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Yoshiyuki Sakuraba

Publications and source records attributed to Yoshiyuki Sakuraba.

8 recordsLinked to original sources
PubMed2006

A series of ENU-induced single-base substitutions in a long-range cis-element altering Sonic hedgehog expression in the developing mouse limb bud.

Mammal-fish-conserved-sequence 1 (MFCS1) is a highly conserved sequence that acts as a limb-specific cis-acting regulator of Sonic hedgehog (Shh) expression, residing 1 Mb away from the Shh coding sequence in mouse. Using gene-driven screening of an ENU-mutagenized mouse archive, we obtained mice with three new point mutations in MFCS1: M101116, M101117, and M101192. Phenotype analysis revealed that M101116 mice exhibit preaxial polydactyly and ectopic Shh expression at the anterior margin of the limb buds like a previously identified mutant, M100081. In contrast, M101117 and M101192 show no marked abnormalities in limb morphology. Furthermore, transgenic analysis revealed that the M101116 and M100081 sequences drive ectopic reporter gene expression at the anterior margin of the limb bud, in addition to the normal posterior expression. Such ectopic expression was not observed in the embryos carrying a reporter transgene driven by M101117. These results suggest that M101116 and M100081 affect the negative regulatory activity of MFCS1, which suppresses anterior Shh expression in developing limb buds. Thus, this study shows that gene-driven screening for ENU-induced mutations is an effective approach for exploring the function of conserved, noncoding sequences and potential cis-regulatory elements.

Animals↗
PubMed2005

Molecular characterization of ENU mouse mutagenesis and archives.

The large-scale mouse mutagenesis with ENU has provided forward-genetic resources for functional genomics. The frozen sperm archive of ENU-mutagenized generation-1 (G1) mice could also provide a "mutant mouse library" that allows us to conduct reverse genetics in any particular target genes. We have archived frozen sperm as well as genomic DNA from 9224 G1 mice. By genome-wide screening of 63 target loci covering a sum of 197 Mbp of the mouse genome, a total of 148 ENU-induced mutations have been directly identified. The sites of mutations were primarily identified by temperature gradient capillary electrophoresis method followed by direct sequencing. The molecular characterization revealed that all the identified mutations were point mutations and mostly independent events except a few cases of redundant mutations. The base-substitution spectra in this study were different from those of the phenotype-based mutagenesis. The ENU-based gene-driven mutagenesis in the mouse now becomes feasible and practical.

Animals↗
PubMed2005

Srs2 and RecQ homologs cooperate in mei-3-mediated homologous recombination repair of Neurospora crassa.

Homologous recombination and post-replication repair facilitate restart of stalled or collapsed replication forks. The SRS2 gene of Saccharomyces cerevisiae encodes a 3'-5' DNA helicase that functions both in homologous recombination repair and in post-replication repair. This study identifies and characterizes the SRS2 homolog in Neurospora crassa, which we call mus-50. A knockout mutant of N.crassa, mus-50, is sensitive to several DNA-damaging agents and genetic analyses indicate that it is epistatic with mei-3 (RAD51 homolog), mus-11 (RAD52 homolog), mus-48 (RAD55 homolog) and mus-49 (RAD57 homolog), suggesting a role for mus-50 in homologous recombination repair. However, epistasis evidence has presented that MUS50 does not participate in post-replication repair in N.crassa. Also, the N.crassa mus-25 (RAD54 homolog) mus-50 double mutant is viable, which is in contrast to the lethal phenotype of the equivalent rad54 srs2 mutant in S.cerevisiae. Tetrad analysis revealed that mus-50 in combination with mutations in two RecQ homologs, qde-3 and recQ2, is lethal, and this lethality is suppressed by mutation in mei-3, mus-11 or mus-25. Evidence is also presented for the two independent pathways for recovery from camptothecin-induced replication fork arrest: one pathway is dependent on QDE3 and MUS50 and the other pathway is dependent on MUS25 and RECQ2.

DNA Helicases↗
PubMed2005

Enamelin (Enam) is essential for amelogenesis: ENU-induced mouse mutants as models for different clinical subtypes of human amelogenesis imperfecta (AI).

Amelogenesis imperfecta (AI) is a group of commonly inherited defects of dental enamel formation, which exhibits marked genetic and clinical heterogeneity. The genetic basis of this heterogeneity is still poorly understood. Enamelin, the affected gene product in one form of AI (AIH2), is an extracellular matrix protein that is one of the components of enamel. We isolated three ENU-induced dominant mouse mutations, M100395, M100514 and M100521, which caused AI-like phenotypes in the incisors and molars of the affected individuals. Linkage analyses mapped each of the three mutations to a region of chromosome 5 that contained the genes encoding enamelin (Enam) and ameloblastin (Ambn). Sequence analysis revealed that each mutation was a single-base substitution in Enam. M100395 (Enam(Rgsc395)) and M100514 (Enam(Rgsc514)) were putative missense mutations that caused S to I and E to G substitutions at positions 55 and 57 of the translated protein, respectively. Enam(Rgsc395) and Enam(Rgsc514) heterozygotes showed severe breakage of the enamel surface, a phenotype that resembled local hypoplastic AI. The M100521 mutation (Enam(Rgsc521)) was a T to A substitution at the splicing donor site in intron 4. This mutation resulted in a frameshift that gave rise to a premature stop codon. The transcript of the Enam(Rgsc521) mutant allele was degraded, indicating that Enam(Rgsc521) is a loss-of-function mutation. Enam(Rgsc521) heterozygotes showed a hypomaturation-type AI phenotype in the incisors, possibly due to haploinsufficiency of Enam. Enam(Rgsc521) homozygotes showed complete loss of enamel on the incisors and the molars. Thus, we report here that the Enam gene is essential for amelogenesis, and that mice with different point mutations at Enam may provide good animal models to study the different clinical subtypes of AI.

