濃尾地震における震裂波動線生成の解明

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Evidence for Muon Neutrino Oscillation in an Accelerator-Based Experiment

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医薬シーズとしての特異的結合性ペプチド（ペプチドアプタマー）の開発

The Saitama Prefecture-led City-Area Project (Saitama Mid-zone) supported by MEXT during June 2007-March 2010 has been finished. Herein, we report on the result of Theme 2 (Peptide Aptamer Research), which fortunately ended in finding of novel functional peptides.textapplication/pdfdepartmental bulletin pape

GLD-1 Has the Opposite Sex Determination Function in C. elegans and C. briggsae

<div><p>For (A) and (B) the distal end of the gonad arm is indicated by the asterisk, and regions of the germline are delimited by dashed vertical lines as follows: M, mitotic zone; TZ, transition zone; P, pachytene; Pa, abnormal pachytene; and S, spermatocytes. For both (A) and (B) staining indicated is as follows: DAPI, blue, nuclear DNA; GLD-1, green; and MSP, red.</p> <p>(A) RNAi of <i>C. briggsae gld-1</i> results in masculinization of the germline. Paired DAPI-stained (left) and GLD-1- and MSP-stained (right) images of dissected young adult hermphrodite germlines. Top four panels illustrate the similarity between C. elegans and C. briggsae germline morphology and polarity (DAPI, blue; GLD-1, green; MSP, red). In both species, sperm (“sperm” arrow) are produced first before switching to oogenesis (“oocytes” arrow), and the pattern of cytoplasmic GLD-1 accumulation (green) is identical. GFP-injected controls were identical to wild-type animals. <i>C. briggsae gld-1</i> RNAi animals exhibit masculinization of the germline (lower panels). A vast excess of sperm extends to the loop region (“sperm” arrows), and spermatogenesis extends further distally (solid line). Masculinization is confirmed by a corresponding extension in MSP staining beyond the loop (compare lower right to controls above).</p> <p>(B) RNAi of <i>gld-1</i> and <i>fog-3</i> in C. elegans and C. briggsae results in a similar tumorous germline phenotype. C. elegans (top) and C. briggsae (bottom) have normal mitotic, transition, and entry into pachytene, but abnormal progression through pachytene, based on DAPI morphology. Both MSP and GLD-1 staining were below the level of detection in both cases.</p></div

The Highly Diverged FOG-2 C-Terminal Region Is Responsible for GLD-1 Interaction in C. elegans

<div><p>(A) Dot plot of FOG-2/FTR-1, with the black diagonal line delimiting regions of greater than 70% identity based on a 10-aa sliding window. The dashed horizontal line at the C-terminus indicates a region of low identity. The arrow indicates the final exon 4 boundary.</p> <p>(B) Protein sequence alignment of FOG-2 and FTR-1 encoded by exon 4. Differences are shaded in black and illustrate the abrupt breakdown in sequence conservation. The dashed line marks the region required for GLD-1 interaction.</p> <p>(C) Nucleotide alignment of <i>fog-2</i> and <i>ftr-1</i> EST coding regions expanded from a portion of the protein sequence alignment, with vertical lines delimiting the reading frame relative to <i>fog-2.</i> Amino acid sequence for FOG-2 (above) and changes in FTR-1 (below) are below the alignment. Frame-shifting indels are indicated by the large open arrowheads.</p> <p>(D) The C-terminal FOG-2 region is required for GLD-1 interaction in the yeast two-hybrid system. Full-length FOG-2 (black) and FTR-1 (grey) constructs were tested for interaction with GLD-1. FOG-2 interacts with GLD-1 (++++) whereas FTR-1 does not (−). Progressive C-terminal deletions (black) in FOG-2 were generated to identify FOG-2 requirements for GLD-1 interaction. Binding to GLD-1 was completely eliminated with the removal of the C-terminal 64 aa of FOG-2 exon 4. Transfer of exon 4 to FTR-1 (grey/black chimera) resulted in the transfer of GLD-1 binding to FTR-1. Control interactions to test for the production of functional proteins were performed with the Skp1 homolog SKR-1, which binds to the F-box region (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030006#s4" target="_blank">Materials and Methods</a>). Searches for C. elegans and C. briggsae proteins with homology to the 64-aa FOG-2 region required for GLD-1 interaction (or FOG-2 exon 4) failed to identify any predicted proteins with significant homology (>35% or e-value = 0.01) other than FTR-1, which cannot bind GLD-1 and does not compensate for FOG-2 in sex determination.</p> <p>(E) Sliding-window (100-nt window, 25-nt shift) estimation of K_a/K_s ratio for <i>fog-2/ftr-1</i> using full-length average K_s. The K_a/K_s ratio is highest at the C-terminal end of the Duf38/FTH domain, reaching a peak of 2.2 in window 37. The position of the F-box and Duf38/FTH domain are indicated by grey shading. The bold horizontal line is at the K_a/K_s = 1 threshold. The dashed vertical line indicates the boundary between exon 3 and exon 4.</p></div

