Solution structure of the RWD domain of the mouse GCN2 protein

GCN2 is the alpha-subunit of the only translation initiation factor (eIF2alpha) kinase that appears in all eukaryotes. Its function requires an interaction with GCN1 via the domain at its N-terminus, which is termed the RWD domain after three major RWD-containing proteins: RING finger-containing proteins, WD-repeat-containing proteins, and yeast DEAD (DEXD)-like helicases. In this study, we determined the solution structure of the mouse GCN2 RWD domain using NMR spectroscopy. The structure forms an alpha + beta sandwich fold consisting of two layers: a four-stranded antiparallel beta-sheet, and three side-by-side alpha-helices, with an alphabetabetabetabetaalphaalpha topology. A characteristic YPXXXP motif, which always occurs in RWD domains, forms a stable loop including three consecutive beta-turns that overlap with each other by two residues (triple beta-turn). As putative binding sites with GCN1, a structure-based alignment allowed the identification of several surface residues in alpha-helix 3 that are characteristic of the GCN2 RWD domains. Despite the apparent absence of sequence similarity, the RWD structure significantly resembles that of ubiquitin-conjugating enzymes (E2s), with most of the structural differences in the region connecting beta-strand 4 and alpha-helix 3. The structural architecture, including the triple beta-turn, is fundamentally common among various RWD domains and E2s, but most of the surface residues on the structure vary. Thus, it appears that the RWD domain is a novel structural domain for protein-binding that plays specific roles in individual RWD-containing proteins.     

Computational analysis of full-length mouse cDNAs compared with human genome sequences

Although the sequencing of the human genome is complete, identification of encoded genes and determination of their structures remain a major challenge. In this report, we introduce a method that effectively uses full-length mouse cDNAs to complement efforts in carrying out these difficult tasks. A total of 61,227 RIKEN mouse cDNAs (21,076 full-length and 40,151 EST sequences containing certain redundancies) were aligned with the draft human sequences. We found 35,141 non-redundant genomic regions that showed a significant alignment with the mouse cDNAs. We analyzed the structures and compositional properties of the regions detected by the full-length cDNAs, including cross-species comparisons, and noted a systematic bias of GENSCAN against exons of small size and/or low GC-content. Of the cDNAs locating the 35,141 genomic regions, 3,217 did not match any sequences of the known human genes or ESTs. Among those 3,217 cDNAs, 1,141 did not show any significant similarity to any protein sequence in the GenBank non-redundant protein database and thus are candidates for novel genes. Received: 18 January 2001 / Accepted: 17 May 2001     

Simultaneous determination of ascorbic acid and dehydroascorbic acid in fish tissues by high-performance liquid chromatography

An high-performance liquid chromatographic method with post-column derivatization has been developed for the simultaneous determination of ascorbic acid (AA) and dehydroascorbic acid (DHAA) in fish tissues. Extracted AA and DHAA were separated by a Shim-pack SCR-101H column within 20 min, reacted with sodium hydroxide containing sodium borohydride and monitored at 300 nm. The detection limits for both AA and DHAA were 0.1 μg/ml.     

Inactivation of Dnmt3b in mouse embryonic fibroblasts results in DNA hypomethylation, chromosomal instability, and spontaneous immortalization

DNA hypomethylation is a hallmark of many types of solid tumors. However, it remains elusive how DNA hypomethylation may contribute to tumorigenesis. In this study, we have investigated how targeted disruption of the DNA methyltransferases Dnmt3a and Dnmt3b affects the growth of mouse embryonic fibroblasts (MEFs). Our studies led to the following observations. 1) Constitutive or conditional deletion of Dnmt3b, but not Dnmt3a, resulted in partial loss of DNA methylation throughout the genome, suggesting that Dnmt3b, in addition to the major maintenance methyltransferase Dnmt1, is required for maintaining DNA methylation in MEF cells. 2) Dnmt3b-deficient MEF cells showed aneuploidy and polyploidy, chromosomal breaks, and fusions. 3) Inactivation of Dnmt3b resulted in either premature senescence or spontaneous immortalization of MEF cells. 4) The G(1) to S-phase checkpoint was intact in primary and spontaneously immortalized Dnmt3b-deficient MEFs because the p53 protein was inducible by DNA damage. Interestingly, protein levels of the cyclindependent kinase inhibitor p21 were increased in immortalized Dnmt3b-deficient MEFs even in the absence of p53 induction. These results suggest that DNA hypomethylation may induce genomic instability, which in turn leads to spontaneous immortalization or premature senescence of Dnmt3b-deficient MEFs via a p53-independent mechanism.     

Sensitive detection of EGFR mutations using a competitive probe to suppress background in the SMart Amplification Process.

In a previous study, a single nucleotide polymorphism (SNP) diagnostic system named the SMart Amplification Process version 2 (SMAP 2) was reported, which enabled rapid gene diagnostics from crude samples such as whole blood. The asymmetric primer design and use of Taq MutS were reported as innovative background suppression technologies employed by SMAP 2, but Taq MutS is known to display differential affinities for various mismatch combinations, and hence may not be entirely effective for all possible applications. To address this issue we developed another approach using a competitive probe (CP) to enhance background suppression technology instead of Taq MutS. CP is a 3'-end aminated oligonucleotide that competes with 3'-end of a discrimination primer or the self-priming elongation site on intermediate product 2 (IM2) for non-target sequences, such as the alternative allele. The preferred hybridization kinetics for the full-match CP on the non-target sequence results in effective background suppression in SMAP 2 assays. By using a CP, we demonstrated the sensitive detection of EGFR gene mutations in purified genomic DNA from mixed cell populations. The CP approach is another tool enhancing the effectiveness and versatility of SMAP 2 assays, expanding its potential applications, and reinforcing its position as a highly effective technology for molecular diagnostics.     

The Riken mouse genome encyclopedia project

The Riken mouse genome encyclopedia a comprehensive full-length cDNA collection and sequence database. High-level functional annotation is based on sequence homology search, expression profiling, mapping and protein-protein interactions. More than 1000000 clones prepared from 163 tissues were end-sequenced and classified into 128000 clusters, and 60000 representative clones were fully sequenced representing 24000 clear protein-encoding genes. The application of the mouse genome database for positional cloning and gene network regulation analysis is reported.     

Therapeutic Strategy for Ischemic Stroke

Possible strategies for treating ischemic stroke include: (1) Neuroprotection: preventing damaged neurons from undergoing apoptosis in the acute phase of cerebral ischemia; (2) Stem cell therapy: the repair of broken neuronal networks with newly born neurons in the chronic phase of cerebral ischemia. Firstly, we studied the neuroprotective effect of a calcium channel blocker, azelnidipine, or a by-product of heme degradation, biliverdin, in the ischemic brain. These results revealed both azelnidipine and biliverdin had a neuroprotective effect in the ischemic brain through their anti-oxidative property. Secondly, we investigated the role of granulocyte colony-stimulating factor (G-CSF) by administering G-CSF to rats after cerebral ischemia and found G-CSF plays a critical role in neuroprotection. Lastly, we developed a restorative stroke therapy with a bio-affinitive scaffold, which is able to provide an appropriate environment for newly born neurons. In the future, we will combine these strategies to develop more effective therapies for treatment of strokes. Special issue article in honor of Dr. Akitane Mori.