Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1999-08-06
2001-10-09
Wang, Andrew (Department: 1635)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S387100, C530S387300, C530S388100
Reexamination Certificate
active
06300479
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention provides novel genes, proteins, and uses thereof including, methods for diagnosing and treating cancer, particularly metastatic cancer.
Most eukaryotic cells (execptions include red blood cells and adult muscles) contain high concentrations, i.e., up to ~250 &mgr;mol/l, of momomeric actin. How such actin remains unpolymerized in the cytoplasm has remained a problem in cell biology (Nachmiar, V.,
Current Opinion in Cell Biology
, 1993, 5:56). Profilin, originally thought to be the actin-sequestering protein, is not present in sufficient amounts to account for more than part of the monomeric actin levels observed. Recently, an actin-sequestering 5 kD peptide was discovered in high concentration in human platelets (Safer, et al.,
Proc. Natl. Acad. Sci
USA 1990 87:2536-2540) and shown to be identical to a previously known peptide (Safter, et al.,
J. BIol. Chem
., 1991, 268:4029-4032) originally thought to be the thymic hormone, thymosin &bgr;
4
(T&bgr;
4
) (D. Safer,
J. Muscle Res. Cell Motil
, 1992.13:269-271). A detailed kinetic study of the interaction of T&bgr;
4
and actin (Weber, et al.,
Biochemistry
1992, 31:6179-6185)), together with other studies (Yu, et al.,
J. BIol. Chem
., 1993, 268:502-509 and Cassimelds, et al.,
J. Cell Biol
., 1992, 119:1261-1270) support the hypothesis that T&bgr;
4
and T&bgr;
10
function primary as G-actin buffers. Unpublished data (E. Hannappel) extend the function to several other, &bgr; thymosins. T&bgr;
4
has also been shown to inhibit nucleotide exchange by actin, whereas profilin increases the rate of exchange (Coldschmidt-Clermont, et al.,
Mol. Cell Bio
., 1992, 3:1015-1025).
All vertebrates studied contain one or often two, &bgr;-thymosins. Thus, the members of the &bgr;-thymosin family are believed to be important in all species. Three new family members (Low, et al.,
Arch. Biochem. Biophys
., 1992, 293:32-39 and Schmid, B., Ph.D Thesis, University of Tubingen 1989) have been found in perch, trout and in sea urchin, the first non-vertebrate source. The sequences are well conserved suggesting that actin sequestration is probably a property of all &bgr;-thymosins. However, when T&bgr;
4
was discovered and its sequence first determined in 1981 (Low, et al.,
Proc. Natl. Acad. Sci
., USA 1981, 78:1162-1166), data were presented that suggested two extracellular functions (Low, et al. supra and Rebar, et al., Science 1981, 214:669-671). Two recent papers indicate a different and unexpected effect of a tetrapeptide which may be derived from the amino terminus of T&bgr;
4
.
Several reports demonstrate regulation of T&bgr;
4
or T&bgr;
10
synthesis at the transcriptional or translational level. An interferon-inducible gene (Cassimelds, et al.,
J. Cell. Biol
. 1992, 119:1261-1270 and Sanders, et al.,
Proc. Natl. Acad. Sci
. USA 1992, 89:4678-4682) is identical to the cDNA of human T&bgr;
4
, and there are several genes for T&bgr;
4
in humans. (Clauss, et al.,
Genomies
1991, 9:75-180 and Gomez-Marquez, et al.,
J. Immunol
. 1989, 143:2740-2744)
It would be desirable to identify new members of the &bgr;-thymosin family, particularly in humans.
Bao and Zetter reported in an abstract presented at the American Association for Cancer Research annual meeting (Mar. 18-22, 1995) the differential expression of a novel mRNA expressed in high-metastatic rat tumor cell lines, but not in a low metastatic variant. cDNA was isolated and was reported to encode a protein with 68% identity to the rat thymosin,64. However, the nucleotide sequence and the deduced amino acid sequence were not reported.
