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Gamete Biology |
ri3
Laboratory of Zoology,3 Graduate School of Agriculture, Kyushu University, Higashiku Hakozaki 6-10-1, Fukuoka 812-8581, Japan
Department of Anatomy,4 Miyazaki Medical College, Miyazaki 889-1692, Japan
Department of Developmental Anatomy,5 Graduate School of Medical Science, Kyushu University, 3-1-1 Maidashi, Fukuoka 812-8582, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gametogenesis, sperm, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Differential display of mRNA is a very useful technique for identifying genes differentially expressed in cultured cells and tissues [3, 4]. Using differential display, we have cloned and sequenced more than 200 cDNA fragments, including several novel and previously identified genes whose expression was developmentally upregulated during rat testis development. By this technique, we previously isolated iba1 (ionized calcium-binding adaptor molecule 1) and spergen-1 (spermatogenic cell-specific gene 1). Iba1 is expressed in the cytoplasm of elongating spermatids and might be involved in reorganization of actin cytoskeleton during spermiogenesis and residual body extrusion [5]. Spergen-1 is a small protein of 154 amino acids, which is associated with mitochondria of both elongating spermatids and matured spermatozoa [6]. It might be involved in mitochondria sheath formation during spermiogenesis by working as an adhesive molecule between mitochondria [7]. Here, we report another gene, spergen-2 (spermatogenic cell-specific gene 2), which encodes a 56-kDa nuclear protein bearing ankyrin repeat motifs.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Investigations were conducted in accordance with the National Research Council Guide for Care and Use of Laboratory Animals.
The mRNA differential display method [3, 4] was carried out using an RNA map kit (GenHunter, Nashville, TN). Total testis RNAs were isolated from Wistar rats 1, 2, 3, 4, 5, 6, 7, and 8 wk of age as described previously [6, 8]. RNAs were reverse transcribed with oligo-(dT) primers anchored to the beginning of the poly(A) tail. The resulting cDNAs were amplified with the polymerase chain reaction (PCR) technique using oligo-(dT) primers and arbitrary primers. The cycling parameters were as follows: 94°C for 30 sec, 40°C for 2 min, and 72°C for 30 sec for 40 cycles. The amplified cDNAs were separated on 6% urea-polyacrylamide gels, fixed, and stained by the silver sequence system (Promega, Madison, WI). Complementary DNA fragments whose expression levels were developmentally increased were recovered directly by cutting out the gel slices. After elution by boiling the gel slices in distilled water for 15 min, cDNA fragments were reamplified by using the same primers used in the initial PCR for differential display. The cDNA fragments were then purified by electrophoresis, cloned into the pGEM easy T-vector (Promega), and sequenced using an automated DNA sequencer (Applied Biosystems, Foster City, CA).
Reverse Transcription PCR
Complementary DNA strands were synthesized from 2 µg of total RNA by using a first-strand synthesis kit (Amersham Pharmacia Biotech, Little Chalfont, U.K.) with random primers. The reverse transcribed cDNA was used as a PCR template to synthesize a gene. The primers used to amplify the gene were 5'-GGC AAC CAG CAG TCT ACA ACC-3' (forward) and 5'-CTC TTC AAT ACA GCC CTT TGT GC-3' (reverse). The PCR-amplified DNA was cloned into pGEM-T easy vector and sequenced using the automated sequencer (Applied Biosystems). The amplified cDNA of 497 base pairs (bp) was used as a probe for plaque hybridization, in situ hybridization, and Northern blot analysis. Primers for the glyceraldehyde-3-phosphate dehydrogenase gene (G3PDH) were 5'-TGA AGG TCG GTG TCA ACG GAT TTG GC-3' (forward) and 5'-CAT GTA GGC CAT GAG GTC CAC CAC-3' (reverse).
Complementary DNA Cloning
To obtain the full-length cDNA encoding the rat gene, plaque hybridization was performed by the standard method [9]. Rat testis 5'-stretch plus cDNA library was obtained from Clontech Laboratories (Palo Alto, CA). The probe for plaque hybridization was the 497-bp cDNA fragment that was labeled with digoxigenin (DIG)-dUTP by DIG High Prime DNA Labeling Kit (Roche Molecular Biochemicals, Mannheim, Germany). The hybridized probe was immunodetected by anti-DIG antibody conjugated with alkaline phosphatase and then recorded on x-ray films with the chemiluminescence substrate CSPD (Roche). Complementary DNAs of isolated clones were sequenced using the automated sequencer.
