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Regular Article |
a Laboratory of Zoology, Graduate School of Agriculture, Kyushu University, Fukuoka 812-8581, Japan
b Department of Developmental Anatomy, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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developmental biology, gametogenesis, sperm maturation, spermatid, spermatogenesis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Differential display of mRNA is a method for studying differential gene expression in cultured cells [3]. In contrast to a subtraction method, the gel display allows many samples to be compared side-by-side, and individual bands that visually indicate differentially regulated mRNA can be isolated and subcloned [4]. Differential display also has a primary advantage over the subtraction method in that it is based on polymerase chain reaction (PCR); therefore, relatively little starting material is required to compare gene expression from different sources. Using differential display, we have cloned and sequenced more than 100 cDNA fragments, which include several novel as well as already identified genes with developmentally up-regulated expression in rat testis. By this technique, we have previously isolated iba1, an ionized calcium-binding adapter molecule-1, which was expressed in haploid spermatids, but not in other germ cells, in rat testis [5].
In the present study, we report another gene isolated by differential display followed by cDNA cloning and that encodes the open-reading frame of 462 base pairs (bp) that codes a small protein of 154 amino acids. The gene was specifically expressed in spermatids of rat testis and, therefore, has been designated as spergen-1 (spermatogenic cell-specific gene-1). A mouse homologue of spergen-1 was found in the recently opened database of the full-length mouse cDNA collection [6]. Here, we describe the expression pattern and distribution of spergen-1 and its gene product in rat testis as well as its intracellular localization in transfected COS-7 cells. We also discuss the functional aspects of spergen-1 with reference to spermiogenesis.
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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 publication Guide for the Care and Use of Laboratory Animals.
The mRNA differential display method [3] was carried out using an RNA map kit (GenHunter, Nashville, TN). Briefly, total testis RNAs were isolated from Wistar rats 1–8 wk of age as described previously [5, 7]. The RNAs were reverse-transcribed with oligo(dT) primers anchored to the beginning of the poly(A) tail. The resulting cDNAs were amplified with 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 then separated on 6% (w/v) urea-polyacrylamide gels, fixed, and stained by the silver sequence system (Promega, Madison, WI). The cDNA fragments with developmentally increased expression levels 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. The cDNA fragments were then purified by electrophoresis, cloned into the pGEM easy T-vector (Promega), and sequenced using a DNA sequencer (Applied Biosystem, Foster City, CA).
5' Rapid Amplification of cDNA End and Reverse Transcription-PCR
The 5' rapid amplification of cDNA end (RACE) was performed using the 5' RACE system kit (Gibco BRL, Rockville, MD). Based on the sequence data of a cDNA fragment isolated by differential display, first-strand cDNA was synthesized from total RNA by SuperScript II reverse transcriptase using a gene-specific primer (5'-GACGCTCCGTCTCACTTGATC-3'). The cDNA was amplified by PCR using a nested primer (5'-CGCTCCTGAATGACGTCATCC-3') and an anchor primer (5'-GGCCACGCGTCGACTAGTAC-3'). A 5' RACE product was cloned into pGEM-T easy vector (Promega) and sequenced using a DNA sequencer (Applied Biosystem).
For reverse transcription (RT)-PCR analysis, cDNA strands were synthesized from 2 µg of total RNA by using a first-strand synthesis kit (Amersham Pharmacia Biotechnology, Buckinghamshire, England) 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'-GCCAAGGAGGCCCTCTGTAAG-3' (forward) and 5'-CGCTCCTGAATGACGTCATCC-3' (reverse). The PCR-amplified DNA was cloned into pGEM-T easy vector and sequenced using a DNA sequencer (Applied Biosystem).
Complementary DNA Cloning
To obtain the full-length cDNA encoding the rat gene, plaque hybridization was performed by the standard method [8]. Rat testis 5'-stretch plus cDNA library was obtained from CLONTECH Laboratories, Inc. (Palo Alto, CA). The probe was the cDNA fragment of 5' RACE that was labeled with digoxigenin (DIG)-deoxyuridine triphosphate (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). The cDNAs of isolated clones were sequenced using a DNA sequencer (Applied Biosystem).
