# Patent

## Title
Testis-specific differentiation-regulatory factor

## Abstract

A gene expressed specifically in the testis has been unexpectedly isolated in the course of studies of the expression of a gene encoding an unknown protein that triggers cell death. The isolated gene was a novel gene sequence that had no significant homologue in the database. This gene was also found to be involved in the regulation of differentiation in the testis.

## Background
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
This application is a divisional of international application number PCT/JP99/03859, with an international filing date of Jul. 16, 1999, which entered the national stage in the U.S. under 35 U.S.C. § 371 as U.S. patent application Ser. No. 09/743,237 on Jun. 4, 2001, now U.S. Pat. No. 6,835,813 and further claims the benefit of Japanese Patent Application No. 10-219856, filed Jul. 17, 1998.


All priority documents, including the specification, drawings, claims and abstract, are incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
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 OF THE INVENTION
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.
SUMMARY OF THE INVENTION
Accordingly, the present invention provides a novel gene and protein involved in differentiation of testicular cells. It also provides a vector and a transformant useful in producing the protein as well as a method of producing the protein. The present invention further provides an oligonucleotide useful in the isolation and the detection of the gene of the invention.
In one embodiment, the invention provides an isolated DNA encoding a protein comprising SEQ ID NO: 4 or 5.
In another embodiment, the invention provides a vector comprising a DNA encoding a protein comprising SEQ ID NO: 4 or 5.
In a further embodiment, the invention provides a transformant comprising a DNA encoding a protein comprising SEQ ID NO: 4 or 5.
In yet another embodiment, the invention provides a method of producing a protein comprising culturing a transformant comprising the DNA encoding a protein comprising SEQ ID NO: 4 or 5 under conditions where the transformant encodes a protein and obtaining the protein from the transformant or the culture supernatant thereof.
In a further embodiment, the invention provides an isolated 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.
In still another embodiment, the invention provides an isolated DNA encoding a protein which comprising an amino acid sequence in which one of several amino acids of SEQ ID NO: 4 or 5 have been replaced, deleted, and/or added, and which is functionally equivalent to a protein comprising SEQ ID NO: 4 or 5.
In yet a further embodiment, the invention provides an isolated DNA encoding a protein which is encoded by a DNA hybridizing under stringent conditions to the DNA comprising the nucleotide sequence of SEQ ID NO: 1 or 3, wherein the protein has testicular differentiation activity.
In a further embodiment, the invention provides a method of producing a protein comprising culturing a transformant comprising a DNA under conditions wherein a protein is expressed, wherein the protein comprises an amino acid sequence in which one of several amino acids of SEQ ID NO: 4 or 5 have been replaced, deleted, and/or added, and wherein the protein is functionally equivalent to a protein comprising SEQ ID NOs: 4 or 5; and obtaining the protein from the transformant or the culture supernatant thereof.
In yet another embodiment, the invention provides a method of producing a protein comprising culturing a transformant comprising DNA which hybridizes under stringent conditions to a DNA comprising a nucleotide of SEQ ID NO: 1 or 3 under conditions where a protein is expressed and obtaining the protein from the transformant or the culture supernatant thereof.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only, and are not restrictive of the invention as claimed.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an electrophoretic photograph showing the results of Northern blot analysis of Tesmin gene expression in various mouse tissues.
FIG. 2 is an electrophoretic photograph showing the results of Northern blot analysis of Tesmin gene expression in various human tissues.
FIG. 3 is an electrophoretic photograph showing the results of the molecular weight detection of mouse Tesmin protein expressed by in vitro translation.
FIG. 4 is an electrophoretic photograph showing the results of Northern blot analysis of Tesmin gene expression in the testis of ICR strain mouse at day 4, 8, 12, 18, and 42 following birth, and in the day 56 testis of W/Wv strain mouse. “MEG1” and “ssH2B” were used as testis differentiation markers. “GAPDH” was used as the control.
FIG. 5 is a photomicrograph showing the results of detection of Tesmin gene expression in testicular tissues by in situ hybridization.
FIG. 6 is a photomicrograph and schematic diagram of the chromosomal location showing the results of the detection of Tesmin gene location in the mouse chromosome using a probe specific to the Tesmin gene.
FIG. 7 is a photomicrograph and schematic diagram of the chromosomal location showing the results of the detection of Tesmin gene location in the human chromosome using a probe specific to the Tesmin gene.
FIG. 8 is a photomicrograph showing the intracellular localization of the complete Tesmin protein and its deletion mutant.
FIG. 9 shows the results of the detection of the Tesmin protein (fusion protein with GST) by Western blotting using the prepared anti Tesmin antibody. Detection using anti GST antibody was also done concurrently. “IPTG+” means the protein detected by adding isopropyl-β-D-thiogalactoside (IPTG) to CDNA-introduced E. coli to induce the expression of the recombinant protein, subjecting the cell lysate to SDS-PAGE and Western-blotting, and “EPTG-” means the protein detected in a lysate of E. coli to which IPTG was not added.