Northern Blot Analysis
A Northern blot membrane loaded with 12 µg total RNA from testis of 2-, 3-, 7-, and 8-wk-old rats was hybridized with the 497-bp PCR fragment, which was gel purified and labeled with DIG-dUTP, according to the Roche instruction manual. Hybridization was performed as previously reported [10, 11]. Messenger RNA hybridized with the probe was immunologically detected as described for plaque hybridization.
In Situ Hybridization
In situ hybridization was carried out as previously reported [5, 6]. Frozen sections of adult rat testis were preincubated for 30 min at 42°C in a hybridization buffer (20 mM Tris-HCl, pH 8.0, 0.3 M NaCl, 2 mM EDTA, 50% formamide, 1 mg/ml BSA, 0.02 % Ficoll, 0.02 % polyvinylpyrolidone, 1 mg/ml herring sperm DNA) and hybridized for 5 h at 42°C in the hybridization buffer containing a DIG-labeled sense or antisense RNA probe of 497 nucleotides. After hybridization, the sections were washed for 1 h in 2x saline sodium citrate with 50% formamide at 42°C and incubated for 30 min at 37°C with RNase A (20 µg/ml), and bound cRNA was detected using anti-DIG alkaline phosphatase-conjugated antibody (1:500 dilution; Roche) and visualized with nitroblue tetrazolium-5-bromocresyl-3-indolylphosphate (Roche).
Antibody Production
The peptide used for raising the antibody is derived from the hydrophilic region of spergen-2 (REDMESRSVPREE). The peptide was coupled to keyhole limpet hemocyanin (Pierce, Rockford, IL) (1 mg total dose) and was dissolved in 1 ml of saline, emulsified with 1 ml of Freund complete adjuvant, and injected at multiple sites on the back of a rabbit as described previously [10, 11]. The antiserum was collected within 2 wk after the third injection. Affinity purification of the antibody was carried out over a matrix of the peptide coupled to 2-fluoro-1-methylpyridinium toluene-4-sulfonate-activated Sephadex (Seikagaku Kougyo, Tokyo, Japan), as described previously [5, 6].
Sample Preparation and Immunoblot Analysis
Spermatozoa, testicular germ cells, and a nucleus-rich fraction of germ cells were prepared for immunoblot analysis. Spermatozoa isolated from epididymides of ether-anesthetized adult Wistar rats were purified by a Percoll density gradient method described previously [7, 8]. For germ cell isolation, seminiferous tubules were taken from testes of adult Wistar rats and subjected to sequential enzyme digestion in PBS containing 1 mg/ml collagenase (Worthington Biochemical Corp., Lakewood, NJ) at 30°C followed by mechanical dissociation. Resultant dispersed cells were collected by centrifugation for 10 min at 1000 x g. To obtain the nucleus-rich fraction of germ cells, seminiferous tubules taken from testes of adult Wistar rats were incubated in a homogenizing buffer (10 mM Tris-HCl, pH 7.3, 5 mM MgCl2, 25 mM KCl) containing 0.25 M sucrose and disrupted with a tight-fitting pestle of a Potter homogenizer until virtually all cells were broken (usually 15–20 strokes). The extent of cell breakage was monitored microscopically. Samples were then separated from the cytoplasmic fraction by sedimentation at 1000 x g for 10 min and washed twice with the same buffer. The resultant pellet, a crude nuclear fraction, was suspended in the homogenizing buffer containing 1.6 M sucrose, and the suspension was layered onto 2.3 M sucrose in the homogenizing buffer. The samples were centrifuged at 100 000 x g for 60 min at 4°C. The pellet at the bottom was collected as the nucleus-rich fraction of germ cells.
Proteins of the samples were extracted by incubation for 30 min on ice in RIPA buffer (50 mM Tris, pH 7.2, 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, 1% Nonidet P-40, protease inhibitors: 1 mU/ml aprotinin, 0.1 mmol/L leupeptin, 0.5 mmol/L PMSF). Extracted proteins were centrifuged for 15 min, and clarified materials were dissolved in SDS-PAGE sample buffer.