Northern Blot Analysis
A Northern blot membrane loaded with 12 µg of total RNA from 3- to 8-wk-old rat testis was hybridized with the 340-bp PCR fragment isolated by differential display or the full-length cDNA, which was gel-purified and labeled with DIG-dUTP according to the instruction manual from Roche Molecular Biochemicals. Hybridization was performed as previously reported [9]. Visualization of mRNA hybridized with the probe was carried out as described for plaque hybridization. Ribosomal RNAs were visualized by staining the membrane with methylene blue.
In Situ Hybridization
In situ hybridization was carried out as previously reported [5]. In brief, 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% (v/v) formamide, 1 mg/ml of BSA, 0.02% (w/v) Ficoll, 0.02% (w/v) polyvinylpyrolidone, and 1 mg/ml of herring sperm DNA) and then hybridized for 5 h at 42°C in the hybridization buffer containing a DIG-labeled sense or antisense RNA probe. After hybridization, the sections were washed for 1 h in 2x SSC (1x SSC: 0.15 M sodium chloride and 0.015 M sodium citrate) with 50% (v/v) formamide at 42°C and then 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 NBT-BCIP (Roche).
Vector Construction
The nucleotide encoding the myc tag peptide (EQKLISEEDL) was engineered to fuse in frame with the C-terminus of spergen-1 cDNA by PCR. The myc-tagged spergen-1 lacking 10 or 20 amino acids of the N-terminus was created by PCR using the primer 5'-CCGGAATTCAAGATGTTGGCCCGGAAGAGCATAGGG-3' and the primer 5'-CCGGAATTCAAGATGCCACCAAGGGTGAACTCGGAC-3', respectively. The spergen-1 (wild-type) and the N-terminal-deleted mutants, both of which were tagged with myc epitope at the C-terminus, were subcloned into the pCDLSR
expression vector [10] for transfection into COS-7 cells grown on cover slips. The N-terminal region of 35 amino acids of spergen-1 was amplified by PCR and fused in frame with Green fluorescence protein (GFP) within pEGFP-N3 expression vector (CLONTECH), which was designated as spergen-1(35)-GFP. Primers used to produce 35 amino acids of spergen-1 were 5'-CCGGAATTCAAGATGATCATTACAACATGGATTATGTAC-3' (forward) and 5'-AACGGATCCAGCCTCAGTTTCTTCAACTTCAAT-3' (reverse). The vector was also introduced into COS-7 cells by transfection.
DNA Transfection and Immunofluorescence Microscopy
Plasmid DNAs were transfected into COS-7 cells using the LIPOFECTAMINE reagent (Gibco BRL) according to the manufacturer's instruction. After 48 h of culture, transfected cells were processed for immunofluorescence microscopy to detect expressed proteins. In some cases, transfected cells were incubated for 60 min in the culture medium containing 2 µM MitoTracker Green FM (a cell-permeable, mitochondria-selective dye; Molecular Probes, Eugene, OR), then washed in PBS and fixed for immunofluorescence microscopy.
Transfected cells were fixed with 3% (w/v) paraformaldehyde in PBS for 15 min, treated with 50 mM NH4Cl for 10 min, and permeabilized in 0.1% (v/v) Triton X-100 for 5 min. After exposure to PBS containing 5% (w/v) skim milk, cells were incubated with the anti-myc antibody (MBL Co., Nagoya, Japan), followed by incubation with Cy3-conjugated goat anti-rabbit IgG (Amersham). The COS-7 cells expressing spergen-1(35)-GFP were examined without immunostaining under a fluorescence microscope after fixation. To stain nuclei of COS-7 cells, immunostained cells were exposed for 30 min to Hoechst 33342 (Sigma Chemical Co., St Louis, MO). The samples were examined by either a fluorescence microscope (Olympus, Tokyo, Japan) or a confocal laser-scanning microscope (LSM-GB 2000; Olympus) as previously described [11].
Antibody Production and Immunoblot Analysis
The peptide used for raising antibody is derived from the hydrophilic region of spergen-1 (NKTEEEASRSI). The peptide was coupled to keyhole limpet hemocyanin (KLH; Pierce, Rockford, IL). The peptide coupled to KLH (total dose, 1 mg) 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 [12]. The antiserum was collected within 2 wk after the final 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 Kogyo, Tokyo, Japan) as described previously [5].