Amelogenesis↗
PubMed2005

Regulated expression and dynamic changes in subnuclear localization of mammalian Rad18 under normal and genotoxic conditions.

Rad18 plays a crucial role in postreplication repair in both lower eukaryotes and higher eukaryotes. However, regulation of the Rad18 expression in higher eukaryotes is largely unknown. We found that the RAD18 transcript is expressed ubiquitously in various tissues and very highly in the testis in mammals. Although human RAD18 (hRAD18) transcription levels fluctuate during the cell cycle, being maximal in the late S and minimal in the early G1, the protein levels remain constant throughout the cell cycle. Following UV-irradiation, hRAD18 transcription levels decrease significantly, but Rad18 protein levels change little. The protein levels are maintained at least in part by enhanced translation rates. hRad18 localizes in the nucleus in two forms: a diffused form and a condensed form forming nuclear dots. These nuclear dots disperse rapidly in the nucleoplasm after treatments with various genotoxic agents, resulting in an enhancement of the intranuclear Rad18 concentration of the diffused form. No de novo protein synthesis is required for this process. These results suggest that in higher eukaryotes, the maintenance and dynamic translocation of Rad18 protein is important for postreplication repair.

Animals↗
PubMed2004

A series of maturity onset diabetes of the young, type 2 (MODY2) mouse models generated by a large-scale ENU mutagenesis program.

Mutant mouse models are indispensable tools for clarifying the functions of genes and for elucidating the underlying pathogenic mechanisms of human diseases. Currently, several large-scale mutagenesis projects that employ the chemical mutagen N-ethyl-N-nitrosourea (ENU) are underway worldwide. One specific aim of our ENU mutagenesis project is to generate diabetic mouse models. We screened 9375 animals for dominant traits using a clinical biochemical test and thereby identified 11 mutations in the glucokinase (Gk) gene that were associated with hyperglycemia. GK is a key regulator of insulin secretion in the pancreatic beta-cell. Approximately 190 heterozygous mutations in the human GK gene have been reported to cause maturity onset diabetes of the young, type 2 (MODY2). In addition, five mutations have been reported to cause permanent neonatal diabetes mellitus (PNDM) when present on both alleles. The mutations in our 11 hyperglycemic mutants are located at different positions in Gk. Four have also been found in human MODY2 patients, and another mutant bears its mutation at the same location that is mutated in a PNDM patient. Thus, ENU mutagenesis is effective for developing mouse models for various human genetic diseases, including diabetes mellitus. Some of our Gk mutant lines displayed impaired glucose-responsive insulin secretion and the mutations had different effects on Gk mRNA levels and/or the stability of the GK protein. This collection of Gk mutants will be valuable for understanding GK gene function, for dissecting the function of the enzyme and as models of human MODY2 and PNDM.

Amino Acid Sequence↗
PubMed2003

The Neurospora crassa mus-19 gene is identical to the qde-3 gene, which encodes a RecQ homologue and is involved in recombination repair and postreplication repair.

An allele called mus-19 was identified by screening temperature-sensitive and mutagen-sensitive mutants of Neurospora crassa. The mus-19 gene was genetically mapped to a region near the end of the right arm of linkage group I, where a RecQ homologue called qde-3 had been physically mapped in the Neurospora database. Complementation tests between the mus-19 mutant and the qde-3(RIP) mutant showed that mus-19 and qde-3 were the same gene. The qde-3 genes of both mutants were cloned and sequenced; and the results showed that they have mutation(s) in their qde-3 genes. The original mus-19 and qde-3(RIP) mutants are defective in quelling, as reported for other qde-3 mutants. The mutants show high sensitivity to methyl methanesulfonate, ethyl methanesulfonate, N-methyl- N'-nitro- N-nitrosoguanidine, tert-butyl hydroperoxide, 4-nitroquinoline-1-oxide, hydroxyurea and histidine. Epistasis analysis indicated that the qde-3 gene belongs both to the uvs-6 recombination repair pathway and the uvs-2 postreplication repair pathway. The qde-3 mutation has no effect on the integration of a plasmid carrying the mtr gene by homologous recombination. In homozygous crosses, the qde-3 mutant is defective in ascospore production.

Adenosine Triphosphatases↗