Protein samples dissolved in SDS-PAGE sample buffer were separated by SDS-PAGE and either stained by Coomassie brilliant blue or transferred to nitrocellulose sheets. The sheets were incubated for 2 h with affinity-purified anti-spergen-2 antibody diluted 1:2000 with a blocking buffer (PBS containing 5% nonfat milk and 0.1% Tween-20), followed by incubation with anti-rabbit IgG conjugated with horseradish peroxidase (BioRad, Richmond, CA) diluted 1:2000 in the same buffer. Antigen-antibody complexes were visualized using an enhanced chemiluminescence detection kit (Amersham).
Preparation of Glutathione S-Transferase Fusion Proteins
The 456-nucleotide fragment of spergen-2 that encodes 152 amino acids was amplified by PCR and cloned in frame to the COOH terminus of glutathione S-transferase (GST) using the pGEX-4T-1 system (Amersham). Recombinant protein was expressed in Escherichia coli and purified onto glutathione-Sepharose (Amersham) as previously described [5, 6]. GST-fused recombinant proteins, Iba1, Mrf-1, Rab3a, Rab6, and Spergen-1, were similarly expressed in E. coli and purified. These recombinant proteins were used for immunoblot analysis.
Immunohistochemistry
Adult rat testes were fixed in 4% paraformaldehyde in PBS at 4°C for 4 h, washed three times in PBS, incubated in PBS containing NH4Cl for 30 min, and then rinsed in PBS. After infiltration of 20% (w/v) sucrose in PBS, the testes were immersed in OCT compound (Tissue-Tek; Miles, Elkhart, IN) and immediately frozen in liquid nitrogen. Frozen sections of 8 µm thickness were cut by a cryostat (CM1850; Leica, Nussloch, Germany). The sections were washed in PBS, incubated with the anti-spergen-2 antibody diluted 1:200 with the blocking buffer, and incubated with goat anti-rabbit IgG conjugated with Cy3 (Amersham). To stain actin filaments, immunostained samples were incubated for 30 min with PBS containing phalloidin conjugated with fluorescein isothiocyanate (phalloidin-FITC; Sigma, St. Louis, MO), which was diluted 1:1000 with PBS. For DNA staining, immunostained samples were incubated for 30 min with PBS containing SYTOX Green (1:1000 dilution; Molecular Probes, Eugene, OR). In some cases, samples were double stained with the anti-spergen-2 antibody and the MN-7 monoclonal antibody followed by incubation with Cy3-conjugated anti-rabbit IgG and FITC-conjugated anti-mouse IgG (Amersham). MN-7 monoclonal antibody recognizes a 90-kDa acrosome protein, Acrin 1, localized in the acrosome [12]. Acrin 1 was used as a marker protein to identify the acrosome of developing spermatids in the immunostained samples. The samples were then washed with PBS and examined with a confocal laser scanning microscope (LSM-GB 2000; Olympus, Tokyo, Japan). For controls, the primary antibody was replaced either by preimmune serum or by antigen-absorbed anti-spergen-2 antibody. For antibody absorption, the anti-spergen-2 antibody (1.0 mg IgG) was incubated with the synthetic peptide (1.5 mg) at 4°C for overnight. After centrifugation at 10 000 rpm for 30 min, the supernatant was used as the antigen-absorbed antibody.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To obtain developmentally upregulated genes in the rat testis, transcripts derived from the testes of 2- to 7-wk-old rats were examined by differential display screening using 40 different combinations of primer pairs. One of the genes, which was approximately 260 bp in length, was expressed after 3 wk of postnatal development (Fig. 1). Based on the sequence data of the cDNA fragment isolated by differential display, we performed 5' rapid amplification of cDNA ends, which led to production of a 1.8-kilobase (kb) PCR fragment (data not shown). We next used reverse transcription (RT)-PCR to examine both the developmental expression of the gene in rat testes and the expression of the gene in various organs of adult rats. The gene was first detectable at 3 wk of postnatal development (Fig. 2), and PCR amplification of the gene was exclusively observed in testis but was undetectable in other organs examined (Fig. 3).
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To determine the length of mRNA for this gene, Northern blot analysis was conducted using the C-terminal 497-bp cDNA fragment of the gene as a probe. A single 1.9-kb transcript was detected in testes of 3-, 7-, and 8-wk-old rats but not in testes of 2-wk-old rats (Fig. 4), suggesting that mRNA of the gene starts to be transcribed in 3-wk-old testis containing spermatocytes executing meiosis. These results suggested that the gene we identified is approximately 1.9 kb in length and is developmentally upregulated and exclusively expressed in testis.
