least 50, 60, 70, 80, 90, 95, 99 or 100% sequence identity with residues 490-494 of the Rim sequence of SEQ ID NO:10.
In preferred embodiments, the Rim fragment includes at least one leucine zipper domain, and preferably about 2 leucine zipper domains, having at least 50, 60, 70, 80, 90, 95, 99 or 100% sequence identity with residues 120-141 or 740-761 of the Rim sequence of SEQ ID NO:10.
In preferred embodiments, the Rim fragment includes at least one nuclear localization signal domain, more preferably 2 to 3 nuclear localization signal domains, and most preferably about 4 nuclear localization signal domains, having at least 50, 60, 70, 80, 90, 95, 99 or 100% sequence identity with residues 355-358, 446-449, 877-880 or 878-881 of the Rim sequence of SEQ ID NO:10.
In preferred embodiments,the fragment can inhibit an interaction, e.g., binding, between Rim and an Rim-interacting protein, e.g., an RB protein.
In a preferred embodiment, the fragment differs in amino acid sequence at up to 1, 2, 3, 5, or 10 residues, from the corresponding residues in SEQ ID NO:10. In other preferred embodiments, the fragment differs in amino acid sequence at up to 1, 2, 3, 5, or 10% of the residues from the corresponding residues in SEQ ID NO:10. In preferred embodiments, the differences are such that the fragment exhibits a Rim biological activity. In other preferred embodiments, the differences are such that the fragment does not have Rim biological activity. In preferred embodiments, one or more, or all of the differences are conservative amino acid changes. In other preferred embodiments, one or more, or all of the differences are other than conservative amino acid changes.
In a preferred embodiment the Rim polypeptide, or fragment thereof, differs in amino acid sequence from the amino acid sequence encoded by EST AA172171 (SEQ ID NO14), EST AA172324 (SEQ ID NO:15), or EST clone 610839.
By xe2x80x9cdiffersxe2x80x9d is meant an amino acid sequence other than the amino acid sequence encoded by EST AA172171 (SEQ ID NO:14), EST AA172324 (SEQ ID NO:15), or EST clone 610839, e.g., it differs from the amino acid sequence encoded by EST AA172171 (SEQ ID NO:14), EST AA172324 (SEQ ID NO:15), or EST clone 610839 by at least one amino acid residue, e.g., the Rim polypeptide is at least one amino acid residue shorter, one amino acid residue longer, differs in sequence at least at one position, has a different N terminus, or has a different C terminus, as compared with the amino acid sequence encoded by EST AA172171 (SEQ ID NO:14), EST AA172324 (SEQ ID NO:15), or EST clone 610839.
Polypeptides of the invention include those which arise as a result of the existence of multiple genes, alternative transcription events, alternative RNA splicing events, and alternative translational and postranslational events. The Rim polypeptide can be expressed in systems, e.g., cultured cells, which result in substantially the same postranslational modifications present when expressed Rim is expressed in a native cell, or in systems which result in the omission of postranslational modifications present when expressed in a native cell.
The invention includes an immunogen which includes a Rim polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for the Rim polypeptide, e.g., a humoral response, an antibody response, or a cellular response. In preferred embodiments, the immunogen comprising an antigenic determinant, e.g., a unique determinant, from a protein represented by SEQ ID NO:10.
The present invention also includes an antibody preparation specifically reactive with an epitope of the Rim immunogen or generally of a Rim polypeptide, preferably an epitope which consists all or in part of residues from the the amino acid sequence of SEQ ID NO:10, or an epitope, which when bound to an antibody, results in the modulation of a biological activity.
In preferred embodiments, the Rim-like polypeptide, as expressed in the cells in which it is normally expressed or in other eukaryotic cells, has a molecular weight of about 102 kDa as predicted from SEQ ID NO:9.
In another embodiment, the Rim polypeptide comprises amino acid residues 1-897 of FIG. 7 (SEQ ID NO:10).
In a preferred embodiment, the recombinant Rim polypeptide has one or more of the following characteristics:
(i) it interacts, e.g., binds to, the retinoblastoma (RB) protein;
(ii) it regulates the cell cycle;
(iii) it has a molecular weight, amino acid composition or other physical characteristic of Rim of SEQ ID NO:10;
(iv) it has an overall sequence similarity of at least 50%, preferably at least 60%, more preferably at least 70, 80, 90, or 95%, with a Rim polypeptide of SEQ ID NO:10;
(v) it is found in all human adult tissues;
(vi) it is found at high levels in the pancreas;
(vii) it is found at high levels in tumor cell lines, e.g., promyelocytic leukemia HL-60, chronic myelogenous leukemia K-562, lymphoblastic leukemia MOLT-4, Burkitt""s lymphoma Raji, and human colorectal carcinoma SW480 cell lines;
(viii) it has at least one, and preferably two potential coiled-coil leucine zipper structures, which is preferably about 70%, 80%, 90% or 95% identical to amino acid residues 120-141 and 740-761 of SEQ ID NO:10;
(ix) it has an RB family binding motif, which is preferably about 70%, 80%, 90% or 95% identical to amino acid residues 153-157 of SEQ ID NO:10;
(x) it has an E1A/CtBP binding motif, which is preferably about 70%, 80%, 90% or 95% identical to amino acid residues 490-494 of SEQ ID NO:10; and
(xi) it has up to four putative nuclear localization sequences which are preferably about 70%, 80%, 90% or 95% identical to amino acid residues 355-358, 446-449 and 877-880 and 878-881 of SEQ ID NO:10.
