Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-12-28
2004-08-24
Falk, Anne-Marie (Department: 1636)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C514S04400A, C435S006120
Reexamination Certificate
active
06780987
ABSTRACT:
BACKGROUND OF THE INVENTION
Many eukaryotic cells necessarily possess the ability to move within their environment in response to certain stimuli (e.g. endothelial cells in response to injury, macrophages during immune response). This property of motility is facilitated by coordinated and dynamic modifications to the structure of the cytoskeleton, resulting in net movement. A major cytoskeletal component of motile eukaryotic cells are actin complexes, whose filamentous structure throughout the cell is complex and highly variable. Numerous isoforms of eukaryotic actin have been identified, many of which are found distributed to specific areas of the cell and its cytoskeleton. The ability to disassemble and reassemble actin polymers in a controlled and orderly fashion is central to proper cellular motility. Polymerized actin exists in equilibrium with the monomeric form. The equilibrium constant for the polymerization and depolymerization reactions of actin can be altered by the presence of actin-binding proteins (ABPs). These proteins play an important role in a cell's ability to control cytoskeletal rearrangements, and hence, promote effective cell motility. Many vital functions are served through the ability of some cells to migrate, including response and repair or tissue damage.
SUMMARY OF THE INVENTION
The present invention is based, at least in part, on the discovery of a novel &bgr;-actin specific binding protein and its family members, referred to herein as “beta-cap73” or “BETA-CAP73” or “&bgr;cap73” or “&bgr;CAP73” or “BCAP73” or “bcap73” nucleic acid and protein molecules. The bcap73 molecules of the present invention are useful as modulating agents to regulate a variety of cellular processes, including but not limited to cell motility, directed migration, subcellular distribution of &bgr;-actin, response to injury, and changes in cellular morphology. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding bcap73 proteins or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of bcap73-encoding nucleic acids.
In one embodiment, a bcap73 nucleic acid molecule of the invention is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 83%, 85%, 90%, 95%, 98%, identical to the nucleotide sequence (e.g., to the entire length of the nucleotide sequence) shown in SEQ ID NO:1 or 3, or a complement thereof.
In a preferred embodiment, the isolated nucleic acid molecule includes the nucleotide sequence shown SEQ ID NO: 1 or 3, or a complement thereof. In another embodiment, the nucleic acid molecule includes SEQ ID NO: 3 and nucleotides 392-4597 of SEQ ID NO: 1. In another preferred embodiment, the nucleic acid molecule consists of the nucleotide sequence shown in SEQ ID NO: 1 or 3. In another preferred embodiment, the nucleic acid molecule includes a fragment of at least 1911 nucleotides (e.g., 1911 contiguous nucleotides) of the nucleotide sequence of SEQ ID NO: 1 or 3, or a complement thereof
In another embodiment, a bcap73 nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence sufficiently identical to the amino acid sequence of SEQ ID NO: 2. In a preferred embodiment, a bcap73 nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 83%, 85%, 90%, 95%, 98% or more identical to the entire length of the amino acid sequence of SEQ ID NO: 2.
In another preferred embodiment, an isolated nucleic acid molecule encodes the amino acid sequence of bovine or human or another vertebrate bcap73. In yet another preferred embodiment, the nucleic acid molecule includes a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO: 2. In yet another preferred embodiment, the nucleic acid molecule is at least 1911 nucleotides in length. In a further preferred embodiment, the nucleic acid molecule is at least 1911 nucleotides in length and encodes a protein having a bcap73 activity (as described herein).
Another embodiment of the invention features nucleic acid molecules, preferably bcap73 nucleic acid molecules, which specifically detect bcap73 nucleic acid molecules relative to nucleic acid molecules encoding non-bcap73 proteins. For example, in one embodiment, such a nucleic acid molecule is at least 1911, 1911-2000, 2000-2250, 2250-2500, 2500-2750, 2750-3000, 3000-3250, 3250-3500, 3500-3750, 3750-4000, 4000-4250, 4250-4500, 4500-4736 or more nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO: 1, or a complement thereof. The lengths of nucleic acid molecules of this invention can be within a range using any one of the aforementioned numbers as the upper or lower limit of the range. For example, the nucleic acid molecules can be of a length between at least 1911 and 3000 nucleotides in length.
In preferred embodiments, the nucleic acid molecules are at least 15 (e.g., contiguous) nucleotides in length and hybridize under stringent conditions to nucleotides 1-357, 2268-3135, 4227-4736, of SEQ ID NO: 1. In other preferred embodiments, the nucleic acid molecules comprise nucleotides 1-357, 2268-3135, 4227-47326, of SEQ ID NO: 1. In other preferred embodiments, the nucleic acid molecules consist of nucleotides 1-357, 2268-3135, 4227-4736, of SEQ ID NO: 1.
In other preferred embodiments, the nucleic acid molecule encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising SEQ ID NO: 1 or 3 under stringent conditions.
Another embodiment of the invention provides an isolated nucleic acid molecule which is antisense to a bcap73 nucleic acid molecule, e.g., the coding strand of a bcap73 nucleic acid molecule.
Another aspect of the invention provides a vector comprising a bcap73 nucleic acid molecule. In certain embodiments, the vector is a recombinant expression vector. In another embodiment, the invention provides a host cell containing a vector of the invention. In yet another embodiment, the invention provides a host cell containing a nucleic acid molecule of the invention. The invention also provides a method for producing a protein, preferably a bcap73 protein, by culturing in a suitable medium, a host cell, e.g., a mammalian host cell such as a human or a non-human mammalian cell, of the invention containing a recombinant expression vector, such that the protein is produced.
Another aspect of this invention features isolated or recombinant bcap73 proteins and polypeptides. In one embodiment, the isolated protein, preferably a bcap73 protein, includes at least one ankyrin repeat domain. In another embodiment, the isolated protein, preferably a bcap73 protein, includes an actin binding domain. In another embodiment, the isolated protein, preferably a bcap73 protein, includes an ezrin binding domain. In yet another embodiment, the isolated protein, preferably a bcap73 protein, includes at least one ankyrin repeat domain and an actin binding domain. In a preferred embodiment, the protein, preferably a bcap73 protein, includes at least one ankyrin repeat domain and has an amino acid sequence at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more identical to the amino acid sequence of SEQ ID NO: 2. In another preferred embodiment, the protein, preferably a bcap73 protein, includes an actin binding domain and has an amino acid sequence at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 83%, 85%, 90%, 95%, 98% or more identical to the amino acid sequence of SEQ ID NO: 2. In a further preferred embodiment, the protein, preferably a bcap73 protein, includes at least one actin binding domain and an ankyrin repeat domain and has an amino acid sequence at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 83%, 85%, 90%, 95%, 98% or m
Herman Ira
Welch Alice Y.
DeConti, Jr. Giulio A.
Falk Anne-Marie
Hanley Elizabeth A.
Lahive & Cockfield LLP
Sullivan Daniel M.
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