Biomedical subjects

S Hirotsune

Publications and source records attributed to S Hirotsune.

At least 19 recordsLinked to original sources

PubMed2002

A new deletion mutation in bovine Claudin-16 (CL-16) deficiency and diagnosis.

Bovine claudin-16/paracellin-1 (CL-16/PCLN-1) deficiency is an autosomal recessive disorder caused by a 37-kb deletion mutation containing the first four exons of the CL-16 gene, which leads to the absence of the CL-16 transcript (type-1 mutation). A PCR-based DNA test for the CL-16 mutation (type-1) was used to screen a herd of Wagyu cattle. A recent report suggested that affected cattle can be bred by dams diagnosed as normal, suggesting the presence of a new mutation in the CL-16 locus. We identified the new mutation as a 56-kb deletion containing exon-1 to -4 and 21-bp of exon-5 of CL-16, and refer to this as a type-2 mutation. A DNA test for specific for this mutation was then established.

Animals↗

PubMed2001

Establishment of a high throughput EST sequencing system using poly(A) tail-removed cDNA libraries and determination of 36,000 bovine ESTs.

We determined 36,310 bovine expressed sequence tag (EST) sequences using 10 different cDNA libraries. For massive EST sequencing, we devised a new system with two major features. First, we constructed cDNA libraries in which the poly(A) tails were removed using nested deletion at the 3'-ends. This permitted high quality reading of sequences from the 3'-end of the cDNA, which is otherwise difficult to do. Second, we increased throughput by sequencing directly on templates generated by colony PCR. Using this system, we determined 600 cDNA sequences per day. The read-out length was >450 bases in >90% of the sequences. Furthermore, we established a data management system for analyses, storage and manipulation of the sequence data. Finally, 16,358 non-redundant ESTs were derived from approximately 6900 independent genes. These data will facilitate construction of a precise comparative map across mammalian species and isolate the functional genes that govern economic traits. This system is applicable to other organisms, including livestock, for which EST data are limited.

Animals↗

PubMed2000

Hippocampal abnormalities and enhanced excitability in a murine model of human lissencephaly.

Human cortical heterotopia and neuronal migration disorders result in epilepsy; however, the precise mechanisms remain elusive. Here we demonstrate severe neuronal dysplasia and heterotopia throughout the granule cell and pyramidal cell layers of mice containing a heterozygous deletion of Lis1, a mouse model of human 17p13.3-linked lissencephaly. Birth-dating analysis using bromodeoxyuridine revealed that neurons in Lis1+/- murine hippocampus are born at the appropriate time but fail in migration to form a defined cell layer. Heterotopic pyramidal neurons in Lis1+/- mice were stunted and possessed fewer dendritic branches, whereas dentate granule cells were hypertrophic and formed spiny basilar dendrites from which the principal axon emerged. Both somatostatin- and parvalbumin-containing inhibitory neurons were heterotopic and displaced into both stratum radiatum and stratum lacunosum-moleculare. Mechanisms of synaptic transmission were severely disrupted, revealing hyperexcitability at Schaffer collateral-CA1 synapses and depression of mossy fiber-CA3 transmission. In addition, the dynamic range of frequency-dependent facilitation of Lis1+/- mossy fiber transmission was less than that of wild type. Consequently, Lis1+/- hippocampi are prone to interictal electrographic seizure activity in an elevated [K(+)](o) model of epilepsy. In Lis1+/- hippocampus, intense interictal bursting was observed on elevation of extracellular potassium to 6.5 mM, a condition that resulted in only minimal bursting in wild type. These anatomical and physiological hippocampal defects may provide a neuronal basis for seizures associated with lissencephaly.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗

PubMed2000

A LIS1/NUDEL/cytoplasmic dynein heavy chain complex in the developing and adult nervous system.