Also included in the invention is a composition which includes a Rim polypeptide (or a nucleic acid which encodes it) and one or more additional components, e.g., a carrier, diluent, or solvent. The additional component can be one which renders the composition useful for in vitro and in vivo pharmaceutical or veterinary use.
In another aspect, the invention provides an isolated or substantially pure nucleic acid having or comprising a nucleotide sequence which encodes a polypeptide, the amino acid of which includes, or is, the sequence of an Rim polypeptide.
In a preferred embodiment, the encoded Rim polypeptide has one or more of the following characteristics:
(i) it interacts, e.g., binds to, the retinoblastoma (RB) protein;
(ii) it regulates the cell cycle;
(iii) it has a molecular weight, amino acid composition or other physical characteristic of Rim of SEQ ID NO:10;
(iv) it has an overall sequence similarity of at least 50%, preferably at least 60%, more preferably at least 70, 80, 90, or 95%, with a Rim polypeptide of SEQ ID NO:10;
(v) it is found in all human adult tissues;
(vi) it is found at high levels in the pancreas;
(vii) it is found at high levels in tumor cell lines, e.g., promyelocytic leukemia HL-60, chronic myelogenous leukemia K-562, lymphoblastic leukemia MOLT-4, Burkitt""s lymphoma Raji, and human colorectal carcinoma SW480 cell lines;
(viii) it has at least one, and preferably two potential coiled-coil leucine zipper structures, which is preferably about 70%, 80%, 90% or 95% identical to amino acid residues 120-141 and 740-761 of SEQ ID NO:10;
(ix) it has an RB family binding motif, which is preferably about 70%, 80%, 90% or 95% identical to amino acid residues 153-157 of SEQ ID NO:10;
(x) it has an E1A/CtBP binding motif, which is preferably about 70%, 80%, 90% or 95% identical to amino acid residues 490-494 of SEQ ID NO:10; and
(xi) it has up to four putative nuclear localization sequences which are preferably about 70%, 80%, 90% or 95% identical to amino acid residues 355-358, 446-449 and 877-880 and 878-881 of SEQ ID NO:10.
In preferred embodiments, the encoded polypeptide has a biological activity, e.g., the polypeptide is either, an agonist or an antagonist, of a biological activity of a naturally occurring Rim.
In preferred embodiments,the encoded polypeptide is a vertebrate, e.g., a mammalian, e.g. a primate, e.g., a human, Rim polypeptide.
In preferred embodiments, the encoded Rim polypeptide includes two potential coiled-coil leucine zipper motifs, an RB family binding motif, an E1/CtBP binding motif, and four putative nuclear localization sequences.
In preferred embodiments, the encoded Rim polypeptide includes a leucine zipper domain. In other preferred embodiments, the encoded Rim polypeptide includes two leucine zipper domains, each one having at least 60, more preferably at least 70, 80, 90, or 100% sequence identity with nucleotides 358-423 and 2218-2283 of SEQ ID NO:9 (or residues 120-141 and 740-161 of SEQ ID NO:10).
In preferred embodiments, the encoded Rim polypeptide includes an RB binding motif, e.g., a motif having the amino acid sequence LXCXE (SEQ ID NO:11), wherein X can be any amino acid, which has at least 60, more preferably at least 70, 80, 90, or 100% sequence identity with nucleotides 457-471 of SEQ ID NO:9 (or residues 153-157 of SEQ ID NO:10).
In preferred embodiments, the encoded Rim polypeptide includes an E1A/CtBP binding motif, which has at least 60, more preferably at least 70, 80, 90, or 100% sequence identity with nucleotides 1568-1582 of SEQ ID NO:9 (or residues 490-494 of SEQ ID NO:10).
In preferred embodiments, the encoded Rim polypeptide includes a nuclear localization signal domain. In other preferred embodiments, the encoded Rim polypeptide includes up to 4 nuclear localization signal domains, each of them having at least 60, more preferably at least 70, 80, 90, or 100% sequence identity with nucleotides 1063-1074, 1336-1347, 2628-2640 and 2632-2643 of SEQ ID NO:9 (or residues 355-358, 446-449, 877-880 and 878-881 of SEQ ID NO:10).
