Biomedical subjects

S Asakawa

Publications and source records attributed to S Asakawa.

At least 37 records · Page 2Linked to original sources

PubMed2000

Cloning and characterization of a putative human glycerol 3-phosphate permease gene (SLC37A1 or G3PP) on 21q22.3: mutation analysis in two candidate phenotypes, DFNB10 and a glycerol kinase deficiency.

Using multiple exons trapped from human chromosome 21 (HC21)-specific cosmids with homology to a putative Arabidopsis thaliana glycerol 3-phosphate permease, we have determined the full-length cDNA sequence of a novel HC21 gene encoding a putative sugar-phosphate transporter (HGMW-approved symbol SLC37A1, aka G3PP). The predicted protein has 12 putative transmembrane domains and is also highly homologous to bacterial glpT proteins. The transcript was precisely mapped to 21q22.3 between D21S49 and D21S113. Comparison of the SLC37A1 cDNA to genomic sequence revealed that the gene encompasses 82 kb, and it is split into 19 coding exons and 7 untranslated exons, which are alternatively spliced in a complex and tissue-specific manner. Glycerol 3-phosphate (G3P) is produced by glycerol kinase (GK) and is found in several biochemical pathways in different cellular compartments, such as the glycerol phosphate shuttle and glycerophospholipid synthesis. Thus SLC37A1 mutations may cause a phenotype similar to GK deficiency. Mutational analyses of SLC37A1 in seven patients with no mutations in the GK gene and low GK activity revealed only nonpathogenetic sequence variants, excluding SLC37A1 as the gene for the phenotype in these patients. SLC37A1 maps in the refined critical region of the autosomal recessive deafness locus, DFNB10, on 21q22.3. Mutation analyses also excluded SLC37A1 as the gene for DFNB10.

Amino Acid Sequence↗

PubMed2000

Genomic organization, chromosomal localization, and the complete 22 kb DNA sequence of the human GCMa/GCM1, a placenta-specific transcription factor gene.

The genomic sequence of the human GCMa/GCM1 gene, a mammalian homologue of Drosophila melanogaster GCM, was determined. Drosophila GCM is a neural transcription factor that regulates glial cell fate. The mammalian homolog however, is a placenta-specific transcription factor that is necessary for placental development. The 22 kb DNA sequence spanning the GCMa gene contains six exons and five introns, encoding a 2.8 kb cDNA. Overall genomic organization is similar for the human and mouse. Several potential binding sites for transcription factors like GATA, Oct-1, and bHLH proteins were found in the 5'-flanking region of the human gene. A DNA motif for GCM protein binding exists in the 5'-flanking region that is highly homologous with that of the mouse gene. The location of this gene was mapped to chromosome 6 using fluorescence in situ hybridization.

Animals↗

PubMed2000

Refined localization of autosomal recessive nonsyndromic deafness DFNB10 locus using 34 novel microsatellite markers, genomic structure, and exclusion of six known genes in the region.

An autosomal recessive nonsyndromic deafness locus, DFNB10, was previously localized to a 12-cM region near the telomere of chromosome 21 (21q22.3). This locus was discovered in a large, consanguineous Palestinian family. We have identified and ordered a total of 50 polymorphic microsatellite markers in 21q22.3, comprising 16 published and 34 new markers, precisely mapped and ordered on BAC/cosmid contigs. Using these microsatellite markers, the locus for DFNB10 has been refined to an area of less than 1 Mb between markers 1016E7.CA60 and 1151C12.GT45. Six previously published cDNAs were mapped to this critical region, and their genomic structures were determined to facilitate mutation analysis in DFNB10. All six genes in this region (in order from centromere to telomere: White/ABCG1, TFF3, TFF2, TFF1, PDE9A, and NDUVF3) have been screened and eliminated as candidates for DFNB10. The new microsatellite markers and single nucleotide polymorphisms identified in this study should enable the refined mapping of other genetic diseases that map to 21q22.3. In addition, the critical region for DFNB10 has been reduced to a size amenable to an intensive positional cloning effort.

Chromosome Mapping↗

PubMed2000

Isolation and characterization of a human chromosome 21q22.3 gene (WDR4) and its mouse homologue that code for a WD-repeat protein.

