Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-12-05
2004-01-13
Myers, Carla J. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S007900, C435S091100, C530S300000, C530S350000, C530S387100, C530S388100, C530S389100, C536S023100, C536S023500, C536S024300, C536S024310, C536S024330
Reexamination Certificate
active
06677119
ABSTRACT:
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
Not applicable.
FIELD OF THE INVENTION
The invention relates generally to the fields of molecular biology, genomics, bioinformatics, pathology, and medicine. More particularly, the invention relates to a gene whose expression is modulated in select cancers.
BACKGROUND
With the recent efforts to sequence the entire human genome, the nucleotide sequences of more than 100,000 human genes are expected to be known within the next few years. See, e.g., Robbins, R. J., J. Computat. Biol., 3: 465-478, 1996; Andrade, M. A. and Sander, C., Curr. Opin. Biotechnol., 8: 675-683, 1997; and Collins et al., Science, 282: 682-689, 1998. Once characterized, these genes are anticipated to be useful for identifying new diagnostic and therapeutic targets for a variety of different diseases. Fannon, M. R., Trends Biotechnol., 14: 294-298, 1996. Already several attempts have been made to identify genes or gene products that are uniquely expressed in diseased tissue. The results of these efforts indicated that pathology correlates more often with the pattern of gene expression in the diseased tissue, rather than simply with the absence or presence of a particular gene.
SUMMARY
The invention relates to the discovery of specific polynucleotide sequences that are upregulated in select cancer cells as compared to non-diseased cells. In particular, several expressed sequence tags (ESTs) more prevalent in cancer tissue libraries than in corresponding non-cancerous tissue libraries were identified. These ESTs were then used to identify specific UniGene clusters associated with cancer. See, Schuler, J. Mol. Med. 75(10), 694-698, 1998; Schuler et al., Science 274, 540-546, 1996; and Boguski & Schuler, Nature Genetics 10, 369-371, 1995. Based on the identified polynucleotide sequences, a partial gene sequence termed C4, whose expression is selectively upregulated in colon tumors was identified. Using this partial sequence, a full length gene, termed CCRG (Colon Carcinoma Related Gene) containing the C4 sequence was isolated and sequenced.
An open reading frame of the CCRG gene encodes a polypeptide, i.e., the CCRG protein, which was predicted to have a signal peptide sequence, and putative phosphorylation, myristylation, and glycosylation sites. Based on comparisons to sequences of known function, the nucleotide sequence of CCRG (and C4) was predicted to encode a prokaryotic lipoprotein binding site and a prenylation site. The C-terminus of the CCRG protein is cysteine rich and contains a motif found in ultra high sulphur matrix protein, hair keratin, metallothionein and cation transporters. Using the secondary structure prediction program provided by the ExPASy proteomics server by the Swiss Institute of Bioinformatics (Geneva), CCRG protein was predicted to contain mostly a mixture of alpha helices, beta strands, and coils. The mature CCRG protein has a theoretical molecular weight of 8.62 kDa and a pI of 8.05. These and other analyses indicated that CCRG protein is a colon tumor associated secreted factor.
Accordingly, the invention features a purified nucleic acid present at higher levels in colon cancer cells than in non-cancerous colon cells and includes a nucleotide sequence that encodes a polypeptide sharing at least 80% sequence identity with SEQ ID NO:7 or with a fragment of SEQ ID NO:7 at least 20 residues in length. The nucleotide sequence can be one that defines a polynucleotide whose complement hybridizes under high stringency conditions to the nucleotide sequence of SEQ ID NO:6. The polypeptide encoded by the nucleic acid can have an amino acid sequence consisting of SEQ ID NO:7 or a fragment of SEQ ID NO:7 at least 20 residues in length. The nucleic acid can include a fragment of the polynucleotide sequence of SEQ ID NO:6 at least 50 residues long (e.g., one including the polynucleotide sequence of SEQ ID NO:6).
Also within the invention is a vector including a purified nucleic acid present at higher levels in colon cancer cells than in non-cancerous colon cells, the purified nucleic acid including a nucleotide sequence that encodes a polypeptide sharing at least 80% sequence identity with SEQ ID NO:7 or with a fragment of SEQ ID NO:7 at least 20 residues in length. The nucleic acid contained within this vector can be operably linked to one or more expression control sequences. In another aspect, the invention features a cell including a vector of the invention. including a purified nucleic acid present at higher levels in colon cancer cells than in non-cancerous colon cells.
The invention also provides a probe including an oligonucleotide and a detectable label attached to the oligonucleotide, the oligonucleotide being at least 15 nucleotides in length and hybridizing under high stringency conditions to the nucleotide sequence of SEQ ID NO:7 or a complement of the nucleotide sequence of SEQ ID NO:7.
A kit for detecting a purified nucleic acid including a nucleotide sequence that encodes a polypeptide sharing at least 80% sequence identity with SEQ ID NO:7 or with a fragment of SEQ ID NO:7 at least 20 residues in length in a cell is also within the invention. The kit includes: a first PCR primer including a first nucleic acid molecule including the nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:9, and a second PCR primer including a second nucleic acid molecule including the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:10.
The invention also features a purified polypeptide expressed at higher levels by colon cancer cells than by non-cancerous colon cells. The purified polypeptide includes an amino acid sequence that shares at least 80% sequence identity with SEQ ID NO:7 or a fragment of SEQ ID NO:7 at least 20 residues in length, e.g., one including a fragment of SEQ ID NO:7 at least 20 residues in length or one including residues 31-111 of the amino acid sequence of SEQ ID NO:7. The purified polypeptide can also include the amino acid sequence of SEQ ID NO:7.
A purified antibody that specifically binds to a polypeptide including an amino acid sequence that shares at least 80% sequence identity with SEQ ID NO:7 or a fragment of SEQ ID NO:7 at least 20 residues in length is featured in the invention. This antibody can include a detectable label.
In further aspect, the invention provides a method of producing a CCRG polypeptide. This method includes the steps of: (a) providing a cell transformed with a purified nucleic acid including a nucleotide sequence that encodes a CCRG polypeptide sharing at least 80% sequence identity with SEQ ID NO:7; (b) culturing the cell under conditions that allow expression of the CCRG polypeptide; and (c) collecting the CCRG polypeptide from the cultured cell.
A screening method for identifying a substance that modulates expression of a gene encoding a CCRG polypeptide sharing at least 80% sequence identity with SEQ ID NO:7 is also within the invention. This method includes the steps of: (a) providing a test cell that includes the gene encoding a CCRG polypeptide sharing at least 80% sequence identity with SEQ ID NO:7; (b) contacting the test cell with a candidate substance; and (c) detecting an increase or decrease in the expression level of the gene encoding the CCRG polypeptide in the presence of the candidate substance, compared to the expression level of the gene encoding CCRG polypeptide in the absence of the candidate substance, as an indication that the candidate substance modulates the level of expression of the gene encoding the CCRG polypeptide.
In addition, the invention provides a method for isolating a substance that binds a CCRG polypeptide sharing at least 80% sequence identity with SEQ ID NO:7. This method includes the steps of: (a) providing a sample of the CCRG polypeptide immobilized on a substrate;(b) contacting a mixture containing the CCRG polypeptide-binding substance with the immobilized CCRG polypeptide; (c) separating unbound components of the mixture from bound components of the mixture; and (d) recovering the CCRG polypeptide-binding substance from the immobilized CCRG polypeptide.
A meth
Akerman & Senterfitt
Florida Atlantic University
Kim Stanley
Myers Carla J.
Spiegler Alexander H.
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