In a preferred embodiment, the encoded Rim polypeptide differs in amino acid sequence at up to 1, 2, 3, 5, or 10 residues, from the sequence in SEQ ID NO:10. In other preferred embodiments, the Rim polypeptide differs in amino acid sequence at up to 1, 2, 3, 5, or 10% of the residues from a sequence in SEQ ID NO:10. In preferred embodiments,the differences are such that the Rim polypeptide exhibits an Rim biological activity. In other preferred embodiments the differences are such that the Rim polypeptide does not have Rim biological activity. In preferred embodiments,one or more, or all of the differences are conservative amino acid changes. In other preferred embodiments one or more, or all of the differences are other than conservative amino acid changes. A position differs if it is a different amino acid, is deleted, or is an insertion, as compared to the sequence of SEQ ID NO:10.
In preferred embodiments, the encoded Rim polypeptide includes an Rim sequence described herein as well as other N-terminal and/or C-terminal amino acid sequence.
In yet other preferred embodiments, the encoded Rim polypeptide is a recombinant fusion protein having a first Rim portion and a second polypeptide portion, e.g., a second polypeptide portion having an amino acid sequence unrelated to Rim. The second polypeptide portion can be, e.g., any of glutathione-S-transferase, a DNA binding domain, or a polymerase activating domain. In preferred embodiment the fusion protein can be used in a two-hybrid assay.
In preferred embodiments, there is at least 70, 80, 90, 95, 99, or 100% sequence identity between the encoded Rim polypeptide and the amino acid sequence of SEQ ID NO:10.
The encoded polypeptide can be a fragment of a full length Rim polypeptide, e.g., a fragment of a naturally occurring Rim polypeptide, e.g., the polypeptide encoded in SEQ ID NO:10.
In preferred embodiments: the encoded fragment is at least 5, 10, 20, 50, 100, or 150 amino acids in length; the encoded fragment is equal to or less than 200, 150, 100, 50 amino acid residues in length; the encoded fragment has a biological activity of a naturally occurring Rim; the encoded fragment is either, an agonist or an antagonist, of a biological activity of a naturally occurring Rim; the encoded fragment can inhibit, e.g., competitively or non competitively inhibit, the binding of Rim to an Rim interacting protein,e.g., an RB protein.
In preferred embodiments, the encoded fragment it has at least 60, and more preferably at least 70, 80, 90, 95, 99, or 100% sequence identity with the corresponding amino acid sequence of SEQ ID NO:10.
In preferred embodiments, the encoded fragment is a fragment of a vertebrate, e.g., a mammalian, e.g. a primate, e.g., a human, Rim polypeptide.
In preferred embodiments, the encoded Rim fragment includes: amino acid residues 120-141, or a sequence that has 80, 90, 95, 99% sequence identity with 120-30 141, of SEQ ID NO:10; amino acid residues 740-761, or a sequence that has 80, 90, 95, 99% sequence identity with 740-761 of SEQ ID NO:I0; amino acid residues 153-157, or a sequence that has 80, 90, 95, 99% sequence identity with 153-157, of SEQ ID NO:10; amino acid residues 490-494, or a sequence that has 80, 90, 95, 99% sequence identity with 490-494, of SEQ ID NO:10; amino acids 355-358, or a sequence that has 35 80, 90, 95, 99% sequence identity with 355-358, of SEQ ID NO:10; amino acids 446-449, or a sequence that has 80, 90, 95, 99% sequence identity with 446-449, of SEQ ID NO:10; amino acids 877-880, or a sequence that has 80, 90, 95, 99% sequence identity with 877-880, of SEQ ID NO:10; or amino acids 878-881, or a sequence that has 80, 90, 95, 99% sequence identity with 878-88 1, of SEQ ID NO:10.
In preferred embodiments, the encoded fragment includes a region which has at least 50, 60, 70, 80, 90, 95, 99 or 100% sequence identity with a Rim polypeptide. In preferred embodiments,the encoded fragment (which term includes terminal and internal deletions) lacks at least one domain found in naturally occurring Rim.
In preferred embodiments, the encoded fragment can inhibit an interaction, e.g., binding, between Rim and an Rim interacting protein. In preferred embodiments,the encoded fragment does not inhibit an interaction, e.g., binding, between Rim and an Rim interacting protein, e.g., an RB protein.
In a preferred embodiment, the encoded fragment differs in amino acid sequence at up to 1, 2, 3, 5, or 10 residues, from the corresponding residues in SEQ ID NO:10. In other preferred embodiments, the encoded fragment differs in amino acid sequence at up to 1, 2, 3, 5, or 10% of the residues from the corresponding residues in SEQ ID NO:10. In preferred embodiments, the differences are such that the encoded fragment exhibits an Rim biological activity. In other preferred embodiments, the differences are such that the encoded fragment does not have Rim biological activity. In preferred embodiments,one or more, or all of the differences are conservative amino acid changes. In other preferred embodiments one or more, or all of the differences are other than conservative amino acid changes.