Mutations in mammalian Lis1 (Pafah1b1) result in neuronal migration defects. Several lines of evidence suggest that LIS1 participates in pathways regulating microtubule function, but the molecular mechanisms are unknown. Here, we demonstrate that LIS1 directly interacts with the cytoplasmic dynein heavy chain (CDHC) and NUDEL, a murine homolog of the Aspergillus nidulans nuclear migration mutant NudE. LIS1 and NUDEL colocalize predominantly at the centrosome in early neuroblasts but redistribute to axons in association with retrograde dynein motor proteins. NUDEL is phosphorylated by Cdk5/p35, a complex essential for neuronal migration. NUDEL and LIS1 regulate the distribution of CDHC along microtubules, and establish a direct functional link between LIS1, NUDEL, and microtubule motors. These results suggest that LIS1 and NUDEL regulate CDHC activity during neuronal migration and axonal retrograde transport in a Cdk5/p35-dependent fashion.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗

PubMed2000

Null mutation of PCLN-1/Claudin-16 results in bovine chronic interstitial nephritis.

Inherited chronic renal diseases are associated with failures in glomerular filtration and tubular resorption. Such failures invariably result from defects in selective filtration and absorption in surface renal epithelium. Recently, we described an autosomal recessive chronic interstitial nephritis with diffuse zonal fibrosis (CINF) in cattle. Bovine CINF, characterized by increased blood urea nitrogen, creatinine, and urinary proteins, leads to lethality before puberty, usually within the first 6 months or year of life. Here, we demonstrate that the first four exons of PCLN-1/Claudin-16 (CL-16), which encodes a member of the claudin family of tight junction proteins, were deleted in CINF-affected cattle. CL-16 was expressed preferentially in kidneys of normal cattle, but transcripts were totally absent in affected offspring. This observation suggests that the lack of CL-16 protein contributes to the dysfunction of paracellular renal transport systems.

Amino Acid Sequence↗

PubMed1999

Murine modelling of classical lissencephaly.

Classical lissencephaly is a severe human neuronal migration disorder characterized by a smooth cerebral surface and a paucity of gyri. Isolated lissencephaly sequence (ILS, OMIM 601545) and Miller-Dieker syndrome (MDS, OMIM 247200) are human malformation syndromes characterized by classical lissencephaly. MDS and some cases of ILS are caused by haploinsufficiency at chromosome 17p13.3. Recent evidence suggests that mutations or deletions of the LIS1 gene, within band 17p13.3, are responsible for classical lissencephaly. LIS1 codes for a subunit of platelet-activating factor acetylhydrolase isoform 1b (PAFAH1B1 or LIS1). To investigate the pathophysiological mechanisms responsible for these two developmental defects, we have undertaken strategies to model these neuronal migration disorders in the mouse. We present a brief review of MDS and ILS, several mouse mutants with cortical neuronal migration defects, and our strategies to model ILS and MDS in the mouse.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗

PubMed1999

Deletion of 150 kb in the minimal DiGeorge/velocardiofacial syndrome critical region in mouse.

Deletions or rearrangements of human chromosome 22q11 lead to a variety of related clinical syndromes such as DiGeorge syndrome (DGS) and velo--cardiofacial syndrome (VCFS). In addition, patients with 22q11 deletions have an increased incidence of schizophrenia and several studies have mapped susceptibility loci for schizophrenia to this region. Human molecular genetic studies have so far failed to identify the crucial genes or disruption mechanisms that result in these disorders. We have used gene targeting in the mouse to delete a defined region within the conserved DGS critical region (DGCR) on mouse chromosome 16 to prospectively investigate the role of the mouse DGCR in 22q11 syndromes. The deletion spans a conserved portion ( approximately 150 kb) of the proximal region of the DGCR, containing at least seven genes ( Znf74l, Idd, Tsk1, Tsk2, Es2, Gscl and Ctp ). Mice heterozygous for this deletion display no findings of DGS/VCFS in either inbred or mixed backgrounds. However, heterozygous mice display an increase in prepulse inhibition of the startle response, a manifestation of sensorimotor gating that is reduced in humans with schizophrenia. Homozygous deleted mice die soon after implantation, demonstrating that the deleted region contains genes essential for early post-implantation embryonic development. These results suggest that heterozygous deletion of this portion of the DGCR is sufficient for sensorimotor gating abnormalities, but not sufficient to produce the common features of DGS/VCFS in the mouse.