The C. elegans XX Hermaphrodite Germline Sex Determination Pathway

<div><p>(A) Genetic pathway for gene activity, where arrows represent positive regulation and bars represent negative regulation. The key genes <i>tra-2</i> and <i>fem-3</i> and the upstream regulators of <i>tra-2</i> that are the focus of this work, <i>fog-2</i> and <i>gld-1,</i> are in large bold font. The upstream genes <i>fog-2</i> and <i>gld-1,</i> which are key regulators of <i>tra-2</i> and addressed in this work, are also in large bold font. The gene activities at each level in the hierarchy are indicated below as “ACTIVE” in bold or “inactive” in grey. In L3 and L4 hermaphrodites the activities of <i>fog-2</i> and <i>gld-1</i> are high, leading to repression of <i>tra-2</i> activity (also see [B]) and the de-repression of <i>fem-3,</i> resulting in the onset of spermatogenesis. In L4 and adult hermaphrodites the activity of <i>fog-2</i> and <i>gld-1</i> are low, leading to high <i>tra-2</i> activity and the repression of <i>fem-3,</i> resulting in oogenesis. The shift in <i>tra-2</i>/<i>fem-3</i> balance allows for the switch from spermatogenesis to oogenesis in an otherwise female somatic gonad in the hermaphrodite.</p> <p>(B) C. elegans FOG-2/GLD-1/<i>tra-2</i> mRNA ternary complex. Current data indicates that FOG-2 and GLD-1 are required for the translational repression of the <i>tra-2</i> mRNA [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030006#pbio-0030006-b25" target="_blank">25</a>]. GLD-1 binds as a dimer to the <i>tra-2</i> mRNA 3′UTR at two 28 nucleotide direct repeat elements (TGE/DRE, blocks) and FOG-2 makes contact with GLD-1 [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030006#pbio-0030006-b32" target="_blank">32</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030006#pbio-0030006-b34" target="_blank">34</a>]. All three components are required for the proper specification of hermaphrodite spermatogenesis.</p></div

GLD-1-Mediated Translational Repression of <i>rme-2</i> mRNA in C. elegans and C. briggsae

<p>In both C. elegans and C. briggsae wild-type (WT) animals (left panels), GLD-1 (green) and RME-2 (red) have mutually exclusive accumulation patterns. In C. elegans (upper right), <i>gld-1</i> and <i>fog-3</i> RNAi results in a germline tumor with ectopic RME-2 accumulation (red expanded). In C. briggsae (lower right), RNAi of <i>gld-1</i> and <i>fog-3</i> also results in germline tumor with ectopic RME-2 accumulation (red expanded). The germline tumor and expansion of RME-2 expression due to ectopic translation are similar between the two species (compare right top and bottom, DAPI [blue]). The distal end of the gonad arm is indicated by the asterisk, and regions of the germline are delimited by dashed vertical lines. DAPI, blue, nuclear DNA; GLD-1, green; RME-2, red; M, mitotic zone; TZ, transition zone; P, pachytene; Pa, abnormal pachytene.</p