In preferred embodiments, the encoded fragment includes an Rim sequence described herein as well as other N-terminal and/or C-terminal amino acid sequence.
In a preferred embodiment, the nucleic acid encodes a polypeptide or fragment thereof which differs by at least one amino acid residue from the amino acid sequence encoded by those EST AA172171 (SEQ ID NO:13), EST AA172324 (SEQ ID NO:14), or EST clone 610839.
In a preferred embodiment, the nucleic acid differs by at least one nucleotide from the nucleotide sequence in those EST AA172171 (SEQ ID NO:13), EST AA172324 (SEQ ID NO:14), or EST clone 610839.
By xe2x80x9cdiffersxe2x80x9d is meant a nucleic acid sequence other than a nucleic acid sequence which encodes the amino acid sequence encoded by those EST AA172171 (SEQ ID NO:13), EST AA172324 (SEQ ID NO:14), or EST clone 610839, e.g., it differs from the sequence encoded by those EST AA172171 (SEQ ID NO:13), EST AA172324 (SEQ ID NO:14), or EST clone 610839, by at least one nucleotide, e.g., the Rim encoding nucleic acid is at least one nucleotide shorter, one nucleotide longer, differs in sequence at least one position, has a different 5xe2x80x2 terminus, or has a different 3xe2x80x2 terminus, as compared with a sequence encoded by those EST AA172171 (SEQ ID NO:13), EST AA172324 (SEQ ID NO:14), or EST clone 610839.
In preferred embodiments, the subject Rim nucleic acid includes a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the Rim gene sequence, e.g., to render the Rim gene sequence suitable for use as an expression vector.
A preferred embodiment of the invention features a nucleic acid molecule having a nucleotide sequence at least about 85% sequence identity to a nucleotide sequence of SEQ ID NO:9. In other preferred embodiments, the Rim polypeptide is encoded by a nucleic acid molecule having a nucleotide sequence with at least about 90% to about 95%, and more preferably about 98% to about 99% sequence identity to the nucleotide sequence from SEQ ID NO:9. In another preferred embodiment, the Rim polypeptide is encoded by the nucleic acid molecule of SEQ ID NO:9.
In preferred embodiments, the subject Rim nucleic acid will include a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the Rim gene sequence, e.g., to render the Rim gene sequence suitable for use as an expression vector.
In yet a further preferred embodiment, the nucleic acid which encodes a Rim polypeptide of the invention, hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 12 consecutive nucleotides of SEQ ID NO:9. In preferred embodiments,the purified nucleic acid is at least 10, more preferably 20, 30, 40, 50 or 100 nucleotides in length.
The invention also provides a probe or primer which includes or comprises a substantially purified oligonucleotide. The oligonucleotide includes a region of nucleotide sequence which hybridizes under stringent conditions to at least 10 consecutive nucleotides of sense or antisense sequence from SEQ ID NO:9, or naturally occurring mutants thereof. In preferred embodiments, the probe or primer further includes a label group attached thereto. The label group can be, e.g., a radioisotope, a fluorescent compound, an enzyme, and/or an enzyme co-factor. Preferably the oligonucleotide is at least 10 and less than 20, 30, 50, 100, or 150 nucleotides in length.
The invention involves nucleic acids, e.g., RNA or DNA, encoding a Rim polypeptide of the invention. This includes double stranded nucleic acids as well as coding and antisense single strands.
In another aspect, the invention features a cell or purified preparation of cells which include a Rim transgene, or which otherwise misexpress a Rim gene. The cell preparation can consist of human or non human cells, e.g., rodent cells, e.g., mouse or rat cells, rabbit cells, or pig cells. In preferred embodiments, the cell or cells include a Rim transgene, e.g., a heterologous form of a Rim gene, e.g., a gene derived from humans (in the case of a non-human cell). The Rim transgene can be misexpressed, e.g., overexpressed or underexpressed. In other preferred embodiments, the cell or cells include a gene which misexpress an endogenous Rim gene, e.g., a gene the expression of which is disrupted, e.g., a knockout. Such cells can serve as a model for studying disorders which are related to mutated or mis-expressed Rim alleles or for use in drug screening.
In another aspect, the invention features a transgenic Rim organism, e.g., an animal, e.g., a rodent, e.g., a mouse or a rat, a rabbit, a pig, a goat, or a cow; or a plant. In preferred embodiments, the transgenic animal includes (and preferably expresses) a heterologous form of a Rim gene, e.g., a gene derived from humans. In a further embodiment, the Rim transgene includes a tissue specific promoter. In other preferred embodiments, the animal has an endogenous Rim gene which is misexpressed, e.g., a knockout. Such a transgenic animal can serve as a model for studying disorders which are related to mutated or mis-expressed Rim alleles or for use in drug screening.