Animals↗

PubMed1998

Graded reduction of Pafah1b1 (Lis1) activity results in neuronal migration defects and early embryonic lethality.

Heterozygous mutation or deletion of the beta subunit of platelet-activating factor acetylhydrolase (PAFAH1B1, also known as LIS1) in humans is associated with type I lissencephaly, a severe developmental brain disorder thought to result from abnormal neuronal migration. To further understand the function of PAFAH1B1, we produced three different mutant alleles in mouse Pafah1b1. Homozygous null mice die early in embryogenesis soon after implantation. Mice with one inactive allele display cortical, hippocampal and olfactory bulb disorganization resulting from delayed neuronal migration by a cell-autonomous neuronal pathway. Mice with further reduction of Pafah1b1 activity display more severe brain disorganization as well as cerebellar defects. Our results demonstrate an essential, dosage-sensitive neuronal-specific role for Pafah1b1 in neuronal migration throughout the brain, and an essential role in early embryonic development. The phenotypes observed are distinct from those of other mouse mutants with neuronal migration defects, suggesting that Pafah1b1 participates in a novel pathway for neuronal migration.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗

PubMed1997

Genomic organization of the murine Miller-Dieker/lissencephaly region: conservation of linkage with the human region.

Several human syndromes are associated with haploinsufficiency of chromosomal regions secondary to microdeletions. Isolated lissencephaly sequence (ILS), a human developmental disease characterized by a smooth cerebral surface (classical lissencephaly) and microscopic evidence of incomplete neuronal migration, is often associated with small deletions or translocations at chromosome 17p13.3. Miller-Dieker syndrome (MDS) is associated with larger deletions of 17p13.3 and consists of classical lissencephaly with additional phenotypes including facial abnormalities. We have isolated the murine homologs of three genes located inside and outside the MDS region: Lis1, Mnt/Rox, and 14-3-3 epsilon. These genes are all located on mouse chromosome 11B2, as determined by metaphase FISH, and the relative order and approximate gene distance was determined by interphase FISH analysis. The transcriptional orientation and intergenic distance of Lis1 and Mnt/Rox were ascertained by fragmentation analysis of a mouse yeast artificial chromosome containing both genes. To determine the distance and orientation of 14-3-3 epsilon with respect to Lis1 and Mnt/Rox, we introduced a super-rare cutter site (VDE) that is unique in the mouse genome into 14-3-3 epsilon by gene targeting. Using the introduced VDE site, the orientation of this gene was determined by pulsed field gel electrophoresis and Southern blot analysis. Our results demonstrate that the MDS region is conserved between human and mouse. This conservation of linkage suggests that the mouse can be used to model microdeletions that occur in ILS and MDS.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗

PubMed1996

Construction of high-resolution physical maps from yeast artificial chromosomes using restriction landmark genomic scanning (RLGS).

We have established a new system for chromosome-specific yeast artificial chromosome (YAC) contig construction using restriction landmark genomic scanning (RLGS-based YAC contig mapper). RLGS is a powerful tool for detecting more than 1000 restriction landmarks distributed on an entire genome in one procedure. In this system, RLGS is applied to sorted chromosomes to cover the target chromosome. Using these landmarks as guideposts, chromosome-specific YAC clones are then ordered. In this paper, we report the construction of a map for a human chromosome 21 YAC contig spanning q22.1 using this new approach. Applying RLGS to sorted chromosomes 21 enables detection of approximately 1400 spots (equivalent of 1050 PacI landmarks), covering the entire region of this chromosome. We constructed the 2.5-Mb YAC contig encompassing 21q22.1 with 66 spots (equivalent of 50 PacI landmarks). With this contig map, we could detect two deleted regions and chimerism in the YAC insert DNA. Our results demonstrated the usefulness of this approach for finding DNA alterations of YACs, such as deletions and chimerism.

Chimera↗

PubMed1996

Atm-deficient mice: a paradigm of ataxia telangiectasia.