Protein samples dissolved in SDS-PAGE sample buffer were separated on SDS-PAGE and transferred to nitrocellulose sheets. The sheets were incubated for 2 h with affinity-purified anti-spergen-1 antibody diluted 1:1000 with a blocking buffer (PBS containing 5% (w/v) nonfat milk and 0.1% (v/v) Tween-20), followed by incubation with anti-rabbit IgG conjugated with horse radish peroxidase (Bio-Rad, Richmond, CA) diluted 1:2000 in the same buffer. Antigen-antibody complexes were visualized using an enhanced chemiluminescence detection kit (Amersham Pharmacia).
Preparation of Glutathione S-Transferase-Fusion Proteins
Full-length spergen-1 was 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, 7]. The GST-fused recombinant proteins, Iba1, Mrf-1, Rab3a, Rab3d, and Rab6, were similarly expressed in E coli and purified. These recombinant proteins were used for immunoblot analysis.
Subcellular Fractionation and Sperm Isolation
Testes were removed from ether-anesthetized adult Wistar rats and decapsulated. Seminiferous tubules were washed in PBS three times and homogenized with a loosely fitting Potter homogenizer (Asahi tekunogurasu, Tokyo, Japan) (five strokes) in a homogenization buffer (10 mM Hepes [pH 7.4], 1 mM EDTA, 0.25 M sucrose, and protease-inhibitor cocktail [Sigma]). An aliquot of washed seminiferous tubules were dissolved in SDS-PAGE buffer for immunoblot analysis. The homogenates were centrifuged at 700 x g for 10 min, and supernatants were further centrifuged at 7000 x g for 15 min. The precipitates were suspended in the homogenization buffer, layered onto a continuous gradient of 39%–62% (w/v) sucrose, and then centrifuged at 110 000 x g for 2 h, which resulted in the appearance of two distinct bands. The top band (cell debris-rich fraction) and the second band (mitochondria-rich fraction) were collected by pipettes, diluted in the homogenization buffer, and centrifuged at 10 000 x g for 60 min. Precipitates were processed either for immunoblot analysis or for electron microscopy as described previously [9].
Spermatozoa were isolated from the epididymis of ether-anesthetized adult Wistar rats by a Percoll density-gradient method as described previously [7, 11], dissolved in SDS-PAGE buffer, and used for immunoblot analysis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To identify developmentally up-regulated 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. During this screening process, we identified a cDNA of approximately 340 bp in length, the expression of which was developmentally up-regulated (Fig. 1). When the developmental expression of the gene in rat testes was examined by RT-PCR, it was first detectable at 4 wk of postnatal development, and its expression increased thereafter (Fig. 2). To confirm the RT-PCR data, Northern blot analysis was conducted using the 340-bp cDNA fragment or the full-length cDNA as a probe. As shown in Figure 3, a single 0.8-kilobase (kb) transcript was detected in 4-, 5-, 6-, and 8-wk-old rat testes, but not in 3-wk-old rat testes. We next examined by RT-PCR the expression of the gene in various organs of adult rats. It was highly expressed in testis, but it was undetectable in other organs examined (Fig. 4). These data indicate that the gene isolated by differential display is developmentally up-regulated during rat testis development, and that it is highly expressed in the testis.