DETAILED DESCRIPTION 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 35S-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, PCR-mediated site-specific-mutation-induction system (GIBCO BRL, Gaithersburg, Maryland), 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 amino acids, preferably within six amino acids, and more preferably within three amino acids.
Herein, “functionally equivalent” means that the mutant protein has a biochemical and/or biological activity equivalent to the natural protein. As such activities, for example, the binding activity between the protein and metal, and the testicular cell differentiation-inducing activity can be given.
The metal-binding activity can be detected, for example, as follows. First, the recombinant Tesmin protein is EDTA-treated to remove heavy metals that may be bound to the Tesmin protein. Next, EDTA is removed by gel filtration, and then, the heavy metals (for example, Zn2+, Cd2+, Cu2+, etc.) to be examined are added and reacted with the recombinant Tesmin protein. After reacting, the presence or absence of a metal bond is detected as CD spectra using a CD spectropolarimeter (J-500C by Jasco) (refer Presta A. et al., Eur. J. Biochem January 15; 227(1-2):226-240).
The testicular cell differentiation-inducing activity can be detected, for example, as follows. First, spermatogoniums, spermatogenous cells, and primary spermatocytes are isolated from mouse testis by centrifugation. Next, Tesmin gene is incorporated into an expression vector (e.g., pBK-CMV vector, Stratagene), and the gene incorporated is introduced to cells isolated by lipofectAMINE (GIBCO BRL). After culturing the cells from a few hours to a few days, the expression of a genetic marker that identifies the differentiation stage (e.g., MEG1, ssH2B, etc.) is verified by the RT-PCR method.
The hybridization technique (Sambrook, J et al., Molecular cloning 2nd ed. 9.47-9.58, Cold Spring Harbor Lab. press, 1989) is well known to a skilled artisan as an alternative method for isolating a functionally equivalent protein. In other words, it is a general procedure for a skilled artisan to isolate DNA having a high homology to the whole or part of the DNA encoding the mouse or human Tesmin protein (a DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 1 to 3) and to obtain a protein functionally equivalent to the mouse or human Tesmin protein from the isolated DNA. Therefore, the protein of the present invention also includes a protein encoded by DNA hybridizing to DNA encoding the mouse or human-derived Tesmin protein, which is functionally equivalent to these proteins. When isolating the hybridizing DNA from other organisms, there is no restriction as to the organisms used, although testicular tissues from, for example, rats, rabbits, and cattle are suitable for the isolation. DNA isolated by hybridization techniques usually has a high homology to DNA encoding the mouse- and human-derived Tesmin protein (DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 1 to 3). “High homology” means, a sequence identity at the amino acid level of at least 40% or more, preferably 60% or more, more preferably 80% or more, and even more preferably, 95% or more. The homology of a sequence can be calculated,: for example, by the method described in Proc. Natl. Acad. Sci. USA (1983) 80:726-730.
An example of hybridization conditions (stringent) for isolating a DNA high in homology is as follows. Namely, after conducting a prehybridization at 68° C. for 30 min or more using the “Rapid-hyb buffer” (Amersham LIFE SCIENCE), a labeled probe is added, and hybridization is done by incubating at 68° C. for 1 hr or more. After that, washing is done three times within 2×SSC/0.01% SDS for 20 min at room temperature, and next, three times within 1×SSC/0.1% SDS, at 37° C. for 20 min, followed by, two times within 1×SSC/0.1% SDS, at 50° C. for 20 min.