SUMMARY OF THE INVENTION
We have now discovered that humans have a gene that encodes a novel protein of the thymosin &bgr; family. This novel protein, herein referred to as thymosin &bgr;15, has the ability to bind and sequester G-actin, like other members of the thymosin &bgr; family, but unlike what is known about other members it also directly regulates cell motility in prostatic carcinoma cells. We have isolated a cDNA of the human thymosin &bgr;15 gene (SEQ ID NO: 1) and have deduced the amino acid sequence (SEQ ID NO: 2). We have shown that enhanced transcripts (mRNA) and expression of the thymosin &bgr;15 gene in non-testicular cells has a high correlation to disease state in a number of cancers, such as prostate, lung, melanoma and breast cancer, particularly metastatic cancers. Accordingly, discovering enhanced levels of transcript or gene product in non-testicular tissues can be used in not only a diagnostic manner, but a prognostic manner for particular cancers.
The present invention provides isolated nucleic acids (polynucleotides) which encode thymosin &bgr;15 having the deduced amino acid sequence of SEQ ID. NO: 2 or a unique fragment thereof. The term “unique fragment” refers to a portion of the nucleotide sequence or polypeptide of the invention that will contain sequences (either nucleotides or amino acid residues) present in thymosin &bgr;15 (SEC ID NO: 2) but not in other member of the thymosin family. This can be determined when the hybridization profile of that fragment under stringent conditions is such that it does not hybridize to other members of the thymosin family. Such fragments can be ascertained from
FIG. 3. A
preferred set of unique fragments are those that contain, or contain polynucleotides that encode, amino acid 7 to 12 of SEQ ID NO: 2, amino acid 21 to 24 of SEQ ID NO: 2 and amino acid 36 to 45 of SEQ ID NO: 2. Preferably, the unique nucleotide sequence fragment is 10 to 60 nucleotides in length, more preferably, 20 to 50 nucleotides, most preferably, 30 to 50 nuceotides. Preferably, the unique polypeptide sequence fragment is 4 to 20 amino acids in length, more preferably, 6 to 15 amino acids, most preferably, 6 to 10 amino acids.
The polynucleotides of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double-stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand. The coding sequence which encodes the mature polypeptides may be identical to the coding sequence shown in SEQ ID NO: 1 or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same protein as the DNA of SEQ ID NO: 1.
The polynucleotide may have a coding sequence which is a naturally occurring allelic variant of the coding sequence shown in SEQ ID NO: 1. As known in the art, an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded protein.
The present invention also provides an isolated polynucleotide segment which hybridize under stringent conditions to a unique portion of the hereinabove-described polynucleotides, particularly SEQ ID NO:1. The segment preferably comprises at least 10 nucleotides. As herein used, the term “stringent conditions” means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. These isolated segments may be used in nucleic acid amplification techniques, e.g., PCR, to identify and/or isolate polynucleotides encoding thymosin &bgr;15.
As used herein a polynucleotide “substantially identical” to SEQ ID NO:1 is one comprising at least 90% homology, preferably at least 95% homology, most preferably 99% homology to SEQ ID NO: 1. The reason for this is that such a sequence can encode thyfnosin &bgr;15 in multiple mammalian species.
The present invention further provides an isolated and purified human thymosin &bgr;15 having the amino acid sequence of SEC ID NO: 2, or a unique fragment thereof, as well as polypeptides comprising such unique fragments, including, for example, amino acid 7 to 12 of SEQ ID NO: 2, amino acid 21 to 24 of SEQ ID NO: 2 and amino acid 36 to 45 of SEQ ID NO: 2.
In accordance with yet another aspect of the present inventi
Bao Lere
Zetter Bruce R.
Children's Medical Center Corporation
Nixon & Peabody LLP
Wang Andrew
LandOfFree
Antibodies specific for human thymosin &bgr;15 protein and... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Antibodies specific for human thymosin &bgr;15 protein and..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Antibodies specific for human thymosin &bgr;15 protein and... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2586230