Shiro Okuno

A radiation hybrid map of the rat genome containing 5,255 markers

A whole-genome radiation hybrid (RH) panel was used to construct a high-resolution map of the rat genome based on microsatellite and gene markers. These include 3,019 new microsatellite markers described here for the first time and 1,714 microsatellite markers with known genetic locations, allowing comparison and integration of maps from different sources. A robust RH framework map containing 1,030 positions ordered with odds of at least 1,000:1 has been defined as a tool for mapping these markers, and for future RH mapping in the rat. More than 500 genes which have been mapped in mouse and/or human were localized with respect to the rat RH framework, allowing the construction of detailed rat-mouse and rat-human comparative maps and illustrating the power of the RH approach for comparative mapping.

Cloning and chromosomal mapping of a novel ABC transporter gene (hABC7), a candidate for X-linked sideroblastic anemia with spinocerebellar ataxia

AbstractWe isolated a novel human ATP-binding cassette (ABC) transporter cDNA, determined its nucleotide sequence, and designated it human ABC7 (hABC7). The nucleotide sequence was highly homologous to the ATM1 gene in yeast, which encodes an ABC transporter (yAtm1p) located in the mitochondrial inner membrane. The deduced human product, a putative half-type transporter, consists of 752 amino acids that are 48.9% identical to those of yAtm1p. A computer-assisted protein structural and localization analysis revealed that the mitochondrial targeting signal of yAtm1p is conserved in the N-terminal region of the primary sequence of the hABC7 protein, and therefore this product is also likely to be located in the mitochondrial inner membrane. The evidence strongly suggests that the hABC7 gene is a counterpart of ATM1 and that its product is probably involved in heme transport. We mapped the hABC7 gene to chromosome Xq13.1–q13.3 by fluorescence in-situ hybridization. As band Xq13 has been implicated in X-linked sideroblastic anemia with spinocerebellar ataxia, hABC7 becomes a candidate gene for this heritable disorder.

Adhalin gene mutations in patients with autosomal recessive childhood onset muscular dystrophy with adhalin deficiency.

Homozygous adhalin gene mutations were found in three patients from two consanguineous families with autosomal recessive childhood onset muscular dystrophy. Muscle biopsies from patients in each family showed complete absence of adhalin. Sequencing of adhalin cDNA prepared from skeletal muscle by reverse transcription PCR demonstrated a cytosine to thymidine substitution at nt 229 in the patient in family 1 and an adenine to guanine substitution at nt 410 and a 15-base insertion between nt 408 and 409 in the two patients in family 2. Sequencing of genomic DNA prepared from peripheral blood leukocytes by PCR confirmed these mutations. The parents in each family were found to be heterozygous for the respective mutations. These adhalin gene mutations are presumed to be responsible for the absence of adhalin in the skeletal muscle. Adhalin deficiency likely causes disruption of the muscle cell membrane, resulting in dystrophic changes in the skeletal muscle similar to dystrophin deficiency in Duchenne muscular dystrophy.

MUTATED G-PROTEIN-COUPLED RECEPTOR GPR10 IS RESPONSIBLE FOR THE HYPERPHAGIA/DYSLIPIDAEMIA/OBESITY LOCUS OF Dmo1 IN THE OLETF RAT

1. We have confirmed the Diabetes Mellitus OLETF type I (Dmo1) effect on hyperphagia, dyslipidaemia and obesity in the Otsuka Long‐Evans Tokushima Fatty (OLETF) strain. The critical interval was narrowed down to 570 kb between D1Got258 to p162CA1 by segregation analyses using congenic lines.

Cloning, expression, and mapping of UBE2I, a novel gene encoding a human homologue of yeast ubiquitin-conjugating enzymes which are critical for regulating the cell cycle.

From a human fetal-brain cDNA library we isolated a novel gene sharing significant homology with two yeast genes, UBC9 and hus5, which encode ubiquitin-conjugating enzyme 9 (UBC9). In yeast this protein is critical for normal mitosis, and seems to be closely involved in progression of G2 to M phase of the cell cycle. The human UBC9 (h-UBC9) cDNA, (gene symbol UBE2I), contained an open reading frame of 474 nucleotides encoding 158 amino acids. Its predicted peptide showed respectively 56% and 66% identity (75% and 82% similarity) with the products of UBC9 and hus5. Northern-blot analysis revealed expression of three transcripts, 6.4 kb, 3.3 kb, and 1.35 kb, in all human tissues examined. This gene, UBE2I, was mapped to chromosome band 16p13.3 by FISH.