In another aspect, a method of modulating, e.g., increasing or decreasing, the cell cycle in a subject animal or cell. The method includes modulating expression and/or activity of Rim gene or Rim polypeptide by contacting said cell with a Rim agent. The Rim agent can be a Rim agonist, e.g., a Rim polypeptide or nucleic acid encoding a Rim polypeptide, or a Rim antagonist, e.g., a drug; e.g., a competitive inhibitor of the interaction of Rim with an RB protein; an antisense oligonucleotide; or an antibody against Rim. The method can be performed in vivo, or in vitro. In in vivo methods the Rim agent is administered to the subject. The administration can be directed to the site where a decrease in cellular stress response is desired, e.g., by topical application or by injection, or administered in a systemic fashion.
In preferred embodiments, the Rim agent is exogenous (e.g., administered to a subject) or is recombinant.
The administration of the Rim agent can be repeated.
In preferred embodiment, the cell is a mammalian cell or a human cell. Exemplary cells include e.g., tumor cells, e.g., leukemic or carcinoma cells, e.g., a cell found in colorectal adenocarcinoma or lung carcinoma, colorectal cancer, melanoma, neuroblastoma, or lung cancer, e.g., small cell lung cancer.
In another aspect, the invention provides, a method of treating or preventing in a subject a Rim-related disorder. The method includes: administering to the subject an effective amount of Rim agent, effective to treat or prevent the Rim-related disorder in the subject. The Rim agent can be an agonist, e.g., a Rim polypeptide or nucleic acid encoding an Rim polypeptide, or an antagonist of Rim activity. The administration can be directed to the site where treatment or prevention is desired, e.g., by topical application or by injection, or administered in a systemic fashion.
In preferred embodiments, a Rim-related disorder includes, e.g., a disorder associated with the misexpression of Rim; a disorder associated with oncogenic transformation, e.g., a cancer e.g., an osteosarcoma, a retinoblastoma, carcinoma, e.g., colorectal adenocarcinoma or lung carcinoma, colorectal cancer, melanoma, neuroblastoma, or lung cancer, e.g., small cell lung cancer, bladder cancer, a leukemia, e.g., promyelocytic leukemia, chronic myelogenous leukemia, Burkitt""s lymphoma; a disorder associated with a genetic lesion at chromosome 18, region q11.2; and a disorder associated with cell cycle deregulation.
In preferred embodiments, the subject is a mammal, e.g., human or non-human.
In preferred embodiments, the Rim agent is exogenous (e.g., administered to a subject) or is recombinant.
The administration of the Rim agent can be repeated.
In another aspect, the invention provides, a method of determining if a subject is at risk for a disorder related to a lesion in or the misexpression of a gene which encodes a Rim described herein.
Such disorders include, e.g., a disorder associated with the misexpression of Rim; a disorder associated with oncogenic transformation, e.g., a cancer e.g., an osteosarcoma, a retinoblastoma, a carcinoma, e.g., colorectal adenocarcinoma or lung carcinoma, colorectal cancer, melanoma, neuroblastoma, or lung cancer, e.g., small cell lung cancer, bladder cancer, a leukemia, e.g., promyelocytic leukemia, chronic myelogenous leukemia, Burkitt""s lymphoma; a disorder associated with a genetic lesion at chromosome 18, region q11.2; and a disorder associated with cell cycle deregulation.
The method includes one or more of the following:
detecting, in a tissue of the subject, the presence or absence of a mutation which affects the expression of the Rim gene, or other gene which encodes a fragment of Rim, e.g., detecting the presence or absence of a mutation in a region which controls the expression of the gene, e.g., a mutation in the 5xe2x80x2 control region;
detecting, in a tissue of the subject, the presence or absence of a mutation which alters the structure of the Rim gene;
detecting, in a tissue of the subject, the misexpression of the Rim gene, at the mRNA level, e.g., detecting a non-wild type level of a Rim mRNA;
detecting, in a tissue of the subject, the misexpression of the Rim gene, at the protein level, e.g., detecting a non-wild type level of a Rim polypeptide.
In preferred embodiments,the method includes: ascertaining the existence of at least one of: a deletion of one or more nucleotides from the Rim gene; an insertion of one or more nucleotides into the gene, a point mutation, e.g., a substitution of one or more nucleotides of the gene, a gross chromosomal rearrangement of the gene, e.g., a translocation, inversion, or deletion.
For example, detecting the genetic lesion can include: (i) providing a probe/primer including an oligonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence from SEQ ID NO:9, or naturally occurring mutants thereof or 5xe2x80x2 or 3xe2x80x2 flanking sequences naturally associated with the Rim gene; (ii) exposing the probe/primer to nucleic acid of the tissue; and detecting, by hybridization, e.g., in situ hybridization, of the probe/primer to the nucleic acid, the presence or absence of the genetic lesion.