To identify candidate genes for Down syndrome phenotypes or disorders that map to human chromosome 21q22.3, trapped exons are being used to isolate full-length transcripts. We isolated a full-length cDNA (WDR4) encoding a novel WD-repeat protein and its mouse homologue. Two RNA species of 1.5 and 2.1 kb were observed in human, with the 1.5-kb transcript being produced by a splicing event after the stop codon, and thus both transcripts encode the same putative 412-amino-acid protein containing four guanine nucleotide-binding WD repeats. The more highly expressed 1.5-kb transcript was expressed mainly in fetal tissues while the 2.1-kb transcript showed a faint expression in most tissues. Two additional alternative splicing events of 270 and 52 nt within the coding region were observed. The WDR4 gene spans 37 kb and is divided into 11 coding exons. WDR4 maps between PDE9A and NDUFV3, a region where several genetic disorders, including a form of manic-depressive psychosis, also map, and seven sequence variants observed in the WDR4 gene could be used in association studies.

Alternative Splicing↗

PubMed2000

Characterization and chromosomal mapping of a novel human gene, ANKHZN.

Ankhzn (ankyrin repeats hooked to a zinc finger motif) was originally isolated by means of the gene trap method, as a novel cytoplasmic protein on mouse embryonic stem cells. The Ankhzn protein is ubiquitously expressed in a spatiotemporal-specific manner and is located on endosomes. In the present study, we have cloned human ANKHZN cDNA by PCR using candidate EST clones exhibiting a high homology to mouse Ankhzn cDNA. The human ANKHZN cDNA encoded a 1166aa protein exhibiting 84.9% identity to the mouse one. The size of the transcript was found to be about 7kb on a Northern blot analysis, and ANKHZN mRNA was found to be ubiquitously expressed in human tissues on RT-PCR analysis. Western blot analysis showed that a 130kDa protein was detected at various levels in human tissues and also present in both membrane and soluble fractions obtained on subcellular fractionation. Human ANKHZN is a single copy gene consisting of predicted 25 exons in the human genome, and has been mapped to human chromosome 17p13 by radiation hybrid panel and fluorescence in-situ hybridization.

Amino Acid Sequence↗

PubMed2000

Isolation of two novel genes, DSCR5 and DSCR6, from Down syndrome critical region on human chromosome 21q22.2.

We have isolated two novel genes, designated DSCR5 and DSCR6, from the Down syndrome critical region (DSCR) on chromosome 21q22.2 which has been defined as minimal overlapping region of partial trisomy 21 patients and located between t(4;21) break point and ERG (approximately 1.6 Mb). DSCR5 and DSCR6 genes consist of 6 and 5 exons, respectively. Alternative use of transcription start sites and alternative splicing events produce different RNA species and proteins from both genes. Three different transcripts of DSCR5 gene encode three putative transmembrane proteins of 158, 134, and 108 amino acids, while 4 different transcripts of DSCR6 gene encode two forms of proteins with 190 and 106 amino acids. The DSCR5 gene is expressed in various human tissues examined, whereas the DSCR6 gene is expressed only in limited tissues at low level. Both DSCR5 and DSCR6 genes are candidates for the pathogenesis of Down syndrome, although the function of these genes remains to be elucidated.

Alternative Splicing↗

PubMed2000

The DNA sequence of human chromosome 21.

Chromosome 21 is the smallest human autosome. An extra copy of chromosome 21 causes Down syndrome, the most frequent genetic cause of significant mental retardation, which affects up to 1 in 700 live births. Several anonymous loci for monogenic disorders and predispositions for common complex disorders have also been mapped to this chromosome, and loss of heterozygosity has been observed in regions associated with solid tumours. Here we report the sequence and gene catalogue of the long arm of chromosome 21. We have sequenced 33,546,361 base pairs (bp) of DNA with very high accuracy, the largest contig being 25,491,867 bp. Only three small clone gaps and seven sequencing gaps remain, comprising about 100 kilobases. Thus, we achieved 99.7% coverage of 21q. We also sequenced 281,116 bp from the short arm. The structural features identified include duplications that are probably involved in chromosomal abnormalities and repeat structures in the telomeric and pericentromeric regions. Analysis of the chromosome revealed 127 known genes, 98 predicted genes and 59 pseudogenes.

Base Sequence↗

PubMed2000

Evaluation of the applicability of the bacterial endotoxin test to antibiotic products.

Fundamental conditions for applying the bacterial endotoxin test to antibiotic products were investigated so as not to affect the level of regulation by the rabbit pyrogen test. According to accuracy evaluation of test methods, the kinetic-turbidimetric and kinetic-colourimetric assays were shown to allow more accurate measurement and, therefore, more sensitive detection of interference to the bacterial endotoxin test than the gel-clot method. In total, 102 antibiotic products were evaluated on their interfering effect to show that the antibiotics could be categorized into three groups depending on intensity of the interference. Although the test was shown to be applicable even to the group showing the strongest interference, it was assumed to be crucial to use appropriate reagents and an accurate test method for avoiding approval of a pyrogenic product. Accordingly, lists of antibiotics are presented to provide limits of concentration for eliminating interference and endotoxin limits for approval to facilitate effective bacterial endotoxin tests.