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Complementary DNA Cloning
Based on the sequence data of the cDNA fragment isolated by differential display, we performed 5' RACE, which led to production of a 480-bp PCR fragment. The 480-bp cDNA fragment was used as a probe for plaque hybridization to isolate the full-length cDNAs from the rat testis cDNA library. We have obtained two positive clones containing the cDNA encoding the open-reading frame of 462 bp that coded a small protein of 154 amino acids. Figure 5 shows the cDNA and the deduced amino acid sequence of the gene. A poly(A) signal, 5'-AATAAA-3', was located 157-bp downstream from the termination codon (TAA). The total length of the gene obtained by 5' RACE and cDNA cloning, which includes 5' and 3' untranslated region, is 764 bases, which is close to the 0.8 kb estimated by Northern blot analysis (Fig. 3). The predicted molecular mass and pI were 18 041 Da and 5.63, respectively. Both hydrophobicity plot analysis and PSORT (a computer program used to predict the sorting and localization of proteins; http://psort.ims.u-tokyo.ac.jp/) suggested the presence of a hydrophobic region at the N-terminal in the protein. No transmembrane domain was detected in the sequence. Because the gene is specifically expressed in spermatogenic cells in rat testis (see below), it was designated as spergen-1 (spermatogenic cell-specific gene-1; accession no. AB057408). Several months after cloning of the gene, the RIKEN Mouse Gene Encyclopedia Project offered a full-length mouse cDNA collection containing more than 21 000 clones [6], and we found in that database a mouse gene (accession no. AK005610, clone name 1700001L23) homologous to rat spergen-1. Figure 6 shows the alignment of the deduced amino acid sequences of rat spergen-1 and its mouse counterpart. The amino acid sequence of rat spergen-1 was 87% identical to that of its mouse counterpart; the identity at the nucleic acid level was 91%.
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In Situ Localization of spergen-1 mRNA
We performed in situ hybridization to determine the cell types expressing spergen-1 mRNA in rat testis. Frozen sections of adult rat testis were hybridized either with a RNA probe having the antisense sequence of spergen-1 mRNA or with a sense probe as control. Hybridization with the antisense probe created strong signals in the inner half-layer of the seminiferous epithelium of adult rat testis, although the intensity of the hybridization signals markedly varied between the seminiferous tubules, probably due to the cycles of the seminiferous epithelium (Fig. 7A). The seminiferous tubules at stages V–XI showed positive signal, whereas the signal was weak or undetectable in the tubules at stages I–IV and XII–XIV (Fig. 7A). Hybridization with the sense probe for spergen-1 gave no signal (Fig. 7B). At higher magnification, spergen-1 mRNA was found to be present in round spermatids (steps 5–7) located in the inner half-layer of the seminiferous epithelium as well as in early elongate spermatids (steps 8–11), the cytoplasm of which protruded into the tubular lumen (Fig. 7C). Both early round spermatids (steps 1–4) and more advanced elongate spermatids (steps 12–19) showed faint or no staining. Somatic Sertoli cells and less-mature germ cells (spermatogonia, primary spermatocytes) located in the outer half-layer of the seminiferous epithelium were negative (Fig. 7, A and C). These data indicate that spergen-1 mRNA is expressed specifically in spermatids of steps 5–11 in the seminiferous epithelium of the rat testis.
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Sorting of Expressed Spergen-1 to Mitochondria in COS-7 Cells
Spergen-1 protein has a hydrophobic sequence interrupted by several basic amino acid residues at the N-terminus. To investigate whether the sequence plays a role as a targeting signal, such as an endoplasmic reticulum-targeting signal (a signal sequence) or a mitochondria-targeting signal, myc-tagged spergen-1 was expressed in COS-7 cells by transfection. Deleted mutants that lacked 10 or 20 N-terminal amino acids of spergen-1 were also transfected into COS-7 cells. After 48 h of culture, transfected cells were either fixed for immunofluorescence microscopy or incubated for an additional 60 min in the medium containing MitoTracker Green FM to visualize mitochondria before immunostaining.
Both myc-tagged spergen-1 protein and N-terminal-deleted mutant proteins expressed in COS-7 cells were detected by anti-myc antibody followed by incubation with Cy3-conjugated secondary antibody. The COS-7 cells transfected with myc-tagged spergen-1 contained a lot of thread-, circular-, or ellipse-like cell organelles that were strongly labeled with anti-myc antibody (Fig. 8A). Because those immunostained cell organelles seemed to be mitochondria, transfected cells incubated with MitoTracker Green FM were processed for immunostaining, followed by examination with a confocal laser-scanning microscope. As shown in Figure 8, B and C, the staining image for myc-tagged spergen-1 (red color) was almost overlapped by that of mitochondria labeled by MitoTracker (green color), indicating the localization of expressed spergen-1 on mitochondria. We next examined the localization of the mutated proteins expressed in COS-7 cells. In contrast to wild-type spergen-1 (Fig. 8D), expression of deleted mutant proteins, which lacked 10 or 20 N-terminal amino acid residues, resulted in diffuse cytoplasmic staining in COS-7 cells (Fig. 8, E and F), suggesting that the mutant proteins are deprived of the mitochondria-targeting signal. To confirm that the N-terminus of spergen-1 has the mitochondria-targeting signal, the N-terminal 35 amino acid residues fused in frame with GFP, designated as spergen-1(35)-GFP, were expressed in COS-7 cells. In control, wild GFP was expressed in COS-7 cells. As shown in Figure 8H, spergen-1(35)-GFP was localized in mitochondria, whereas wild GFP was diffusely distributed in the cells (Fig. 8G). Taken together, these data strongly suggest that spergen-1 protein has the N-terminal-targeting signal for mitochondria.