In preferred embodiments,detecting the misexpression includes ascertaining the existence of at least one of: an alteration in the level of a messenger RNA transcript of the Rim gene; the presence of a non-wild type splicing pattern of a messenger RNA transcript of the Rim gene; or a non-wild type level of Rim.
Methods of the invention can be used prenatally or to determine if a subject""s offspring will be at risk for a disorder.
In preferred embodiments,the method includes determining the structure of a Rim gene, an abnormal structure being indicative of risk for the disorder.
In preferred embodiments,the method includes contacting a sample form the subject with an antibody to the Rim protein or a nucleic acid which hybridizes specifically with the Rim gene.
In another aspect, the invention features, a method of evaluating a compound for the ability to interact with, e.g., to bind to, a subject Rim polypeptide, e.g., Rim or a fragment thereof, e.g., a leucine zipper motif, an RB family binding motif, an E1A/CtBP binding motif, and/or a nuclear localization sequence, of Rim. The method includes: contacting the compound with the subject Rim polypeptide; and evaluating ability of the compound to interact with, e.g., to bind to, or form a complex with the subject Rim polypeptide. This method can be performed in vitro, e.g., in a cell free system, or in vivo, e.g., in a two-hybrid interaction trap assay. This method can be used to identify naturally occurring molecules which interact with the subject Rim polypeptide. It can also be used to find natural or synthetic inhibitors of the subject Rim polypeptide.
In another aspect, the invention features, a method of evaluating a compound, e.g., a polypeptide, e.g., a naturally occurring ligand of or a naturally occuring substrate which binds to a subject Rim polypeptide, e.g., Rim or a fragment thereof, e.g., a leucine zipper motif, an RB family binding motif, an E1A/CtBP binding motif, and/or a nuclear localization sequence, of Rim, for the ability to bind a subject Rim polypeptide. The method includes: contacting the compound with the subject Rim polypeptide; and evaluating the ability of the compound to interact with, e.g., to bind to, or form a complex with the subject Rim polypeptide, e.g., the ability of the compound to inhibit a subject Rim polypeptide/ligand interaction. This method can be performed in vitro, e.g., in a cell free system, or in vivo, e.g., in a two-hybrid interaction trap assay. This method can be used to identify compounds, e.g., fragments or analogs of a subject Rim polypeptide, which are agonists or antagonists of a subject Rim polypeptide.
In another aspect, the invention features, a method of evaluating a first compound, e.g., a subject Rim polypeptide, e.g., Rim or a fragment thereof, e.g., a leucine zipper motif, an RB family binding motif, an E1A/CtBP binding motif, and/or a nuclear localization sequence, of Rim, for the ability to bind a second compound, e.g., a second polypeptide, e.g., a naturally occurring ligand of or substrate which binds to a subject Rim polypeptide. The method includes: contacting the first compound with the second compound; and evaluating the ability of the first compound to form a complex with the second compound. This method can be performed in vitro, e.g., in a cell free system, or in vivo, e.g., in a two-hybrid interaction trap assay. This method can be used to identify compounds, e.g., fragments or analogs of a subject Rim polypeptide, which are agonists or antagonists of a subject Rim polypeptide.
In yet another aspect, the invention features a method for evaluating a compound, e.g., for the ability to modulate an interaction, e.g., the ability to inhibit an interaction of a subject Rim polypeptide, e.g., Rim or a fragment thereof, e.g., a leucine zipper motif, an RB family binding motif, an E1A/CtBP binding motif, and/or a nuclear localization sequence, of Rim, with a second polypeptide, e.g., a polypeptide, e.g., a natural ligand of the Rim polypeptide or a substrate which binds to a subject Rim polypeptide, or a fragment thereof. The method includes the steps of (i) combining the second polypeptide (or preferably a purified preparation thereof), a subject Rim polypeptide, (or preferably a purified preparation thereof), and a compound, e.g., under conditions wherein in the absence of the compound, the second polypeptide, and the subject Rim polypeptide, are able to interact, e.g., to bind or form a complex; and (ii) detecting the interaction, e.g., detecting the formation (or dissolution) of a complex which includes the second polypeptide, and the subject Rim polypeptide. A change, e.g., a decrease or increase, in the formation of the complex in the presence of a compound (relative to what is seen in the absence of the compound) is indicative of a modulation, e.g., an inhibition or promotion, of the interaction between the second polypeptide, and the subject Rim polypeptide. In preferred embodiments: the second polypeptide, and the subject Rim polypeptide, are combined in a cell-free system and contacted with the compound; the cell-free system is selected from a group consisting of a cell lysate and a reconstituted protein mixture; the subject Rim polypeptide, and the second polypeptide are simultaneously expressed in a cell, and the cell is contacted with the compound, e.g. in an interaction trap assay (e.g., a two-hybrid assay).