A murine model of ataxia telangiectasia was created by disrupting the Atm locus via gene targeting. Mice homozygous for the disrupted Atm allele displayed growth retardation, neurologic dysfunction, male and female infertility secondary to the absence of mature gametes, defects in T lymphocyte maturation, and extreme sensitivity to gamma-irradiation. The majority of animals developed malignant thymic lymphomas between 2 and 4 months of age. Several chromosomal anomalies were detected in one of these tumors. Fibroblasts from these mice grew slowly and exhibited abnormal radiation-induced G1 checkpoint function. Atm-disrupted mice recapitulate the ataxia telangiectasia phenotype in humans, providing a mammalian model in which to study the pathophysiology of this pleiotropic disorder.

Animals↗

PubMed1996

Identification and chromosomal localization of Atm, the mouse homolog of the ataxia-telangiectasia gene.

Atm, the mouse homolog of the human ATM gene defective in ataxia-telangiectasia (A-T), has been identified. The entire coding sequence of the Atm transcript was cloned and found to contain an open reading frame encoding a protein of 3066 amino acids with 84% overall identity and 91% similarity to the human ATM protein. Variable levels of expression of Atm were observed in different tissues. Fluorescence in situ hybridization and linkage analysis located the Atm gene on mouse chromosome 9, band 9C, in a region homologous to the ATM region on human chromosome 11q22-q23.

Amino Acid Sequence↗

PubMed1996

Identification of Grf1 on mouse chromosome 9 as an imprinted gene by RLGS-M.

Normal mammalian development requires a diploid combination of both haploid parental genomes. Uniparental disomy for certain segments of specific chromosomes results in aberrant development or prenatal lethality, indicating that the parental genomes have undergone modifications during gametogenesis. These modifications result in parent-of-origin specific expression for some genes, a phenomenon called genomic imprinting. Recent work with DNA methyltransferase deficient mice showed that differential methylation is the probable basis of the imprinted character of several genes. Screening for endogenous imprinted loci using restriction landmark genomic scanning with methylation sensitive enzymes (RLGS-M) identified eight imprinted RLGS (Irigs) candidate loci. Molecular analysis of the genomic region of one of the loci (Irigs2) resulted in the discovery of the paternally imprinted U2afbp-rs gene within a previously identified imprinted region on mouse chromosome 11 (refs 5, 7). This paper describes the characterisation of a novel imprinted RLGS-M locus, Irigs3, on mouse chromosome 9 (ref. 6). Within this locus we identified the Grf1 (also called Cdc25Mm) gene, which is homologous to the RAS-specific guanine nucleotide exchange factor gene, CDC25, in Saccharomyces cerevisiae. Grf1 is located about 30 kb downstream of the methylation imprinted site, identified by RLGS-M, and shows paternal allele specific expression in mouse brain, stomach and heart. Our results indicate that imprinting may have a role in regulating mitogenic signal transduction pathways during growth and development.

Animals↗

PubMed1995

Direct detection and isolation of restriction landmark genomic scanning (RLGS) spot DNA markers tightly linked to a specific trait by using the RLGS spot-bombing method.

We have developed a technique for isolating DNA markers tightly linked to a target region that is based on RLGS, named RLGS spot-bombing (RLGS-SB). RLGS-SB allows us to scan the genome of higher organisms quickly and efficiently to identify loci that are linked to either a target region or gene of interest. The method was initially tested by analyzing a C57BL/6-GusS mouse congenic strain. We identified 33 variant markers out of 10,565 total loci in a 4.2-centimorgan (cM) interval surrounding the Gus locus in 4 days of laboratory work. The validity of RLGS-SB to find DNA markers linked to a target locus was also tested on pooled DNA from segregating backcross progeny by analyzing the spot intensity of already mapped RLGS loci. Finally, we used RLGS-SB to identify DNA markers closely linked to the mouse reeler (rl) locus on chromosome 5 by phenotypic pooling. A total of 31 RLGS loci were identified and mapped to the target region after screening 8856 loci. These 31 loci were mapped within 11.7 cM surrounding rl. The average density of RLGS loci located in the rl region was 0.38 cM. Three loci were closely linked to rl showing a recombination frequency of 0/340, which is < 1 cM from rl. Thus, RLGS-SB provides an efficient and rapid method for the detection and isolation of polymorphic DNA markers linked to a trait or gene of interest.