This invention also provides a DNA encoding the Tesmin protein. The DNA of the present invention includes genomic DNA, synthetic DNA, and such, as well as cDNA, as long as such DNA encodes the Tesmin protein of the invention. The DNA of the invention can be used, for example, for producing recombinant proteins. Namely, the recombinant proteins can be prepared by inserting the DNA of the invention (e.g., SEQ ID NOs: 1 and 2) into a suitable expression vector, introducing this into a suitable cell, culturing the resulting transformant, and purifying the protein expressed. Cells used for the production of recombinant proteins are, for example, mammalian cells such as COS cells, CHO cells, and NIH3T3 cells; insect cells such as Sf9 cells; yeast cells; and E. coli, but there is no restriction as to the cells used. The vector for expressing the recombinant protein within cells varies according to the host cell, and, for example, pcDNA3 (Invitrogen), and pEF-BOS (Nucleic Acids. Res. 1990, 18 (17), p5322) and such are given as vectors for mammalian cells, Bac-to-BAC baculovirus expression system (GIBCO BRL) and such for insect cells, Pichia Expression Kit (Invitrogen) and such for yeast cells, and pGEX-5×-1 (Pharmacia) and QIAexpress system (Qiagen) and such for E. coli. Vectors can be introduced into hosts for example, by the calcium phosphate method, DEAE dextran method, the method using cationic liposome DOTAP (Boehringer Mannheim), electroporation method, calcium chloride method, etc. Transformants can be cultured according to their properties using methods well known to skilled artisans. Recombinant proteins can be purified from transformants by methods well known to skilled artisans, for example, the methods described in reference “The Qiaexpressionist handbook, Qiagen, Hilden, Germany.”
The present DNA can be used for gene therapy of diseases caused by mutations of the gene. The Tesmin gene especially may be the causative of the genetic disease of infertile mice, and therefore, is expected to be applied in the gene therapy of infertility. When using for gene therapy, the DNA of the invention is inserted into, for example, a viral vector such as an adenovirus vector (e.g. pAdexLcw) and a retrovirus vector (e.g. pZIPneo), or a non-viral vector, and administered to a target site of the body. The method of administration may be ex vivo or in vivo.
The present invention also provides an antibody that binds to the protein of the invention. The antibody of the present invention includes polyclonal antibodies and monoclonal antibodies. These antibodies can be prepared by following methods well known to skilled artisans. Polyclonal antibodies can be made by, for example, obtaining the serum of small animals such as rabbits immunized with the protein (or a partial peptide) of the present invention, and purifying by, for example, ammonium sulfate precipitation, a protein A or protein G column, etc. Monoclonal antibodies can be made by immunizing small animals such as mice with the protein (or a partial peptide) of the present invention, excising the spleen from the animal, homogenizing the organ into cells, fusing the cells with mouse myeloma cells using a reagent such as polyethylene glycol, selecting clones that produce antibodies against the protein of the invention from the fused cells (hybridomas), transplanting the obtained hybridomas into the abdominal cavity of a mouse, and collecting ascites from the mouse. The obtained monoclonal antibodies can be purified by, for example, ammonium sulfate precipitation, a protein A or protein G column, etc. The antibody thus prepared can be applied for antibody therapy and such, other than for the purification and detection of the protein of the invention. When administrating the antibody to humans with the aim of antibody therapy, humanized antibodies are effective in decreasing immunogenicity. Antibodies can be humanized by, for example, cloning the antibody gene from monoclonal antibody producing cells and using the CDR graft method which transplants the antigen-recognition site of the gene into a known human antibody. Human antibodies can also be prepared like ordinary monoclonal antibodies by immunizing a mouse whose immune system has been replaced by a human immune system with the protein of the invention.
This invention also provides a DNA specifically hybridizing to DNA encoding the Tesmin protein and comprising at least 15 nucleotides. The term “specifically hybridizing” as used herein indicates that cross-hybridization does not significantly occur with DNA encoding proteins other than the Tesmin protein, under the usual hybridization conditions, preferably under stringent hybridization conditions. Such DNA can be used as a probe for detecting or isolating DNA encoding the Tesmin protein, or as a primer for amplification. Tesmin gene is expressed only in the testis, and even in the testis, it is expressed for a limited period. Therefore, the DNA can be used as a testis differentiation marker (a test drug). Also, there is a possibility that the Tesmin gene is the causative gene of the genetic disease of infertile mice, and therefore, the DNA may be used for the testing of infertility.
INDUSTRIAL APPLICABILITY