Clinical, pathological, and genetic features of limb‐girdle muscular dystrophy type 2A with new calpain 3 gene mutations in seven patients from three Japanese families

We report on the clinical, pathological, and genetic features of 7 patients with limb‐girdle muscular dystrophy type 2A (LGMD2A) from three Japanese families. The mean age of onset was 9.7 ± 3.1 years (mean ± SD), and loss of ambulance occurred at 38.5 ± 2.1 years. Muscle atrophy was predominant in the pelvic and shoulder girdles, and proximal limb muscles. Muscle pathology revealed dystrophic changes. In two families, an identical G to C mutation at position 1080 the in calpain 3 gene was identified, and a frameshift mutation (1796insA) was found in the third family. The former mutation results in a W360R substitution in the proteolytic site of calpain 3, and the latter in a deletion of the Ca2+‐binding domain.

Cloning, expression, and mapping of TCTEL1 a putative human homologue of murine Tcte1, to 6q

From a human fetal-brain cDNA library we isolated a putative human homologue of the murine Tcte1 gene. The cDNA, designated TCTEL1, contained an open reading frame of 339 nucleotides encoding 113 amino acids. The predicted peptides of TCTEL1 showed 94% and 55% identity (100% and 94% similarity) with those of murine Tcte1 and human RP3. Northern-blot analysis revealed a 0.9-kb transcript in all tissues examined. This gene was mapped by FISH to chromosome bands 6q25.2 --> q25.3, the syntenic region of the murine t-complex locus of chromosome 17.

Isolation and mapping of karyopherin α3 (KPNA3), a human gene that is highly homologous to genes encoding Xenopus importin, yeast SRP1 and human RCH1

From a human fetal-brain cDNA library, we isolated and characterized a novel gene (KPNA3) encoding a protein highly homologous to certain nuclear transport proteins of Xenopus and human. The complete cDNA clone, designated karyopherin alpha 3, contained an open reading frame of 1,563 nucleotides encoding 521 amino acids. The predicted amino acid sequence showed 48%, 45% and 48% identity with Xenopus importin, yeast SRP1 and human RCH1, respectively. The similarities among these proteins suggest that karyopherin alpha 3 may be involved in the nuclear transport system. Eight repeats of the arm motif were well conserved among these proteins. The N-terminal region of the predicted karyopherin alpha 3 product was highly basic and the C-terminal region was strongly acidic. A 4.3-kb transcript was expressed in all adult human tissues examined by Northern blotting. The cDNA clone was assigned to chromosome band 13q14.3 by fluorescence in situ hybridization.

Cloning and chromosome assignment to 1q32 of a human cDNA (RAB7L1) encoding a small GTP-binding protein, a member of the RAS superfamily

A full-length cDNA homologous to RAB7, a member of the RAB-related GTP-binding protein subfamily, was isolated from a human placenta cDNA library. This cDNA, designated RAB7L1, has an open reading frame of 609 nucleotides encoding 203 amino acids. Northern analysis showed that the mRNA is ubiquitously expressed in human tissues, although signal intensities were different among the various organs examined. This gene was located on chromosome band 1q32 by fluorescence in situ hybridization.

Characterization of newly developed SSLP markers for the rat.

Abstract. We have isolated more than 12,000 clones containing microsatellite sequences, mainly consisting of (CA)n dinucleotide repeats, using genomic DNA from the BN strain of laboratory rat. Data trimming yielded 9636 non-redundant microsatellite sequences, and we designed oligonucleotide primer pairs to amplify 8189 of these. PCR amplification of genomic DNA from five different rat strains yielded clean amplification products for 7040 of these simple-sequence-length-polymorphism (SSLP) markers; 3019 markers had been mapped previously by radiation hybrid (RH) mapping methods (Nat Genet 22, 27–36, 1998). Here we report the characterization of these newly developed microsatellite markers as well as the release of previously unpublished microsatellite marker information. In addition, we have constructed a genome-wide linkage map of 515 markers, 204 of which are derived from our new collection, by genotyping 48 F2 progeny of (OLETFxBN)F2 crosses. This map spans 1830.9 cM, with an average spacing of 3.56 cM. Together with our ongoing project of preparing a whole-genome radiation hybrid map for the rat, this dense linkage map should provide a valuable resource for genetic studies in this model species.