Animals↗

PubMed1995

A spot cloning method for restriction landmark genomic scanning.

We introduce two new methods for target cloning of DNA fragments corresponding to spots on the two-dimensional profile of restriction landmark genomic scanning (RLGS). One is a restriction trapper-based method and the other is a polymerase chain reaction (PCR) mediated method. Both are designed to select the target DNA fragments from a large amount of unlabeled background DNA fragments in the RLGS gel which produce background clones. The restriction trapper method is simple, with a cloning efficiency that is not biased by the length of the target DNA nor by its GC content. On the other hand, the PCR-mediated method is efficient for cloning DNA fragments from a small amount of starting materials. These methods provide us with powerful tools for isolating DNA clones identified by the RLGS system as interesting spots. This paper reports the precise protocols of these methods and discusses their application and usefulness.

Animals↗

PubMed1995

Accessibility to tissue-specific genes from methylation profiles of mouse brain genomic DNA.

The DNA methylation status of a large number of genomic loci is visualized simultaneously and quantitatively as two-dimensional gel spots in the newly developed restriction landmark genomic scanning with a methylation-sensitive restriction enzyme (RLGS-M). Here, we demonstrate that RLGS-M using NorI as a methylation-sensitive enzyme could also scan gene loci of mammalian genomes, since almost all of the NotI loci corresponding to randomly chosen RLGS-M spots were located near or in transcriptional units (6 out of 7 NotI-linking clones) when mouse brain genomic DNA was used. This supports the previous prediction that most NotI sites are located in CpG islands (Lindsay and Bird, Nature 1987, 327, 336-338). Furthermore, beginning with RLGS-M spots we examined how to approach their corresponding RNA messages, whose expression may be associated with methylation. We compared RLGS-M patterns among various developmental stages of the mouse brain from embryonic day 9.5 to postnatal 8 weeks or among in vitro cell lines, and detected alterations of RLGS-M spots which were due to methylation of NotI sites. Two experiments using NotI-linking clones or polymerase chain reaction (PCR) were carried out to approach to their corresponding RNA messages. Consequently, we isolated two PCR-amplified clones (# 15 and # 91) which corresponded to methylatable loci and gave positive signals to mRNA from the adult brain. Furthermore, we identified two NotI-linking clones (C211 and C198) whose corresponding NotI loci localized near or at transcriptional units and were methylated in cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

The reeler gene encodes a protein with an EGF-like motif expressed by pioneer neurons.

We have identified a strong candidate cDNA for the mouse reeler gene. This 5 kb transcript encodes a 99.4 kD protein consisting of 881 amino acids and possessing two EGF-like motifs. We assayed two independent mutant alleles--'Jackson reeler', which has a deletion of the entire gene, and 'Orleans reeler' which exhibits a 220 bp deletion in the open reading frame, including the second EGF-like motif and resulting in a frame shift. In situ hybridization reveals that the transcript is detected exclusively in the pioneer neurons which guide neuronal cell migration along the radial array. Our findings offer an explanation for how the reeler mutant phenotype causes a disturbance of the complex architecture of the neuronal network.

Amino Acid Sequence↗

PubMed1994

Spot mapping on the standard profile of restriction landmark genomic scanning (RLGS) of sorted chromosome 20 using methylation-insensitive enzyme.

We established the spot mapping system on a restriction landmark genomic scanning (RLGS) profile using sorted chromosome as RLGS material. In this mapping system, we can mapped RLGS spots physically, regardless of their polymorphism, using methylation-insensitive enzymes in all RLGS steps. Here, we report that we identified 28 spots derived from human chromosome 20 on an RLGS profile, and that number was in good agreement with the number predicted from the length of the chromosome 20.

Cell Line↗