The present invention provides the Tesmin protein comprising a metal-binding site, which is closely associated with the differentiation of testicular cells, and the gene thereof. The Tesmin protein and the gene thereof are involved in the differentiation during spermatogenesis, and Tesmin gene expression is not seen in infertile mice. Therefore, this gene may also be the causative gene of the genetic disease of infertile mice. Hence, it is anticipated that gene therapy of infertility would be possible by introducing the Tesmin gene into the body or cells. Moreover, Tesmin is expressed in the testis only, and even in the testis, the expression is seen only at limited stages. Therefore, Tesmin may also be applied as a test drug for determining the differentiation stage of testicular cells. Tesmin is also thought to contain a metal-binding site similar to metallothionein, and therefore, can also be applied as a metal-poison neutralizing agent similar to metallothionein. It is also expected to be utilized in applied studies such as those analyzing the importance of metal binding in the testis.
The following examples are given to illustrate the present invention. It should be understood, however, that the present invention is not to be limited to the specific embodiments described in these examples. It will be apparent to those skilled in the art that various modifications and variations can be made to the embodiments of the present invention without departing from the spirit or scope of the present invention. Thus, it is intended that the present invention covers other modifications and variations of this invention within the scope of the appended claims and their equivalents.
EXAMPLE 1
Isolation of Tesmin Gene Fragment Using RT-PCR
The expression of the novel substance WF-1 (a function-unknown novel gene comprising 1700 bp) in each organ was analyzed by the RT-PCR method. Specifically, total RNA was extracted from the brain, liver, spleen, kidney, heart, and testis of ICR strain mice (Clea Japan) using ISOGEN (NIPPON GENE). After denaturing RNA at 65° C., cDNA was prepared using reverse transcriptase: superscript 2 (GIBCO BRL). Using cDNA from each organ, and the oligo primers for WF-1 amplification described in SEQ ID NOs: 6 and 7, PCR reaction was conducted for 32 cycles of 94° C. for 1 min, 58° C. for 2 min, and 72° C. for 3 min. The control GAPDH was amplified by PCR using the oligo primers described in SEQ ID NOs: 8 and 9 under the condition of 30 cycles of 94° C. for 1 min, 58° C. for 2 min, and 72° C. for 3 min. As a result, a gene specifically expressed only in the testis was unexpectedly found through a detection using oligo primers for WF-1 amplification described in SEQ ID NOs: 6 and 7. This cDNA fragment was isolated, and the gene encoded by the cDNA was named “Tesmin” (first named “Testin,” but later changed to “Tesmin”).
EXAMPLE 2
Cloning and Sequencing of Mouse Tesmin cDNA
The sequence of the above cDNA fragment was determined by the dideoxy chain termination method and analyzed by the ABI377 auto sequencer. As a result of a database search for the determined sequence, this sequence was revealed to be a novel gene that did not have a homology to genes in the databank. This cDNA fragment was 32P radio-labeled to prepare a probe, and using this, a mouse testis library was screened. As a result, a clone having an approximately 1.7 kb length was obtained.
Moreover, 5′-RACE was conducted to determine the 5′ end sequence. In 5′-RACE, three antisense primers specific to the Tesmin gene, namely, SP1 (SEQ ID NO: 10), SP2 (SEQ ID NO: 11), and SP3 (SEQ ID NO: 12), and mouse testis-derived 5′-Marathon RACE cDNA were used. The 5′-RACE method was conducted following the protocol provided in the Marathon-ReadyTM cDNA kit (mouse testis), which is commercially available from Clontech. The whole nucleotide sequence of Tesmin cDNA obtained is shown in SEQ ID NOs: 1 (2241 bp) and 2 (1861 bp). These two cDNAs are thought to be splicing variants arising from a difference in splicing at the point of transcription.
When the database was searched using these cDNA sequences, no sequence comprising a significant homology was found in the databank. These cDNAs encode the same protein comprising 295 amino acids (pI-7.64), and no significantly homologous proteins were found in the protein database as well.
EXAMPLE 3
Cloning and Sequencing of Human Tesmin cDNA