<i>fog-2</i> Is Likely Absent in C. briggsae

<div><p>Low-stringency Southern blotting (A) and conservation of synteny (B and C) were used in an attempt to identify a potential <i>fog-2</i> gene in C. briggsae.</p> <p>(A) A total of 2–20 ug of digested genomic DNA was used in low-stringency Southern blotting. <i>C. elegans fem-2</i> probe (<i>Ce</i>_<i>fem-2</i>) was able to detect <i>fem-2</i> on both same-species and cross-species blots (first two panels). The <i>C. elegans fog-2</i> probe (<i>Ce_fog-2</i>), which detects both <i>fog-2</i> and <i>ftr-1</i> on the 5.8-kb XhoI fragment, produced a signal with C. elegans but not C. briggsae genomic DNA (next two panels). <i>fog-2</i> cross-species blot integrity was verified by stripping and reprobing with same-species <i>C. briggsae fem-2</i> (final panel). Same-species exposures were 4 h and cross-species were 4 d. The <i>C. elegans fem-2</i> probe is 70% identical to the C. briggsae genomic sequence.</p> <p>(B) Scale diagram of the C. elegans Chromosome 5 region containing <i>fog-2</i>. A 82.6-kb enlargement below, indicated by the dashed lines, shows the <i>fog-2</i> cluster containing five canonical FTR genes, one FTR gene with divergent structure, and 16 non-FTR genes (also see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030006#st001" target="_blank">Table S1</a>).</p> <p>(C) C. briggsae contig from the genome assembly containing flanking regions with conserved synteny. A 194.4-kb enlargement below, indicated by the dashed lines, covers the C. briggsae region that is predicted to contain a putative <i>fog-2</i> ortholog. The conserved genes used to identify the C. briggsae contig are indicated by the arrowheads, with the genes flanking <i>fog-2</i> indicated by the large arrowheads.</p> <p>Each gene from the C. briggsae contig with an ortholog defined as a reciprocal best BLAST hit is present on both maps (B and C), and blocks of synteny defined by the C. elegans organization are in the same color. Only one (Y113G7B.11) of the 22 genes from the 82.6-kb <i>fog-2</i> cluster was found to have a reciprocal best BLAST hit in C. briggsae (contig cb25.fpc0129, corresponding to the predicted gene CBG05618; <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030006#st001" target="_blank">Table S1</a>). No FTR genes or genes related to those in the <i>fog-2</i> cluster were found within 50-kb on either side of <i>CBG05618,</i> indicating that this region does not share conserved synteny with the <i>fog-2</i> cluster. Instead, the potential C. briggsae ortholog of Y113G7B.11 is located on a C. briggsae contig region that shows extensive conserved synteny with a different portion of C. elegans Chromosome 5 not involving the <i>fog-2</i> cluster (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030006#st002" target="_blank">Table S2</a>).</p></div

The FTR Gene Family in C. elegans and C. briggsae

<p>A radial phylogram showing the relationships of 30 C. elegans and C. briggsae FTR genes closely related to FOG-2 was generated using neighbor-joining. C. elegans and C. briggsae protein predictions with complete F-box and Duf38/FTH (FTR proteins) were identified using BLAST and HMMs, aligned using CLUSTALW, trimmed, de-gapped, and realigned (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030006#s4" target="_blank">Materials and Methods</a>). A clear separation of C. elegans (below dashed line) and C. briggsae (above dashed line) FTR proteins is indicated by the phylogeny. The branch containing FOG-2 and FTR-1 is in bold. Tree is unrooted, and branch lengths are proportional to divergence (also see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030006#sg001" target="_blank">Figure S1</a>). Bar represents 0.1 substitutions per site. FOG-2 and FTR-1, across their entire length, are more similar to each other than to any other gene in C. elegans. Comparison of the diverged approximately 40aa C-terminal region from both proteins to the closely related FTR genes in the FOG-2 cluster reveals 48% average pairwise identity between these FTRs and FTR-1 and 22% average pairwise identify between these FTRs and FOG-2 (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030006#sg002" target="_blank">Figure S2</a>). One interpretation of this greater similarity is that FTR-1 may be ancestral; however, it is not clear whether the slight increase in similarity over about 40aa is significant or whether selection rather than evolutionary history produced the sequence similarity observed.</p

Analysis of Prostate-Specific Antigen Transcripts in Chimpanzees, Cynomolgus Monkeys, Baboons, and African Green Monkeys

<div><p>The function of prostate-specific antigen (PSA) is to liquefy the semen coagulum so that the released sperm can fuse with the ovum. Fifteen spliced variants of the <i>PSA</i> gene have been reported in humans, but little is known about alternative splicing in nonhuman primates. Positive selection has been reported in sex- and reproductive-related genes from sea urchins to <i>Drosophila</i> to humans; however, there are few studies of adaptive evolution of the <i>PSA</i> gene. Here, using polymerase chain reaction (PCR) product cloning and sequencing, we study <i>PSA</i> transcript variant heterogeneity in the prostates of chimpanzees (<i>Pan troglodytes</i>), cynomolgus monkeys (<i>Macaca fascicularis</i>), baboons <i>(Papio hamadryas anubis),</i> and African green monkeys <i>(Chlorocebus aethiops)</i>. Six <i>PSA</i> variants were identified in the chimpanzee prostate, but only two variants were found in cynomolgus monkeys, baboons, and African green monkeys. In the chimpanzee the full-length transcript is expressed at the same magnitude as the transcripts that retain intron 3. We have found previously unidentified splice variants of the <i>PSA</i> gene, some of which might be linked to disease conditions. Selection on the <i>PSA</i> gene was studied in 11 primate species by computational methods using the sequences reported here for African green monkey, cynomolgus monkey, baboon, and chimpanzee and other sequences available in public databases. A codon-based analysis (dN/dS) of the <i>PSA</i> gene identified potential adaptive evolution at five residue sites (Arg45, Lys70, Gln144, Pro189, and Thr203).</p></div