Kumiko Yoshinobu

Structure and expression of human mitochondrial adenylate kinase targeted to the mitochondrial matrix

The previously isolated cDNA encoding human adenylate kinase (AK) isozyme 3 was recently renamed AK4. Consequently, human AK3 cDNA remains to be identified and we have little information about the functional relationship between human AK3 and AK4. In pursuit of the physiological roles of both the AK3 and AK4 proteins, we first isolated an authentic human AK3 cDNA and compared their expression. Nucleotide sequencing revealed that the cDNA encoded a 227-amino-acid protein, with a deduced molecular mass of 25.6 kDa, that shares greater homology with the AK3 cDNAs isolated from bovine and rat than that from human. We named the isolated cDNA AK3. Northern-blot analysis revealed that AK3 mRNA was present in all tissues examined, and was highly expressed in heart, skeletal muscle and liver, moderately expressed in pancreas and kidney, and weakly expressed in placenta, brain and lung. On the other hand, we found that human AK4 mRNA was highly expressed in kidney, moderately expressed in heart and liver and weakly expressed in brain. Western-blot analysis demonstrated expression profiles of AK3 and AK4 that were similar to their mRNA expression patterns in each tissue. Over expression of AK3, but not AK4, in both Escherichia coli CV2, a temperature-sensitive AK mutant, and a human embryonic kidney-derived cell line, HEK-293, not only produced significant GTP:AMP phosphotransferase (AK3) activity, but also complemented the CV2 cells at 42 degrees C. Subcellular and submitochondrial fractionation analysis demonstrated that both AK3 and AK4 are localized in the mitochondrial matrix.

Region-specific expression of murine Hox genes implies the Hox code-mediated patterning of the digestive tract

Hox genes encode transcription factors which are involved in the establishment of regional identities along the anteroposterior (AP) body axis. To elucidate the AP patterning of the digestive tract, we have systematically examined the expression patterns of Hox genes belonging to paralogue groups 6, 7, 8 and 9 by whole‐mount in situ hybridization and by section in situ hybridization analyses.

A novel murine gene, Sickle tail, linked to the Danforth's short tail locus, is required for normal development of the intervertebral disc

We established the mutant mouse line, B6;CB-SktGtAyu8021IMEG (SktGt), through gene-trap mutagenesis in embryonic stem cells. The novel gene identified, called Sickle tail (Skt), is composed of 19 exons and encodes a protein of 1352 amino acids. Expression of a reporter gene was detected in the notochord during embryogenesis and in the nucleus pulposus of mice. Compression of some of the nuclei pulposi in the intervertebral discs (IVDs) appeared at embryonic day (E) 17.5, resulting in a kinky-tail phenotype showing defects in the nucleus pulposus and annulus fibrosus of IVDs in SktGt/Gt mice. These phenotypes were different from those in Danforths short tail (Sd) mice in which the nucleus pulposus was totally absent and replaced by peripheral fibers similar to those seen in the annulus fibrosus in all IVDs. The Skt gene maps to the proximal part of mouse chromosome 2, near the Sd locus. The genetic distance between them was 0.95 cM. The number of vertebrae in both [Sd +/+ SktGt] and [Sd SktGt/+ +] compound heterozygotes was less than that of Sd heterozygotes. Furthermore, the enhancer trap locus Etl4lacZ, which was previously reported to be an allele of Sd, was located in the third intron of the Skt gene.

Database for exchangeable gene trap clones: Pathway and gene ontology analysis of exchangeable gene trap clone mouse lines

Gene trapping in embryonic stem (ES) cells is a proven method for large‐scale random insertional mutagenesis in the mouse genome. We have established an exchangeable gene trap system, in which a reporter gene can be exchanged for any other DNA of interest through Cre/mutant lox‐mediated recombination. We isolated trap clones, analyzed trapped genes, and constructed the database for Exchangeable Gene Trap Clones (EGTC) [http://egtc.jp]. The number of registered ES cell lines was 1162 on 31 August 2013. We also established 454 mouse lines from trap ES clones and deposited them in the mouse embryo bank at the Center for Animal Resources and Development, Kumamoto University, Japan. The EGTC database is the most extensive academic resource for gene‐trap mouse lines. Because we used a promoter‐trap strategy, all trapped genes were expressed in ES cells. To understand the general characteristics of the trapped genes in the EGTC library, we used Kyoto Encyclopedia of Genes and Genomes (KEGG) for pathway analysis and found that the EGTC ES clones covered a broad range of pathways. We also used Gene Ontology (GO) classification data provided by Mouse Genome Informatics (MGI) to compare the functional distribution of genes in each GO term between trapped genes in the EGTC mouse lines and total genes annotated in MGI. We found the functional distributions for the trapped genes in the EGTC mouse lines and for the RefSeq genes for the whole mouse genome were similar, indicating that the EGTC mouse lines had trapped a wide range of mouse genes.

Primary structures of sperm-specific basic nuclear proteins and gene expression in Japanese newt, Cynops pyrrhogaster

Electrophoretic analyses of acid extracts from mature sperm of newt, Cynops pyrrhogaster, on acid/urea/Triton X‐100 polyacrylamide gel showed the exclusive occurrence of sperm‐specific nuclear basic proteins (SBPs), which moved faster than somatic histones on the gel. These SBPs were eluted separately by reversed phase‐high‐performance liquid chromatography as two large peaks and a few small peaks. Of these, only the small peaks disappeared with treatment of the acid extracts with alkaline phosphatase before they were injected into the column, so that there were only two distinct components: NP1 and NP2. Determination of amino acid sequences by the Edman method as well as by sequencing of cDNA for both components indicated that each protein consisted of 43 (NP1) or 48 (NP2) amino acid residues, rich in arginine residues (53.5% in NP1; 47.9% in NP2), forming the clusters. They had molecular masses of 5,386 Da (NP1) and 5,748 Da (NP2), respectively. Northern blot analysis using cDNAs as probes indicated that mRNAs for both NP1 and NP2 occurred not in primary spermatocytes but in round spermatids. In situ hybridization analyses using antisense RNA for NP1 as a probe clearly showed the first appearance of NP1 mRNA at the late stage of round spermatid. Mol. Reprod. Dev. 46:243–251, 1997.

Development of an efficient screening system to identify novel bone metabolism-related genes using the exchangeable gene trap mutagenesis mouse models.

Despite numerous genetic studies on bone metabolism, understanding of the specific mechanisms is lacking. We developed an efficient screening system to identify novel genes involved in bone metabolism using mutant mouse strains registered with the Exchangeable Gene Trap Clones (EGTC) database. From 1278 trap clones in the EGTC database, 52 candidate lines were selected in the first screening, determined based on “EST profile”, “X-gal”, “Related article”, and “Novel gene”. For the second screening, bone morphometric analysis, biomechanical strength analysis, bone X-gal staining, etc. were performed on candidate lines. Forty-two male trap lines (80.8%) showed abnormalities with either bone morphometric analysis or biomechanical strength analysis. In the screening process, X-gal staining was significantly efficient (P = 0.0057). As examples, Lbr and Nedd4 trap lines selected using the screening system showed significant bone decrease and fragility, suggesting a relationship with osteoblast differentiation. This screening system using EGTC mouse lines is extremely efficient for identifying novel genes involved in bone metabolism. The gene trap lines identified as abnormal using this screening approach are highly likely to trap important genes for bone metabolism. These selected trap mice will be valuable for use as novel bio-resources in bone research.