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Cloning of cDNA
The 497-bp cDNA fragment was used as a probe for plaque hybridization to obtain the full-length cDNAs from the rat testis cDNA library. After four rounds of screening, we isolated seven positive clones, and the longest is 1.9 kb in length, the same size as the mRNA described above. The full-length cDNA sequence and deduced amino acid sequence are shown in Figure 5. The identified cDNA contains a single open reading frame (ORF) of 1500 nucleotides with 104 nucleotides of 5' untranslated region (UTR) and 274 nucleotides of 3' UTR. A poly(A) tail is located 263 nucleotides downstream from the termination codon (TGA). Although a consensus poly(A) signal (AAUAAA) was not found in the sequence, a putative poly(A) signal in germ cell transcripts (AAUAGA) is located 25 nucleotides upstream of the poly(A) site. Because the gene is specifically expressed in spermatogenic cells in rat testis, it was designated as spergen-2 (spermatogenic cell-specific gene 2, accession AB095021).
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The ORF of spergen-2 encodes a protein of 500 amino acids, with a predicted molecular mass of 56 218 Da and a pI of 4.95. Both hydrophobicity plot and SOSUI (http://sosui.proteosome.bio.tua.ac.jp/sosuiframe0.html) analysis suggested that the gene encodes a soluble protein with no transmembrane region. PSORT analysis (a computer program used to predict the sorting and localization of proteins, http://psort.ims.u.tokyo.ac.jp/) suggested the presence of a nuclear localization signal (NLS) at amino acid residues 65–68 (RKKR)(Fig. 5), and a search in the Pfam protein family database (http://www.sanger.ac.uk/Software/Pfam/search.shtml) revealed five tandem ankyrin repeats at amino acid residues 68–98, 99–131, 132–164, 165–197, and 198–230 in spergen-2 protein (Fig. 5). A search of the NCBI database employing BLAST programs revealed that N-terminal 292-amino acid fragment of spergen-2 protein shares 87.7% amino acid identity with the mouse ankyrin-like protein (accessions AK005925, NM023816, and AF294328), whereas the C-terminal 208 amino acids of spergen-2 have no match in the databases (Fig. 6). The ankyrin-like protein consists of 300 amino acid residues with a predicted molecular mass of 33 706 Da and a pI of 6.89 and possesses both ankyrin repeat motifs and an NLS (RKKR). Expression, localization, and characterization of the ankyrin-like protein, however, have not yet been reported.
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In Situ Localization of spergen-2 mRNA
We performed in situ hybridization to determine cell types expressing spergen-2 mRNA in rat testis. Frozen sections of adult rat testis were hybridized either with a cRNA probe having the antisense sequence of spergen-2 mRNA or with a sense probe as a control. DIG-labeled probes were synthesized using PCR-amplified 497-bp cDNA as the template (see Fig. 5). Hybridization with the antisense probe created positive signals in spermatocytes and round spermatids (steps 1–6) in seminiferous tubules (ST) (Fig. 7A). Within seminiferous epithelium, the strongest signal for spergen-2 was observed in spermatocytes, and the signal gradually weakened as differentiation of spermatogenic cell proceeded (Fig. 7, B and C). Faint or no signal was detected in somatic Sertoli cells and spermatogonia and in elongating and elongated spermatids (steps 8–19) (Fig. 7, B and C), although it was sometimes difficult to determine positive cell types because precipitation of hybridized probe obscured cellular boundaries in the epithelium. Hybridization with the sense probe for spergen-2 gave faint or no signal (Fig. 7D). These results suggested that spergen-2 mRNA is expressed in spermatocytes and round spermatids (steps 1–6).
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Specificity of Anti-Spergen-2 Antibody and Immunoblot Analysis
To examine the expression and localization of spergen-2 protein in rat testis, a polyclonal antibody was raised against the synthetic peptide (REDMESRSVPREE) corresponding to amino acid residues 391–404 of spergen-2 (see Fig. 5), which was chosen to distinguish spergen-2 from the putative ankyrin-like protein. The anti-spergen-2 antibody was used after affinity purification. Specificity of the anti-spergen-2 antibody was examined on the blot to which GST-fused truncated spergen-2 protein (39 kDa) and several GST-fused recombinant proteins were transferred (Fig. 8A). The anti-spergen-2 antibody specifically recognized GST-fused truncated spergen-2 protein but did not react with other GST-fusion proteins (Fig. 8B), indicating that the antibody is specific for spergen-2 protein.
