Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
2001-06-04
2004-12-28
Wax, Robert A. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S400000
Reexamination Certificate
active
06835813
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a protein and its gene involved in the differentiation of testicular cells and belongs to the field of bioscience, specifically, developmental biology.
BACKGROUND ART
In the developmental process, reproductive cells carry out spermatogenesis via a differentiation process that includes meiosis. This differentiation process is different from that of somatic cells and consists of three main steps. The first step is the proliferation of spermatogenous cells and differentiation into primary spermatocytes. The second is the meiosis of primary spermatocytes, and the third is the transformation into sperms.
Owing to the progress in Molecular Biology, recent years have seen the isolation of several genes specifically expressed in these stages. For example, Hox-1.4 (Propst, F. et al. (1988) Oncogene 2:227-33), ferT (Sarge, K. D. et al. (1994) Biol Reprod 50:1334-1343) of the HSP70 family, and TESK1 (Toshima, J. et al. (1995) J. Biol. Chem. 270:31331-31337) that is a serine-threonine kinase, have been reported as genes specific to primary spermatocytes. However, still very little is known about the biological and physical roles of their gene products.
Genes expressing specifically in the differentiation process of reproductive cells carry a fundamental and vital role that decides the fate of those cells, and thus, defects in these genes are considered to be a cause of diseases such as infertility. Therefore, genes expressing specifically in the differentiation process of reproductive cells are recently gaining wide attention as targets in the development of pharmaceutical drugs. Such drugs can be used for the prevention and treatment of diseases such as infertility caused by defects in reproductive cell differentiation.
DISCLOSURE OF THE INVENTION
The present invention provides a novel protein relating to the differentiation of testicular cells, and the encoding gene. It also provides a vector and transformant used for, for example, producing the protein, and a method of producing the protein. The present invention also provides an oligonucleotide used for the isolation and the detection of the gene of the invention.
The inventors were evaluating the expression of genes encoding unknown proteins that trigger cell death when, irrelevant to their original aim, they unexpectedly succeeded in isolating a novel gene specifically expressed in the testis. When the databases were searched for the isolated gene, it was found to be a novel gene that did not have a significant homologous gene. Structural analysis of the protein encoded by the gene showed that it had in part a structure similar to the metal-binding site of metallothionein, which is known to be a metal-binding factor. Expression analysis in tissues revealed that the gene is extremely specific to the testis, especially to primary spermatocytes. The expression was not seen in the testis of infertile mice. Analysis of the human and mouse chromosomal locations showed that the gene was located in the same site as the gene locus that is known to be defective in infertile mice. Results of these analyses suggest that the protein encoded by the isolated gene is involved in regulating the differentiation of the testis.
The present invention relates to a novel protein involved in the regulation of testicular differentiation having a metal-binding site, and the gene thereof, more specifically:
(1) a protein comprising the amino acid sequence of SEQ ID NO: 4 or 5;
(2) a protein which comprises an amino acid sequence in which one or more amino acids in the amino acid sequence of SEQ ID NO: 4 or 5 have been replaced, deleted, and/or added, and which is functionally equivalent to the protein of (1);
(3) a protein which is encoded by a DNA hybridizing to the DNA comprising the nucleotide sequence of SEQ ID NO: 1 or 3, and which is functionally equivalent to the protein of (1);
(4) a DNA encoding the protein of any one of (1) to (3);
(5) a vector comprising the DNA of (4);
(6) a transformant comprising the DNA of (4) in an expressible manner;
(7) a method of producing the protein of any one of (1) to (3) comprising the steps of culturing the transformant of (6), and collecting the expressed protein from said transformant or the culture supernatant thereof;
(8) an antibody binding to the protein of any one of (1) to (3); and,
(9) a DNA specifically hybridizing to a DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 1 to 3, and comprising at least 15 nucleotides.
The present invention provides the protein Tesmin, which may regulate the differentiation of spermatogenous cells into primary spermatocytes, and the gene thereof.
The inventors isolated two types of Tesmin cDNA of mouse origin arising possibly from splicing differences in the transcriptional process. The nucleotide sequences of these cDNAS are shown in SEQ ID NOs: 1 and 2, and the amino acid sequence of the protein encoded by these cDNAs in SEQ ID NO: 4. The nucleotide sequence of human Tesmin cDNA also isolated by the inventors is shown in SEQ ID NO: 3, and the amino acid sequence of the protein encoded by the cDNA is shown in SEQ ID NO: 5.
As shown in SEQ ID NOs: 1 and 2, mouse-derived Tesmin cDNA has an ORF encoding a protein comprising 295 amino acids. On the other hand, human-derived Tesmin cDNA has an ORF encoding a protein comprising 299 amino acids, as shown in SEQ ID NO: 3. SDS-PAGE analysis of the in vitro translational product of mouse Tesmin using
35
S-labeled methionine showed that mouse Tesmin protein had a molecular weight of 32.5 kDa (FIG.
3
).
Among the tissues within the body, both mouse and human Tesmin genes were expressed only in the testis, as revealed by Northern blot analysis and RT-PCR (FIGS.
1
and
2
). RT-PCR analysis showed that Tesmin gene is hardly expressed in the immature testis up to day 8 following birth, but the expression increases from day 12 when the sperm differentiation starts, and its high expression stabilizes from day 18. In the W/Wv mouse known as an infertile mouse that lacks the growth factor receptor “c-kit” gene, Tesmin gene expression was hardly seen even in the matured testis of day 52 following birth (FIG.
4
). These facts suggest that the Tesmin protein is involved in the differentiation of the testis. The Tesmin protein and its gene can be applied, for example, in the treatment of infertility.
The Tesmin protein of the invention can be prepared by incorporating DNA encoding the protein (e.g., DNA comprising the nucleotide sequence of any one of SEQ ID NO: 1 to 3) into a suitable vector, introducing this into a suitable host cell, and purifying the protein from the transformant obtained. The protein of the present invention can also be prepared as a recombinant protein made using genetic engineering techniques by culturing cells transformed with DNA encoding the Tesmin protein, as mentioned later. The natural protein can be isolated from testicular tissues by methods well known to one skilled in the art, for example, the affinity chromatography later described, using an antibody that binds to the Tesmin protein.
A skilled artisan can prepare not only a natural Tesmin protein, but also a modified protein functionally equivalent to the natural protein by, for example, suitably performing amino acid substitution of the protein using known methods. Amino acid mutations of a protein can occur spontaneously, too. Therefore, the protein of the invention includes a mutant in which the amino acid sequence of the natural protein was mutated by, for example, replacing, deleting, or adding one or several amino acids, and which is functionally equivalent to the natural protein. Methods well known to a skilled artisan for modifying amino acids are, for example, PCR-mediated site-specific-mutation-induction system (GIBCO BRL, Gaithersburg, Md.), oligonucleotide-mediated site-specific-mutagenesis (Kramer, W. and Fritz, H J (1987) Methods in Enzymol. 154:350-367), the Kunkel method (Methods Enzymol. 85:2763-2766 (1988)), and so on. The number of amino acids mutated is normally within ten ami
Kaul Sunil C.
Mitsui Youji
Sugihara Takashi
Wadhwa Renu
Chugai Seiyaku Kabushiki Kaisha
Foley & Lardner LLP
Wax Robert A.
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