huBUB3 gene involved in human cancers

Chemistry: molecular biology and microbiology – Vector – per se

Reexamination Certificate

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C435S069100, C435S325000, C435S455000, C536S023100, C536S023500

Reexamination Certificate

active

06410312

ABSTRACT:

TECHNICAL AREA OF THE INVENTION
The invention relates to the area of cancer diagnostics. More particularly, the invention relates to detection of the loss and or alteration of wild-type huBUB3 genes in tumor tissues.
BACKGROUND OF THE INVENTION
Genes and proteins involved in cell cycle regulation and apoptosis have been found to be important in the development of cancers. There is a continuing need in the art for identification of components of cells which control the cell cycle and apoptosis. These components can be used both diagnostically and therapeutically to identify and detect neoplasms as well as to treat them.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide methods and tools for diagnosing and treating neoplasia. These and other objects of the invention are provided by one or more of the embodiments which are described below.
One embodiment of the invention is an isolated and purified huBUB3 protein having an amino acid sequence which is at least 85% identical to SEQ ID NO:2. Percent identity is determined using a Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 1.
Another embodiment of the invention is an isolated and purified polypeptide comprising at least 8 contiguous amino acids as shown in SEQ ID NO:2.
Even another embodiment of the invention is a huBUB3 fusion protein comprising a first protein segment and a second protein segment fused together by means of a peptide bond. The first protein segment consists of at least 8 contiguous amino acids of a huBUB3 protein as shown in SEQ ID NO:2.
Still another embodiment of the invention is a preparation of antibodies which specifically bind to a huBUB3 protein having an amino acid sequence as shown in SEQ ID NO:2.
A further embodiment of the invention is a cDNA molecule which encodes a huBUB3 protein having an amino acid sequence which is at least 85% identical to SEQ ID NO:2. Percent identity is determined using a Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 1.
Yet another embodiment of the invention is a cDNA molecule which encodes at least 8 contiguous amino acids of SEQ ID NO:2.
Another embodiment of the invention is a cDNA molecule comprising at least 12 contiguous nucleotides of SEQ ID NO:1.
Still another embodiment of the invention is a cDNA molecule which is at least 85% identical to the nucleotide sequence shown in SEQ ID NO:1. Percent identity is determined using a Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 1.
Even another embodiment of the invention is an isolated and purified subgenomic polynucleotide comprising a nucleotide sequence which hybridizes to SEQ ID NO:1 after washing with 0.2×SSC at 65° C. The nucleotide sequence encodes a huBUB3 protein having the amino acid sequence of SEQ ID NO:2.
Yet another embodiment of the invention is a construct comprising a promoter and a polynucleotide segment encoding at least 8 contiguous amino acids of a huBUB3 protein as shown in SEQ ID NO:2. The polynucleotide segment is located downstream from the promoter. Transcription of the polynucleotide segment initiates at the promoter.
Even another embodiment of the invention is a host cell comprising a construct which comprises a promoter and a polynucleotide segment encoding at least 8 contiguous amino acids of a huBUB3 protein having an amino acid sequence as shown in SEQ ID NO:2.
A further embodiment of the invention is a recombinant host cell comprising a new transcription initiation unit. The new transcription initiation unit comprises in 5′ to 3′ order an exogenous regulatory sequence, an exogenous exon, and a splice donor site. The new transcription initiation unit is located upstream of a coding sequence of a huBUB3 gene as shown in SEQ ID NO:1. The exogenous regulatory sequence controls transcription of the coding sequence of the huBUB3 gene.
Still another embodiment of the invention is a pair of single stranded DNA primers. The set allows synthesis of all or part of a huBUB3 gene coding sequence.
Yet another embodiment of the invention is a nucleic acid probe complementary to a wild-type huBUB3 gene as shown in SEQ ID NO:1.
Even another embodiment of the invention is a method of diagnosing a neoplastic tissue of a human. Loss of a wild-type huBUB3 gene or an expression product of the wild-type huBUB3 gene from a tissue suspected of being neoplastic is detected. The wild-type huBUB3 gene has the coding sequence shown in SEQ ID NO:1. The loss indicates neoplasia of the tissue.
Another embodiment of the invention is a method of identifying a neoplastic tissue of a human. Expression of a first huBUB3 gene in a first tissue of a human suspected of being neoplastic is compared with expression of a second huBUB3 gene in a second tissue of the human which is normal. The second huBUB3 gene has the coding sequence shown in SEQ ID NO:1. Decreased expression of the first huBUB3 gene relative to the second huBUB3 gene identifies the first tissue as being neoplastic.
Still another embodiment of the invention is a method to aid in the diagnosis or prognosis of neoplasia in a human. A first huBUB3 gene, mRNA, or protein in a first tissue of a human suspected of being neoplastic is compared with a second huBUB3 gene, mRNA, or protein in a second tissue of a human which is normal. The second huBUB3 gene has the coding sequence shown in SEQ ID NO:1. A difference between the first and second huBUB3 genes, mRNAs, or proteins indicates the presence of neoplastic cells in the first tissue.
Even another embodiment of the invention is a method to aid in detecting a genetic predisposition to neoplasia in a human. A huBUB3 gene, mRNA, or protein in the fetal tissue of a human is compared with a wild-type huBUB3 gene, mRNA, or protein. The wild-type huBUB3 gene has the coding sequence shown in SEQ ID NO:1. A difference between the huBUB3 gene, mRNA, or protein in the fetal tissue of the human and the wild-type huBUB3 gene, mRNA, or protein indicates a genetic predisposition to neoplasia in the human.
Yet another embodiment of the invention is a method of screening test compounds for the ability to interfere with the binding of a huBUB3 protein to a huBUB1 protein. A test compound is contacted with at least a huBUB3-binding domain of a huBUB1 protein as shown in SEQ ID NO:4 and at least a huBUB1-binding domain of a huBUB3 protein as shown in SEQ ID NO:2. The huBUB3-binding domain binds to the huBUB1-binding domain in the absence of the test compound. The amount of the huBUB1-binding domain which is bound or unbound to the huBUB3-binding domain or the amount of the huBUB3-binding domain which is bound or unbound to the huBUB1-binding domain in the presence of the test compound is determined. A test compound which decreases the amount of bound huBUB1- or huBUB3-binding domains or which increases the amount of unbound huBUB1- and huBUB3-binding domains is a potential inducer of mitosis or cell cycle progression.
Even another embodiment of the invention is a method of screening test compounds for the ability to interfere with the binding of a huBUB1 protein to a huBUB3 protein. A cell is with a test compound. The cell comprises a first fusion protein, a second fusion protein, and a reporter gene. The first fusion protein comprises (1) at least a huBUB1-binding domain of a huBUB3 protein as shown in SEQ ID NO:2 and (2) either a DNA binding domain or a transcriptional activating domain. The second fusion protein comprises at least a huBUB3-binding domain of a huBUB1 protein as shown in SEQ ID NO:4. The huBUB1-binding domain binds to the huBUB3-binding domain. If the first fusion protein comprises a DNA binding domain, then the second fusion protein comprises a transcriptional activating domain. If the first fusion protein comprises a transcriptional activating domain, then the second fusion protein comprises a DNA binding domain. The interaction of the

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