In yet another aspect, the invention features a two-phase method (e.g., a method having an in vitro, e.g., in a cell free system, and an in vivo phase) for evaluating a compound, e.g., for the ability to modulate, e.g., to inhibit or promote, an interaction of a subject Rim polypeptide, e.g., Rim or a fragment thereof, e.g., a leucine zipper motif, an RB family binding motif, an E1A/CtBP binding motif, and/or a nuclear localization sequence, of Rim, with a second compound, e.g., a second polypeptide, e.g., a naturally occurring ligand of a Rim polypeptide or a substrate which binds to a subject Rim polypeptide, or a fragment thereof. The method includes steps (i) and (ii) of the method described immediately above performed in vitro, and further includes: (iii) determining if the compound modulates the interaction in vitro, e.g., in a cell free system, and if so; (iv) administering the compound to a cell or animal; and (v) evaluating the in vivo effect of the compound on an interaction, e.g., inhibition, of a subject Rim polypeptide, with a second polypeptide.
In another aspect, the invention features, a method of evaluating a compound for the ability to bind a nucleic acid encoding a subject Rim polypeptide, e.g., Rim or a fragment thereof, e.g., a leucine zipper motif, an RB family binding motif, an E1A/CtBP binding motif, and/or a nuclear localization sequence, of Rim. The method includes: contacting the compound with the nucleic acid; and evaluating ability of the compound to form a complex with the nucleic acid.
In another aspect, the invention features a method of making a Rim polypeptide, e.g., a peptide having a non-wild type activity, e.g., an antagonist, agonist, or super agonist of a naturally occurring Rim polypeptide, e.g., a naturally occurring Rim polypeptide. The method includes: altering the sequence of a Rim polypeptide, e.g., altering the sequence, e.g., by substitution or deletion of one or more residues of a non-conserved region, a domain or residue disclosed herein, and testing the altered polypeptide for the desired activity.
In another aspect, the invention features a method of making a fragment or analog of a Rim polypeptide having a biological activity of a naturally occurring Rim polypeptide. The method includes: altering the sequence, e.g., by substitution or deletion of one or more residues, of a Rim polypeptide, e.g., altering the sequence of a non-conserved region, or a domain or residue described herein, and testing the altered polypeptide for the desired activity.
In another aspect, the invention features, a human cell, e.g., a tumor cell, e.g., a white blood cell, e.g., a lymphocyte, transformed with nucleic acid which encodes a subject Rim polypeptide.
In another aspect, the invention includes: a Rim nucleic acid, e.g., a Rim nucleic acid inserted into a vector; a cell transformed with a Rim nucleic acid; a Rim made by culturing a cell transformed with a Rim nucleic acid; and a method of making a Rim polypeptide including culturing a a cell transformed with a Rim nucleic acid.
A xe2x80x9cheterologous promoterxe2x80x9d, as used herein is a promoter which is not naturally associated with a gene or a purified nucleic acid.
A xe2x80x9cpurifiedxe2x80x9d or xe2x80x9csubstantially purexe2x80x9d or isolated xe2x80x9cpreparationxe2x80x9d of a polypeptide, as used herein, means a polypeptide that has been separated from other proteins, lipids, and nucleic acids with which it naturally occurs. Preferably, the polypeptide is also separated from substances, e.g., antibodies or gel matrix, e.g., polyacrylamide, which are used to purify it. Preferably, the polypeptide constitutes at least 10, 20, 50 70, 80 or 95% dry weight of the purified preparation. Preferably, the preparation contains: sufficient polypeptide to allow protein sequencing; at least 1, 10, or 100 Hg of the polypeptide; at least 1, 10, or 100 mg of the polypeptide.
A xe2x80x9cpurified preparation of cellsxe2x80x9d, as used herein, refers to, in the case of plant or animal cells, an in vitro preparation of cells and not an entire intact plant or animal. In the case of cultured cells or microbial cells, it consists of a preparation of at least 10% and more preferably 50% of the subject cells.
A xe2x80x9ctreatmentxe2x80x9d, as used herein, includes any therapeutic treatment, e.g., the administration of a therapeutic agent or substance, e.g., a drug.
As used herein, the term xe2x80x9csubjectxe2x80x9d refers to human and non-human animals. In preferred embodiments, the subject is a a human, e.g., person, e.g., a person having an Omi related disorder. The term xe2x80x9cnon-human animalsxe2x80x9d of the invention includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, ruminants, birds, amphibians, reptiles.