Mouse Tesmin plasmid (a plasmid in which the Tesmin gene has been inserted into the pBluescript2 vector) was cleaved by SphI-Sa1I, and this 1.7 kb gene fragment was used as a probe to screen the cDNA library prepared by human testis MRNA. Hybridization was done using the “Rapid-hyb buffer” (Amersham LIFE SCIENCE) under the following conditions: (i) a prehybridization at 60° C. for 30 min, (ii) addition of the labeled probe, and (iii) hybridization by incubating at 60° C. for 2 hr. After that, washing is done three times within 2×SSC, 0.01% SDS for 20 min at room temperature, and next, three times within 1×SSC, 0.1% SDS, at 37° C. for 20 min, followed by, two times within 1×SSC, 0.1% SDS, at 50° C. for 20 min.
The nucleotide sequence of thus obtained human Tesmin cDNA is shown in SEQ ID NO: 3. Database search for the determined nucleotide sequence was done but there were no homologous sequences within the databank, similar to the mouse cDNA. The obtained human cDNA had four amino acids more than mouse Tesmin and encoded a protein comprising 299 amino acids (pI-7.71). No significant homology was found in the protein database as well. However, as a result of amino acid sequence analysis by BLAST, the mouse and human Tesmins were found to be cysteine-rich proteins partially having the structure very similar to the metal-binding domain of the metallothionein family.
Metallothionein expression in the liver is induced by heavy metals, and metallothionein is known as a protein that neutralizes metallic poison. However, in the testis, the metallothionein gene is constantly expressed and is not induced by metals. Therefore, it was thought to play some vital roles other than metal binding in the testis. Recent findings showed that the estrogen receptor, which is a zinc-finger transcription factor and a receptor protein, and metallothionein conduct metal transfer in vitro (Cano-Gauci, D. and Sarkar, B. (1996) FEBS Lett 386 (1):1-4). Therefore, the metal-binding site of metallothionein is thought to play a vital role in the regulation of transcription factors. The “Cys-X-Cys-X-X-X-X-X-X-X-X-X-Cys-X-Cys (where it is an arbitrary amino acid)” sequence (SEQ ID NO: 23) having a cysteine structure in the amino acid sequence is thought to be vital for metal binding in the metallothionein family.
This cysteine structure (in mouse, from the 157th to the 171st positions, in human, from the 161st to the 175th positions) was conserved in Tesmin too. However, the metallothionein family members known up to now were relatively low-molecular comprising 60 to 70 amino acids, whereas Tesmin was comparatively longer (mouse: 295 amino acids, human: 299 amino acids). Domain search by PROSITE revealed that mouse Tesmin had a N-myristylation site and a casein kinase 2 phosphorylation site. Human Tesmin had a cAMP and cGMP-dependent kinase phosphorylation site, a protein kinase C phosphorylation site, a N-myristylation site, and a N-glycosylation site. Other than those, a cAMP and cGMP-dependent protein kinase phosphorylation site and a protein kinase phosphorylation site were also present.
Domain search by BLOCKS revealed sites common to mouse and human Tesmins. Namely, “high potential iron-sulfate protein” (from 87th to 103rd positions in mouse, from 87th to 103rd positions in human), “Adenodoxin family” (iron-sulfate binding region) (from 177th to 194th positions in mouse, from 181st to 198th positions in human), “Alpha-2-mavrogloburin family thiolester region” (from 243rd to 252nd positions in mouse, from 247th to 256th in human), “Arrestins proteins” (from 267th to 277th positions in mouse, from 271st to 281st positions in human), “Ribosomal protein L14 proteins” (from 5th to 26th positions in mouse, from 5th to 26th positions in human), “Cooper amine oxidase topaquinone proteins” (from 81st to 109th positions in mouse, from 81st to 109th positions in human), and “VFWC domain proteins” (from 13th to 19th, positions and from 105th to 113th positions in mouse, from 105th to 113th positions in human) were confirmed in mouse and human Tesmins. When sequence features were analyzed by PRINTS, both mouse and human Tesmins had a “Rhodopsin-like GPCR superfamily signature” (from 93rd to 117th positions, from 231st to 252nd positions, and from 232nd to 253rd positions in mouse, from 43rd to 67th positions and from 236th to 257th positions in human).
EXAMPLE 4
Transcription and Translation In Vitro
In vitro translation was done to verify the open reading frame anticipated in mouse Tesmin. Specifically, the cDNA pBluescript-Tesmin cloned from the testis was transcribed and translated for one hour in vitro using the rabbit reticulocyte lysate (Promega Corp.) to which L-[35S] methionine has been added. Translation products were separated by SDS-PAGE, and detected by autoradiography. As a result, a protein of approximately 32.5 kDa was detected (FIG. 3). This product coincided well with the size of the protein thought to be the ORF within the mouse Tesmin sequence.