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Subcellular Distribution of Spergen-1 Protein
The transfection experiments described above suggest that spergen-1 protein expressed in testis might be associated with mitochondria. To investigate this possibility, we first produced antibody against the hydrophilic region of spergen-1 (NKTEEEASRSI). Specificity of the affinity-purified anti-spergen-1 antibody was examined on the blot to which several GST-fused recombinant proteins were transferred. As shown in Figure 9, A and B, the anti-spergen-1 antibody specifically recognized GST-spergen-1 protein, but it did not react with other GST-fusion proteins, indicating that the antibody is specific for spergen-1 protein.
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Using the anti-spergen-1 antibody with confocal laser-scanning microscopy, we intended to determine the cell types expressing spergen-1 protein in frozen sections of adult rat testis. We, however, failed to detect antibody-specific immunostaining signal in the samples. Although the reason for this negative result is not clear at present, it is possible that the antigen epitope in spergen-1 is masked by some factors, which make detecting the protein difficult using the antibody. It is also probable that the expression level of spergen-1 is too low to detect by immunohistochemistry. We, therefore, used the immunoblot approach to detect spergen-1 in the fractionated samples obtained by sucrose density-gradient centrifugation.
The immunoblot analysis was carried out on the blot to which proteins of the cell debris-rich fraction (Fig. 10C), the mitochondria-rich fraction (Fig. 10D), and the seminiferous tubule extract were transferred. The antibody against spergen-1 recognized a protein migrating at approximately 17–18 kDa in the mitochondria-rich fraction and the seminiferous tubule extract (Fig. 10, A and B), which is close to the 18 041 Da calculated from the spergen-1 amino acid sequence deduced from the cDNA sequence. An extra band migrating just below the main band on the blot (Fig. 10B) might be due to degradation of the protein during the fractionation. Spergen-1 was not detected in the cell debris-rich fraction. It was also undetectable in spermatozoa purified from epididymis (not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Using differential display and expression analyses, we have previously discovered that iba1, an ionized calcium-binding adaptor molecule-1, was expressed in haploid spermatids, but not in other germ cells, of the rat testis [5]. Iba1 might be involved in reorganization of actin cytoskeleton during spermiogenesis and residual body extrusion [5, 16]. Another gene, spergen-1, isolated using the same method in the present study, is also up-regulated during postnatal development of the rat testis, and it was found to be specifically expressed in haploid spermatids. The observation that the timing of spergen-1 expression was precisely regulated during postnatal development of rat testis (Figs. 2 and 3) suggests the presence of elaborated transcriptional mechanisms for the gene. The expression pattern of spergen-1 mRNA is similar, but not identical, to that of iba1, but no sequential homology is found between the two genes. Because a mouse homologue of rat spergen-1 is found in the full-length mouse cDNA collection [6], it would be also expressed in the testes of mice and, probably, of other mammals as well.
Spergen-1 protein has a N-terminal hydrophobic region interrupted by several basic amino acid residues, which is also conserved in mouse spergen-1. Transfection experiments followed by immunofluorescence microscopy clearly showed that spergen-1 is transported to mitochondria in COS-7 cells. In addition, we provided evidence that the 10 or 20 N-terminal amino acid residues in spergen-1 protein are essential for transport of the protein to mitochondria. These results indicate that spergen-1 protein has a mitochondria-targeting signal at the N-terminus. We are currently undertaking experiments to investigate in detail the amino acid sequence of spergen-1 required for mitochondrial targeting.