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To detect spergen-2 protein biochemically in testis, we performed immnunoblot analysis on the blots to which proteins extracted from purified spermatozoa, testicular germ cells, and the nucleus-rich fraction of germ cells were transferred (Fig. 9). On the blot, the anti-spergen-2 antibody recognized a single band migrating at approximately 56–57 kDa in the samples of testicular germ cells and the nucleus-rich fraction (Fig. 9B). The size of 56–57 kDa was close to 56 218 Da calculated from the spergen-2 amino acid sequence deduced from the cDNA sequence. Spergen-2 protein was undetectable in the sample prepared from purified spermatozoa.
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Localization of Spergen-2 Protein in Rat Testis
Using the anti-spergen-2 antibody and a confocal laser-scanning microscope, we intended to determine cell types expressing spergen-2 protein in frozen sections of adult rat testis. MN-7 monoclonal antibody was used to identify the acrosome of developing spermatids within seminiferous epithelium, and actin staining by phalloidin-FITC was employed to define the contour of the ST. In a cross section of the testis viewed at low power with the confocal laser-scanning microscope, spergen-2 immunofluorescence signal was detected in nuclei of spermatogenic cells in all ST (Fig. 10A). Counterstain with phalloidin-FITC clarified that nuclei of cells located in the outer layer of ST, i.e., spermatogonia and Sertoli cells and nuclei of spermatozoa present at the luminal surface of ST were negative (Fig. 10B). Neither Leydig cells nor other interstitial cells between ST were stained with the antibody. Immunostaining either with the anti-spergen-2 antibody absorbed with the synthetic peptide (Fig. 10C) or with preimmune serum (not shown) gave no positive signal. To determine in detail cell types immunostained with the anti-spergen-2 antibody, we examined the samples double stained with the anti-spergen-2 antibody and monoclonal MN-7 antibody, which stains acrosomes. MN-7-positive acrosomes of spermatids are green in Figure 10, D–G. Spergen-2 immunoproduct was observed in nuclei of round spermatids at steps 1–3 (Fig. 10D), at steps 5–7 (Fig. 10E), and at steps 9–11 (Fig. 10F). The signal was also detected in nuclei of spermatocytes of late pachytene stage (Fig. 10F) and of late pachytene or diplotene stage (Fig. 10G). MN-7-positive acrosomes associated with spergen-2-positive nuclei were seen in round spermatids (Fig. 10E) and elongating step 9–11 spermatids (Fig. 10F). Spergen-2 immunostaining was undetectable in nuclei of elongated spermatids of steps 12–14 (Fig. 10G) and of steps 15–17 (Fig. 10D). From these data, we concluded that spergen-2 protein is expressed in nuclei of late spermatocytes (stages IX–XIV), of round spermatids (steps 1–7), and of elongating early spermatids (steps 8–11).
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We next used confocal laser scanning microscopy to determine the nucleoplasmic regions where spergen-2 localizes. At high magnification with adequate power of a laser beam, spergen-2 immunoproduct was seen within the nuclei in a punctate or dappled pattern rather than a uniform distribution (Fig. 11A). When the specimens immunostained for spergen-2 were examined after counterstaining with SYTOX Green, which stains nuclear DNA, we found that the regions in which spergen-2 immunoproduct accumulated were areas with relatively low density of DNA in both spermatid nuclei (Fig. 11B) and spermatocyte nuclei (Fig. 11C), except for some areas containing both spergen-2 and SYTOX-labeled DNA.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In rat testis, spergen-2 mRNA was expressed in spermatocytes and round spermatids (steps 1–6), and spergen-2 protein was expressed in late spermatocytes (stages IX–XIV) and spermatids (steps 1–11). Spergen-2 protein was undetectable in late elongated spermatids (steps 12–19) and in leptotene, zygotene, and early pachytene (stages I–VIII) spermatocytes. We interpret this restricted expression pattern as an indicator of a potential role of spergen-2 in spermatogenesis, especially in cell differentiation from late pachytene spermatocytes to spermatids or in early spermatid differentiation.