An xe2x80x9cisolatedxe2x80x9d or xe2x80x9cpure nucleic acidxe2x80x9d, e.g., a substantially pure DNA, is a nucleic acid which is one or both of: not immediately contiguous with either one or both of the sequences, e.g., coding sequences, with which it is immediately contiguous (i.e., one at the 5xe2x80x2 end and one at the 3xe2x80x2 end) in the naturally-occurring genome of the organism from which the nucleic acid is derived; or which is substantially free of a nucleic acid sequence with which it occurs in the organism from which the nucleic acid is derived. The term includes, for example, a recombinant DNA which is incorporated into a vector, e.g., into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other DNA sequences. Substantially pure DNA can also includes a recombinant DNA which is part of a hybrid gene encoding sequence.
xe2x80x9cSequence identity or homologyxe2x80x9d, as used herein, refers to the sequence similarity between two polypeptide molecules or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous or sequence identical at that position. The percent of homology or sequence identity between two sequences is a function of the number of matching or homologous identical positions shared by the two sequences divided by the number of positions comparedxc3x97100. For example, if 6 of 10, of the positions in two sequences are the same then the two sequences are 60% homologous or have 60% sequence identity. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology or sequence identity. Generally, a comparison is made when two sequences are aligned to give maximum homology. Unless otherwise specified xe2x80x9cloop out regionsxe2x80x9d, e.g., those arising from, from deletions or insertions in one of the sequences are counted as mismatches.
The comparison of sequences and determination of percent homology between two sequences can be accomplished using a mathematical algorithim. Preferably, the alignment can be performed using the Clustal Method. Multiple alignment paramethers include GAP Penalty=10, Gap Length Penalty=10. For DNA alignments, the pairwise alignment paramenters can be Htuple=2, Gap penalty=5, Window=4, and Diagonal saved=4. For protein alignments, the pairwise alignment parameters can be Ktuple=1, Gap penalty=3, Window=5, and Diagonals Saved=5.
Additional non-limiting example of a mathematical algorithim utilized for the comparison of sequences is the algorithm of Karlin and Altschul 1990 Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin and Altschul 1993 Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. 1990 J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997 Nucleic Acids Research 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. Another preferred, non-limiting example of a mathematical algorithim utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS 1989. Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
The terms xe2x80x9cpeptidesxe2x80x9d, xe2x80x9cproteinsxe2x80x9d, and xe2x80x9cpolypeptidesxe2x80x9d are used interchangeably herein.
As used herein, the term xe2x80x9ctransgenexe2x80x9d means a nucleic acid sequence (encoding, e.g., one or more subject Nmi, Omi or Rim polypeptides), which is partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal""s genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout). A transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of the selected nucleic acid, all operably linked to the selected nucleic acid, and may include an enhancer sequence.
As used herein, the term xe2x80x9ctransgenic cellxe2x80x9d refers to a cell containing a transgene.
As used herein, a xe2x80x9ctransgenic animalxe2x80x9d is any animal in which one or more, and preferably essentially all, of the cells of the animal includes a transgene. The transgene can be introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA.
As used herein, the term xe2x80x9ctissue-specific promoterxe2x80x9d means a DNA sequence that serves as a promoter, i.e., regulates expression of a selected DNA sequence operably linked to the promoter, and which effects expression of the selected DNA sequence in specific cells of a tissue, such as mammary tissue. The term also covers so-called xe2x80x9cleakyxe2x80x9d promoters, which regulate expression of a selected DNA primarily in one tissue, but cause expression in other tissues as well.
xe2x80x9cUnrelated to an Nmi, Omi or Rim amino acid or nucleic acid sequencexe2x80x9d means having less than 30% sequence identity, less than 20% sequence identity, or, preferably, less than 10% homology with a naturally occuring Nmi, Omi or Rim sequence disclosed herein.
A polypeptide has Nmi, Omi or Rim biological activity if it has one or more of the properties of Nmi, Omi or Rim disclosed herein. A polypeptide has biological activity if it is an antagonist, agonist, or super-agonist of a polypeptide having one of the properties of Nmi, Omi or Rim disclosed herein. xe2x80x9cMisexpressionxe2x80x9d, as used herein, refers to a non-wild type pattern of gene expression, at the RNA or protein level. It includes: expression at non-wild type levels, i.e., over or under expression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of decreased expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the splicing size, amino acid sequence, post-transitional modification, or biological activity of the expressed polypeptide; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the stimulus.
As described herein, one aspect of the invention features a substantially pure (or recombinant) nucleic acid which includes a nucleotide sequence encoding an Nmi, Omi or Rim polypeptide and/or equivalents of such nucleic acids. The term nucleic acid as used herein can include fragments and equivalents. The term equivalent refers to nucleotide sequences encoding functionally equivalent polypeptides. Equivalent nucleotide sequences will include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants, and include sequences that differ from the nucleotide sequences disclosed herein by degeneracy of the genetic code.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are described in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames and S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames and S. J. Higgins eds. 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; the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.