Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-05-08
2002-12-03
Bugalsky, Gabrielle (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C435S252300, C435S252330, C435S254110, C435S325000, C435S410000, C536S023500, C536S024300, C536S024310
Reexamination Certificate
active
06489136
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to the Nmi, Omi and Rim genes, their products, and uses thereof.
SUMMARY OF THE INVENTION
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 inclu
Bugalsky Gabrielle
Fish & Richardson PC
The General Hospital Corporation
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