In spite of the absence of a separating membrane, the nucleoplasm can be divided into three subnuclear compartments, i.e., nucleolus, chromosome territories including euchromatin and telomeric heterochromatin, and interchromosome space [13]. These subnuclear compartments are highly dynamic structures whose size, shape, and location constantly change according to metabolic activities. It has been postulated that the interchromosome space defines a functional compartment for transcription of active genes, splicing of transcripts, and maturation and transport of mRNA [14, 15]. "Speckles," at which the splicing machinery accumulates together with pre- and polyadenylated RNA, localize in the interchromosome space in association with the periphery of the chromosome territories [13]. We demonstrated immunohistochemically and biochemically that spergen-2 is predominantly localized in nuclei of spermatogenic cells. We also found that spergen-2 is not colocalized with a nucleolus-associated protein, nucleolin [16] (data not shown), suggesting that it is not directly involved in rRNA synthesis and processing. Confocal laser scanning microscopy revealed that spergen-2 seemed to localize in the subnuclear regions with low density of DNA, which might be equivalent to the interchromosome space where DNA transcription, splicing, and maturation of mRNA takes place. In view of the restricted expression pattern of spergen-2 during spermatogenesis and its localization in the interchromosome space, spergen-2 might be involved either in DNA transcription or in splicing and maturation of mRNA. Although the biological significance of spergen-2 in spermatogenesis is still obscure, further studies of spergen-2, such as determination of more precise localization within the nucleus and finding of spergen-2-associated nuclear proteins, would provide insights into the biological functions of spergen-2 in spermatogenesis.
The ankyrin repeats are tandem repeats of about 33 amino acids, which consist of pairs of antiparallel 
-helices stacked side by side and connected by a series of intervening ß-hairpin structures [17]. The ankyrin repeats have now been recognized in more than 400 proteins, including cyclin-dependent kinase inhibitors, transcriptional regulators, cytoskeletal organizers, and developmental regulators [18, 19] and a germ cell-specific protein, Gasz [20]. The role of these repeats in mediating protein-protein interactions has been well documented in several cases, such as 53BP2-p53 complex [21] and GABAß-GABA-DNA complex [22]. Spergen-2 has five tandem ankyrin repeats (Fig. 5). The same ankyrin repeats are seen in the putative ankyrin-like protein that shares 87.7% amino acid identity with the N-terminal 292 amino acids of spergen-2. Although biological significance of the ankyrin repeats in spergen-2 remains to be determined, identification of proteins interacting with spergen-2 would provide an important clue to the biological functions of spergen-2 in nuclei of spermatogenic cells.
Translocation of proteins across the nuclear envelope depends on the classical NLS [23], which consists of a cluster of basic residues (monopartite) or two clusters of basic residues separated by 10–12 residues (bipartite) [24, 25]. For transport of proteins across the nuclear envelope, the NLS is recognized by the heterodimer import receptor complex comprising importin and importin ß [26]. The definition of an NLS sequence is, however, somewhat vague because of the diversity of sequence that can apparently act as a functional NLS [23, 27]. Both immunohistochemical and biochemical analyses revealed that spergen-2 is predominantly localized in nuclei of spermatogenic cells, suggesting that it is synthesized in cytoplasm and transported into nuclei across the nuclear envelope. This hypothesis is supported by the presence of a putative monopartite NLS in spergen-2. Investigations using a transfection technique are being conducted to determine whether the NLS in spergen-2 works practically as a signal for transport to the nucleus.
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FOOTNOTES |
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2 Correspondence. FAX: 92 642 2804; iidahiro{at}agr.kyushu-u.ac.jp
Received: 3 December 2002.
First decision: 19 December 2002.
Accepted: 25 February 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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motif, and a basic leucine zipper. Mol Endocrinol 2002 16:1168-1184/ß: an ETS domain-ankyrin repeat heterodimer bound to DNA. Science 1998 279:1037-1041This article has been cited by other articles:
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  Y. Iwamoto, T. Kaneko, J. Ichinose, T. Mori, Y. Shibata, K. Toshimori, and H. Iida Molecular Cloning of Rat Spetex2 Family Genes Mapped on Chromosome 15p16, Encoding a 23-Kilodalton Protein Associated with the Plasma Membranes of Haploid Spermatids Biol Reprod, February 1, 2005; 72(2): 284 - 292. [Abstract] [Full Text] [PDF]
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 H. Iida, H. Yamashita, M. Doiguchi, and T. Kaneko Molecular Cloning of Rat Spergen-3, a Spermatogenic Cell-Specific Gene-3, Encoding a Novel 75-kDa Protein Bearing EF-Hand Motifs J Androl, November 1, 2004; 25(6): 885 - 892. [Abstract] [Full Text] [PDF]
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