EXAMPLE 5
Preparation of Recombinant Tesmin
The open reading frame of mouse Tesmin cDNA was amplified by a PCR reaction using sense (SEQ ID NO: 19) and antisense (SEQ ID NO: 20) primers having an EcoRI site. The fragment amplified by the PCR reaction was cloned to the pGEM-T vector to verify its precise sequence. Next, it was cleaved by EcoRI-EcoRI, and finally cloned to pGEX-2TK vector that produces GST fusion protein. Tesmin product cloned into pGEX-2TK was gene transfected into E. coli JM109, induced using IPTG 0.2 mM at 37° C. for 3 hr, and the E. coli lysate was separated by SDS-PAGE, and detected by Western blotting using the GST antibody. As a result, a 58.5 kDa protein comprising a GST fusion portion with a molecular weight of 26 kDa was synthesized (FIG. 8 left). The recombinant protein had the same size expected by the presumed size of the molecule, similar to the result of in vitro translation.
EXAMPLE 6
Northern Blot Analysis
A membrane loaded with 2 μg/lane of various mouse and human tissue MRNA was purchased (Clontech laboratories, Palo Alto, Calif.), and Northern blot analysis was conducted. The probe was a 1.7 kb gene fragment made by cleaving mouse Tesmin plasmid (a plasmid in which the Tesmin gene has been inserted into pBluescript2 vector) with SphI-Sa1I. Hybridization was done using the “Rapid-hyb buffer” (Amersham LIFE SCIENCE) under the following conditions: (i) a prehybridization at 68° C. for 30 min,(ii) addition of the labeled probe, and, (iii) hybridization by incubating at 68° C. for 2 hr. Next, washing is done three times within 2×SSC, 0.01% SDS for 20 min at room temperature, and next, three times within 1×SSC, 0.1% SDS, at 37° C. for 20 min, followed by, two times within 1×SSC, 0.1% SDS, at 50° C. for 20 min. Detection was done by autoradiography. Similar to the results of RT-PCR, Tesmin gene expression was detectable in the testis only, and gene expression was seen at the 2.4 kb and 2.0 kb locations in mouse (FIG. 1) and in just the 2.4 kb location in human (FIG. 2).
EXAMPLE 7
Involvement in the Differentiation of Reproductive Cells
Total RNA was extracted from the testis of ICR strain mouse at day 4, 8, 12, 18, and 42 following birth, and from the day 56 testis of W/Wv strain mouse (Japan SLC; type WBB6F1-W/Wv known as an infertile mouse deficient of the mouse growth factor S1 receptor c-kit gene; refer Chabot, B. et al. (1988) Nature 335 (6185):88-9, Yoshinaga, K. et al. (1991) Development 113 (2):689-99) using ISOGEN (NIPPON GENE). After denaturing this RNA at 65° C., cDNA was prepared using reverse transcriptase: superscript 2 (GIBCO BRL). Tesmin gene was amplified using the oligo primers described in SEQ ID NOs: 6 and 7, under the conditions of 35 cycles of 94° C. for 1 min, 58° C. for 2 min, and 72° C. for 3 min. The control GAPDH gene was amplified using the oligo primers described in SEQ ID NOs: 8 and 9, under the conditions of 30 cycles of 94° C. for 1 min, 58° C. for 2 min, and 72° C. for 3 min. For the MEG1 PCR reaction, the oligo primers described in SEQ ID NOs: 13 and 14 were used, under conditions of 35 cycles of 94° C. for 1 min, 58° C. for 2 min, and 12° C. for 3 min. For the ssH2B reaction, the oligo primers described in SEQ ID NOs: 15 and 16 were used, under the conditions of 35 cycles of 94° C. for 1 min, 58° C. for 2 min, and 72° C. for 3 min. Marker MEG1 expresses when spermatogenous cells divide into primary spermatocytes (Don, J. and Wolgemuth, D. J. (1992) August; 3 (8):495-505), and marker ssH2B is known to express at spermatogenesis (Unni, E. et al. (1995) Biol Reprod, October; 53 (4):820-826).
PCR analysis showed that Tesmin gene is not expressed until day 8 following birth, having a weak expression at day 12, and taking a stable expression pattern from day 18 (FIG. 4). Tesmin gene expression pattern in the testis was similar to MEG1. Therefore, it was revealed that Tesmin expression is regulated at time points similar to MEG1.
When Tesmin expression in the W/Wv mouse was examined, it was revealed that Tesmin is not expressed in these mice. Since the W/Wv mouse is known to be an infertile mouse, the relationship between Tesmin gene and infertility was strongly suggested (FIG. 4).
EXAMPLE 8
In Situ Hybridization