Animals↗

PubMed2000

PARKIN as a pathogenic gene for autosomal recessive juvenile parkinsonism.

Parkinson's disease is a common neurodegenerative disease with complex clinical features. Recently, we idenfied a novel gene named Parkin to be responsible for the pathogenesis of autosomal recessive juvenile parkinsonism (AR-JP). Various mutations were found in AR-JP patients of Japanese and other ethnic origins, providing a definitive evidence for the Parkin to be a causative gene for AR-JP. The predicted structure of Parkin protein and its mutation provide important clues for studying the functional role of the Parkin protein in leading to selective degeneration of nigral neurons in the brains of AR-JP patients.

Amino Acid Motifs↗

PubMed2000

Molecular cloning, gene expression, and identification of a splicing variant of the mouse parkin gene.

We have isolated mouse cDNA clones that are homologous to human Parkin gene, which was recently found to be responsible for the pathogenesis of autosomal recessive juvenile parkinsonism (AR-JP). One of these cDNA clones had the 1,392-bp open reading frame encoding a protein of 464 amino acids with presumed molecular weight of 51,615. The amino acid sequence of mouse parkin protein exhibits 83.2% identity to human Parkin protein, including the ubiquitin-like domain at the N-terminus (identity = 89.5%) and the RING finger-like domain at the C-terminus (identity = 90.6%). Two other clones had the 783-bp open reading frame encoding a truncated protein of 261 amino acids without RING finger-like domain. It was proved to be a novel splicing variant by 3'-RACE method. Northern blot analysis revealed that mouse parkin gene is expressed in various tissues including brain, heart, liver, skeletal muscle, kidney, and testis. It is notable that mouse parkin gene expression appears evident in 15th day mouse embryo and increases toward the later stage of development. These mouse parkin cDNA clones will be useful for elucidating the essential physiological function of parkin protein in mammals.

Alternative Splicing↗

PubMed2000

Progress in the clinical and molecular genetics of familial parkinsonism.

Parkinson's disease (PD) is a neurodegenerative disease with clinical features resulting from deficiency of dopamine in the nigrostriatal system. Most PD cases are sporadic and the primary cause of the disease is still unknown. Recently, familial PD and parkinsonism have received much attention because these forms of the disease might provide clues to the genetic risk factors involved in the pathogenesis of idiopathic PD. To date, two causative genes, alpha-synuclein and the parkin gene, have been identified. alpha-Synuclein is involved in the pathogenesis of an autosomal dominant form of PD and constitutes a major component of the Lewy body, which is a pathological hallmark of idiopathic PD. In addition, mutations in the parkin gene have been identified as the cause of autosomal recessive juvenile parkinsonism (AR-JP). AR-JP manifests itself as a highly selective degeneration of the substantia nigra and the locus coeruleus, but without Lewy body formation. In addition to these two genes, four chromosomal loci have been linked to other forms of familial PD. Furthermore, there are a number of other pedigrees of familial PD in which linkage to known genetic loci has been excluded. Molecular cloning of these disease genes and elucidation of the function of their gene products will greatly contribute to our understanding of the pathogenesis of idiopathic PD.

Amino Acid Sequence↗

PubMed2000

Genomic bacterial artificial chromosome library of the Japanese flounder Paralichthys olivaceus.

We have constructed a genomic bacterial artificial chromosome (BAC) library from homozygous cloned Japanese flounder Paralichthys olivaceus using the pBAC- lac vector. This BAC library consists of about 49,100 clones and is deposited in 128 microtiter plates with 384 wells. The average size of inserted DNA was calculated to be 165 kb. The BAC library was determined to cover 9 times the Japanese flounder haploid genome. The Japanese flounder genomic BAC library will be useful for gene isolation as well as quantitative trait loci (QTL) analysis.

Journal Article↗

PubMed2000

Familial Parkinson disease gene product, parkin, is a ubiquitin-protein ligase.

