Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-03-13
2001-03-27
Burke, Julie (Department: 1642)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023200, C536S023400, C536S023500, C536S023510, C536S023520, C536S023530, C536S024300, C536S024310, C536S024320, C536S024330, C530S350000, C530S358000
Reexamination Certificate
active
06207812
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to bone malignancies.
Chondrosarcoma, which usually occurs in late adulthood and old age, is the second most common form of bone malignancy. Conventional chondrosarcoma tumors are graded from stage I through stage III, stage III being the most advanced. In addition to conventional chondrosarcoma, there are other types of chondrosarcoma with distinguishing characteristics: myxoid, mesenchymal, clear cell, and dedifferentiated (spindle cell) chondrosarcoma.
Diagnosis and grading of chondrosarcoma has been problematic. For example, the criteria used to distinguish benign enchondroma from low grade chondrosarcoma include parameters which are difficult to quantify such as increased cellularity and more than occasional binucleate cells. The histologic criteria are not absolute, and the diagnosis is frequently made by taking into account clinical features such as pain, rate of growth, location, and radiologic features. Furthermore, the location of the tumor may affect clinical assessment. For example, lesions in the hand can appear aggressive histologically and yet behave benignly. In contrast, lesions occurring in the pelvis are likely to represent a malignancy despite a relatively innocuous histologic appearance. Notwithstanding attempts to integrate clinicopathologic criteria, it has not been possible to predict which tumors will metastasize or recur.
SUMMARY OF THE INVENTION
The invention is based on the discovery of a novel gene which is differentially expressed in chondrosarcoma cells. Accordingly the invention features an isolated nucleic acid (e.g., genomic DNA, cDNA or synthetic DNA) encoding a chondrosarcoma associated (CSA) polypeptide such as human CSA-1. The term “chondrosarcoma associated” refers to the property of differential expression in chondrosarcoma cell compared to normal cartilage cells. For example, a CSA gene product is expressed at a detectably higher or lower level compared to the level at which it is expressed in normal cartilage cells. A CSA gene product may be expressed solely in chondrosarcoma cells (and not in normal cartilage cells).
The nucleic acid molecule contains a nucleotide sequence encoding a polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence of CSA-1 (SEQ ID NO:2). Preferably, the nucleic acid molecule contains the nucleotide sequence of SEQ ID NO:1 or a degenerate variant thereof. For example, the nucleic acid contain the nucleotide sequence of SEQ ID NO:3. The invention also includes a nucleic acid molecule which contains a strand which hybridizes at high stringency to a DNA having the sequence of SEQ ID NO:1, or the complement thereof. A substantially pure DNA having at least 50% sequence identity (preferably at least 70%, more preferably at least 80%, and most preferably at least 90%) to SEQ ID NO:1, and encoding a polypeptide having the differential pattern of expression of a CSA-1 polypeptide is also within the invention. For expression of a CSA polypeptide, a CSA polypeptide encoding nucleic acid molecule is operably linked to regulatory sequences, e.g., a promoter.
The invention also includes a substantially pure CSA polypeptide such as human CSA-1 or a fragment thereof. CSA-1 fragments, e.g., a fragment containing the amino acid sequence of SEQ ID NO: 8), are useful as immunogens for raising anti-CSA antibodies. The CSA polypeptide preferably contains an amino acid sequence that is at least 50% identical to the amino acid sequence of SEQ ID NO:2. More preferably the amino acid sequence of the polypeptide is 75%, 85%, 95%, 98%, and most preferably 100% identical to the amino acid sequence of SEQ ID NO:2. A cell containing a CSA polypeptide-encoding nucleic acid molecule is also within the invention, as is a method of making a CSA polypeptide. Such a method may involve the following steps: (a) providing cell containing a CSA polypeptide-encoding nucleic acid molecule, and (b) culturing it under conditions permitting expression of the nucleic acid molecule.
By “isolated nucleic acid molecule” is meant a nucleic acid molecule that is free of the genes which, in the naturally-occurring genome of the organism, flank a csa gene. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; 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 or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence. The term excludes large segments of genomic DNA, e.g., such as those present in cosmid clones, which contain a gene of interest, e.g., a csa gene, flanked by one or more other genes which naturally flank it in a naturally-occurring genome.
Nucleic acid molecules include both RNA and DNA, including cDNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA. Where single-stranded, the nucleic acid molecule may be a sense strand or an antisense strand. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote at a site other than its natural site; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by polymerase chain reaction (PCR) or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
Hybridization is carried out using standard techniques such as those described in Ausubel et al.,
Current Protocols in Molecular Biology,
John Wiley & Sons, (1989). “High stringency” refers to DNA hybridization and wash conditions characterized by high temperature and low salt concentration, e.g., wash conditions of 65° C. at a salt concentration of approximately 0.1×SSC. “Low” to “moderate” stringency refers to DNA hybridization and wash conditions characterized by low temperature and high salt concentration, e.g. wash conditions of less than 60° C. at a salt concentration of at least 1.0×SSC. For example, high stringency conditions may include hybridization at about 42° C., and about 50% formamide; a first wash at about 65° C., about 2× SSC, and 1% SDS; followed by a second wash at about 65° C. and about 0.1% ×SSC. Lower stringency conditions suitable for detecting DNA sequences having about 50% sequence identity to csa-1 gene are detected by, for example, hybridization at about 42° C. in the absence of formamide; a first wash at about 42° C., about 6× SSC, and about 1% SDS; and a second wash at about 50° C., about 6× SSC, and about 1% SDS.
Where a particular polypeptide or nucleic acid molecule is said to have a specific percent identity to a reference polypeptide or nucleic acid molecule of a defined length, the percent identity is relative to the reference polypeptide or nucleic acid molecule. Thus, a peptide that is 50% identical to a reference polypeptide that is 100 amino acids long can be a 50 amino acid polypeptide that is completely identical to a 50 amino acid long portion of the reference polypeptide. It might also be a 100 amino acid long polypeptide which is 50% identical to the reference polypeptide over its entire length. Of course, many other polypeptides will meet the same criteria. The same rule applies for nucleic acid molecules.
For polypeptides, the length of the reference polypeptide sequence will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids, 50 amino acids, or 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 100 nucleot
Beattie Ingrid A.
Burke Julie
Elrifi Ivor R.
Mintz Levin Cohn Ferris Glovsky and Popeo P.C.
Rhode Island Hospital
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