Labeled RNA probe was prepared from mouse Tesmin plasmid (the pBluescript2 vector in which the Tesmin gene has been inserted) using T7 and T3 polymerases and digoxigenin-duTP. This probe was hybridized to sliced mouse testis tissue within a solution containing 50% formamide, 10% dextran sulfate, and 2×SSC. The slide glass on which hybridization was done was incubated within a solution of anti-digoxigenin antibody bound to alkaline phosphatase, and the signal specific to hybridization was detected using the chromogenic substrate NBT/BCIP. As a result, it was verified that Tesmin is extremely specifically expressed in the testis, especially in primary spermatocytes (FIG. 5).
EXAMPLE 9
Chromosomal Location
Mouse P1 genomic library was obtained by PCR screening the P1 bacteriophage genomic library using a mouse Tesmin-specific sense primer (SEQ ID NO: 6) and an antisense primer (SEQ ID NO: 7). Also, human P1 genomic library was obtained using human Tesmin-specific sense primer (SEQ ID NO: 17) and antisense primer (SEQ ID NO: 18) and conducting a screening similar to mouse. The isolated P1 clones were used to examine the chromosomal localization by fluorescent in situ hybridization (FISH). Mouse and human P1 clone-derived DNA was labeled by nick translation using digoxigenin-dUTP, and this probe was hybridized to mouse and human primary fibroblast-derived metaphase chromosomes within a solution containing 50% formamide, 10% dextran sulfate, and 2×SSC. The slide glass on which hybridization was done was incubated within a solution of fluorescence-labeled anti-digoxigenin antibody, and the signal specific to hybridization was detected by counter staining using 4′6′-diamino-2-phenolindol (DAPI).
As a result, the above P1 clones were found to encode the Tesmin gene since the mouse and human Tesmin-specific probes hybridized to the respective P1 clone. When DAPI staining was done using these P1 clones as probes, the 19th B chromosome and the 11th q13.2 chromosome were specifically labeled in mouse and human, respectively. The above results confirmed that Tesmin was located on the 19th B chromosome (FIG. 6) in mouse, and on the 11th q13.2 chromosome (FIG. 7) in human. The relationship between Tesmin and mouse genetic disease was examined based on these results using the Jackson Laboratory Database to find that there is a study reporting that a mutation on the 19th B chromosome where Tesmin exists causes infertility in mice (Evans, E P. (1977) Mouse News Letter, 17). This suggests the possibility that Tesmin mutations trigger infertility in mice.
EXAMPLE 10
Intracellular Localization
A DNA encoding whole open reading frame of the Tesmin cDNA were prepared, using sense (SEQ ID NO: 19) and antisense (SEQ ID NO: 20) primers having an EcoRI site, and also prepared was a gene designed so that 70 amino acids are deleted from the Tesmin cDNA open reading frame, by using a sense (SEQ ID NO: 19) primer having an EcoRI site and antisense (SEQ ID NO: 21) primer having an Sa1I site. These genes were treated with restriction enzymes and inserted into the C terminal region of GFP ORF of the pEGFC1 vector (Clontech). Using Tfx-50 (Promega), this plasmid that encodes the GFP-Tesmin fusion protein was introduced into COS1 cells growing on a cover glass. The cover glass was fixed by methanol/acetone (1:1) and washed three times with PBS. The cells were observed with Olympus BH-2 Epifluorescent Microscope. As a result, although the protein fused to the full sequence of Tesmin was localized within the cytoplasm, one having the partially deleted Tesmin sequence had migrated into the nucleus (FIG. 8).
EXAMPLE 11
Preparation of a Specific Antibody that Binds to the Tesmin Protein
A peptide antibody against the 18 amino acids presumed by the gene arrangement of Tesmin was prepared. Specifically, an 18 amino acid sequence (SEQ ID NO: 22) was made using a peptide synthesizer. KLH was covalently bound to this obtained peptide with a crosslinking reagent. Next, this peptide was purified by HPLC, and a rabbit was immunized with it. Serum was drawn out at four stages, and finally, all the blood was collected. This serum was purified, using a protein A column to prepare the polyclonal antibody. Tesmin protein fused with GST was separated on a gel by SDS-PAGE. Detection by Western blotting confirmed that this anti Tesmin polyclonal antibody recognizes the Tesmin protein (FIG. 9).
Western blotting was done by inducing recombinant protein expression through isopropyl-β-D-thiogalactoside (IPTG) added to Tesmin-cDNA-introduced E. coli, and subjecting an E. coli lysate to SDS-PAGE (IPTG+, FIG. 9). A detection using a cell lysate of E. coli without IPTG was also done (IPTG−, FIG. 9).