The vast proportion of mitochondrial proteins are synthesized on cytoplasmic ribosomes and posttranslationally imported into mitochondria via mitochondrial import-stimulation factor (MSF)-dependent pathway or heat shock protein 70 (Hsp70)-dependent pathway [17, 18]. The MSF specifically recognizes mitochondria-targeting signals of precursor proteins and transports them to the Toms receptors on the outer membrane of mitochondria [19]. It remains to be determined whether spergen-1 interacts with MSF or Hsp70.
Although we failed to determine the cell types expressing spergen-1 protein by immunocytochemistry, in situ hybridization revealed that spergen-1 mRNA was exclusively expressed in haploid spermatids. Subcellular fractionation followed by immunoblot analysis suggested that the protein is associated with mitochondria, which is consistent with data from the transfection experiments in COS-7 cells. These outcomes suggest that spergen-1 might be a molecule associated with mitochondria in haploid spermatids in testis. It is noteworthy that spergen-1 is highly expressed in the testis, but not in the other organs, that we examined (Fig. 4), indicating that the protein might be involved in germ cell-specific phenomena, probably involving mitochondria-related function in haploid spermatids.
Mitochondria in spermatozoa are different from those in somatic cells in several ways. Mitochondria of spermatogenic cells modify their morphological organization, number, and location during spermatogenesis, and the morphology of mitochondria changes from orthodox type via a condensed form to the intermediate type during differentiation from spermatogonia to haploid spermatids [20]. From step 8–10 haploid spermatids onward, the intermediate type of mitochondria, which shows crescent-shaped cristae and less condensed matrix, begins to elongate and fuse to a chain-like, twisted structure around the flagellum. Part of the mitochondria tagged with ubiquitin leaves the maturating spermatids and is phagocytosed by Sertoli cells [21]. Because spergen-1 containing mitochondria-targeting signal is specifically expressed in haploid spermatids in the testis, it seems to be probable that spergen-1 might be correlated with these structural and biochemical changes of mitochondria in spermatids. Further studies of spergen-1 may shed more light on the molecular mechanisms regulating the morphological changes of spermatids during spermiogenesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by a Grant-in-Aid for Scientific Research of the Japan Society for the Promotion of Science and Narishige Zoological Science Award. 
2 Correspondence: Hiroshi Iida, Laboratory of Zoology, Graduate School of Agriculture, Kyushu University, Higashiku Hakozaki 6-10-1, Fukuoka 812-8581, Japan. FAX: 92 642 2804; iidahiro{at}agr.kyushu-u.ac.jp
Accepted: December 10, 2001.
Received: October 29, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat. Mol Cell Biol 1988; 8:466-472This article has been cited by other articles:
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  D. Rengaraj, D. K. Kim, Y. H. Zheng, S. I. Lee, H. Kim, and J. Y. Han Testis-Specific Novel Transcripts in Chicken: In Situ Localization and Expression Pattern Profiling During Sexual Development Biol Reprod, September 1, 2008; 79(3): 413 - 420. [Abstract] [Full Text] [PDF]
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 S. Tamai, H. Iida, S. Yokota, T. Sayano, S. Kiguchiya, N. Ishihara, J.-I. Hayashi, K. Mihara, and T. Oka Characterization of the mitochondrial protein LETM1, which maintains the mitochondrial tubular shapes and interacts with the AAA-ATPase BCS1L J. Cell Sci., August 1, 2008; 121(15): 2588 - 2600. [Abstract] [Full Text] [PDF]
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F. Suzuki-Toyota, C. Ito, Y. Toyama, M. Maekawa, R. Yao, T. Noda, H. Iida, and K. Toshimori Factors Maintaining Normal Sperm Tail Structure During Epididymal Maturation Studied in Gopc / Mice Biol Reprod, July 1, 2007; 77(1): 71 - 82. [Abstract] [Full Text] [PDF]
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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, A. Urasoko, M. Doiguchi, T. Mori, K. Toshimori, and Y. Shibata Complementary DNA Cloning and Characterization of Rat spergen-2, a Spermatogenic Cell-Specific Gene 2 Encoding a 56-Kilodalton Nuclear Protein Bearing Ankyrin Repeat Motifs Biol Reprod, August 1, 2003; 69(2): 421 - 429. [Abstract] [Full Text] [PDF]
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