## Claims

1. An isolated DNA encoding a protein comprising SEQ ID NO: 4 or 5.

2. A vector comprising the DNA of claim 1.

3. An isolated transformant comprising the DNA of claim 1, wherein said isolated transformant is capable of expressing the DNA.

4. A method of producing a protein comprising:
culturing an isolated transformant comprising the DNA of claim 1 under conditions wherein said isolated transformant expresses said protein; and 
obtaining the protein from said isolated transformant or the culture supernatant thereof.

5. An isolated DNA specifically hybridizing to a DNA sequence comprising the nucleotide sequence of any one of SEQ ID NOs: 1-3 under the following conditions: prehybridize at 68° C. for at least 30 minutes, incubate at 68° C. for at least 1 hour, then wash three times with 2×SSC/0.01% SDS for 20 minutes at room temperature, and next, wash three times with 1×SSC/0.1% SDS, at 37° C. for 20 minutes, followed by two times with 1×SSC/0.1% SDS, at 50° C. for 20 minutes.

6. An isolated DNA encoding a protein comprising an amino acid sequence having 60% or more homology to SEQ ID NO: 4 or 5, wherein the protein has testicular differentiation activity.

7. An isolated DNA encoding a protein which is encoded by a DNA hybridizing under stringent conditions to the DNA comprising the nucleotide sequence of SEQ ID NO: 1 or 3, wherein the protein has testicular differentiation activity.

8. A method of producing a protein comprising:
culturing a transformant comprising the DNA of claim 1 under conditions wherein a protein is expressed, wherein the protein comprises an amino acid sequence having 60% or more homology to SEQ ID NO: 4 or 5, wherein the protein has testicular differentiation activity; and 
obtaining the protein from the transformant or the culture supernatant thereof.

9. A method of producing a protein comprising:
culturing a transformant comprising DNA which hybridizes under stringent conditions to a DNA comprising a nucleotide of SEQ ID NO: 1 or 3 under conditions where a protein is expressed; and 
obtaining the protein from the transformant or the culture supernatant thereof.

10. The isolated DNA of claim 6, wherein the protein comprises an amino acid sequence having 80% or more identity to SEQ ID NO: 4 or 5, and wherein the protein has testicular cell differentiation activity.

11. The isolated DNA of claim 6, wherein the protein comprises an amino acid sequence having 95% or more identity to SEQ ID NO: 4 or 5, and wherein the protein has testicular cell differentiation activity.

12. The isolated DNA of claim 7, wherein the protein is encoded by a DNA hybridizing to a DNA comprising the nucleotide sequence of SEQ ID NO: 1 or 3 under the following conditions: prehybridize at 68° C. for at least 30 minutes, incubate at 68° C. for at least 1 hour, wash three times with 2×SSC/0.01% SDS for 20 minutes at room temperature, wash three times with 1×SSC/0.1% SDS, at 37° C. for 20 minutes, and wash two times with 1×SSC/0.1% SDS, at 50° C. for 20 minutes, and wherein said protein has testicular cell differentiation activity.

13. A vector comprising the DNA of claim 6.

14. A vector comprising the DNA of claim 7.

15. A vector comprising the DNA of claim 10.

16. A vector comprising the DNA of claim 11.

17. A vector comprising the DNA of claim 12.

18. A transformant comprising the DNA of claim 6, wherein the transformant is capable of expressing the DNA.

19. A transformant comprising the DNA of claim 7, wherein the transformant is capable of expressing the DNA.

20. A transformant comprising the DNA of claim 10, wherein the transformant is capable of expressing the DNA.

21. A transformant comprising the DNA of claim 11, wherein transformant is capable of expressing the DNA.

22. A transformant comprising the DNA of claim 12, wherein the transformant is capable of expressing the DNA.

23. A method of producing a protein comprising:
culturing a transformant comprising the DNA of claim 6 under conditions wherein the transformant encodes a protein; and 
obtaining the protein from the transformant or the culture supernatant thereof.

24. A method of producing a protein comprising:
culturing a transformant comprising the DNA of claim 10 under conditions wherein the transformant encodes a protein; and 
obtaining the protein from the transformant or the culture supernatant thereof.

25. A method of producing a protein comprising:
culturing a transformant comprising the DNA of claim 11 under conditions wherein the transformant encodes a protein; and 
obtaining the protein from the transformant or the culture supernatant thereof.

26. A method of producing a protein comprising:
culturing a transformant comprising the DNA of claim 12 under conditions wherein the transformant encodes a protein; and 
obtaining the protein from the transformant or the culture supernatant thereof.