Autosomal recessive juvenile parkinsonism (AR-JP), one of the most common familial forms of Parkinson disease, is characterized by selective dopaminergic neural cell death and the absence of the Lewy body, a cytoplasmic inclusion body consisting of aggregates of abnormally accumulated proteins. We previously cloned PARK2, mutations of which cause AR-JP (ref. 2), but the function of the gene product, parkin, remains unknown. We report here that parkin is involved in protein degradation as a ubiquitin-protein ligase collaborating with the ubiquitin-conjugating enzyme UbcH7, and that mutant parkins from AR-JP patients show loss of the ubiquitin-protein ligase activity. Our findings indicate that accumulation of proteins that have yet to be identified causes a selective neural cell death without formation of Lewy bodies. Our findings should enhance the exploration of the molecular mechanisms of neurodegeneration in Parkinson disease as well as in other neurodegenerative diseases that are characterized by involvement of abnormal protein ubiquitination, including Alzheimer disease, other tauopathies, CAG triplet repeat disorders and amyotrophic lateral sclerosis.

Humans↗

PubMed2000

Construction and evaluation of a porcine bacterial artificial chromosome library.

A porcine bacterial artificial chromosome (BAC) library consisting of 103,488 clones has been constructed. The average insert size in the BAC vector was calculated to be 133 kb based on the examination of 189 randomly selected clones, indicating that the library contained 4.4 genome equivalents. The library can be screened by two-step PCR. The first screening step is performed on 22 superpools, each containing 4704 clones (49 x 96 well plates). In the second screening step, 49 plates comprising a superpool are arrayed in a 7 x 7 matrix and 4D-PCR is performed. Screening of the library superpools by PCR for 125 marker sequences selected from different regions of swine genome revealed 123 sequences, indicating that the library is not biased. Subsequent screenings (4D-PCR) were successfully applied for identification of clones containing each marker sequence. This porcine BAC library and the PCR screening system are useful for isolation of genomic DNA fragments containing desired sequences.

Animals↗

PubMed2000

Autosomal recessive juvenile parkinsonism: a key to understanding nigral degeneration in sporadic Parkinson's disease.

The contribution of genetic factors to the pathogenesis of Parkinson's disease (PD) is supported by the demonstration of the high concordance in twins studies using positron emission tomography (PET), the increased risk among relatives of PD patients in case-control and family studies, and the existence of familial PD and parkinsonism by single gene defect. Recently several genes have been mapped and/or identified. Alpha-synuclein is involved in a rare dominant form of familial PD with dopa-responsive parkinsonism features and Lewy body-positive pathology. In contrast, parkin is responsible for the autosomal recessive form (AR-JP) of early onset PD with Lewy body-negative pathology. The clinical features of this form include early onset (in the 20s), levodopa-responsive parkinsonism, diurnal fluctuation, and slow progression of the disease. Parkin consists of 12 exons and the estimated size is over 1.5 Mb. To date, variable mutations such as deletions or point mutations resulting in missense and nonsense changes have been reported in AR-JP patients. In addition, the localization of parkin indicates that parkin may be involved in the axonal transport system. More recently we have found that parkin interacts with the ubiquitin-conjugating enzyme E2 and is functionally linked to the Ub-proteasome pathway as a ubiquitin ligase, E3. These findings fit the characteristics of a lack of Lewy bodies (these are cytoplasmic inclusions that are considered to be a pathological hallmark). Our findings should enhance the exploration of the mechanisms of neuronal death in PD as well as other neurodegenerative disorders of which variable inclusion bodies are observed.

Chromosomes, Human, Pair 6↗

PubMed2000

Detailed deletion mapping of chromosome band 14q32 in human neuroblastoma defines a 1.1-Mb region of common allelic loss.

Neuroblastoma (NB) is a well-known malignant disease in infants, but its molecular mechanisms have not yet been fully elucidated. To investigate the genetic contribution of abnormalities on the long arm of chromosome 14 (14q) in NB, we analysed loss of heterozygosity (LOH) in 54 primary NB samples using 12 microsatellite markers on 14q32. Seventeen (31%) of 54 tumours showed LOH at one or more of the markers analysed, and the smallest common region of allelic loss was identified between D14S62 and D14S987. This region was estimated to be 1-cM long from the linkage map. Fluorescence in situ hybridization also confirmed the loss. There was no statistical correlation between LOH and any clinicopathologic features, including age, stage, amplification of MYCN and ploidy. We further constructed a contig spanning the lost region using bacterial artificial chromosome and estimated this region to be approximately 1.1-Mb by pulsed-field gel electrophoresis. Our results will contribute to cloning and characterizing the putative tumour-associated gene(s) in 14q32 in NB.

Alleles↗

PubMed2000

Wpkci, encoding an altered form of PKCI, is conserved widely on the avian W chromosome and expressed in early female embryos: implication of its role in female sex determination.

Two W chromosome-linked cDNA clones, p5fm2 and p5fm3, were obtained from a subtracted (female minus male) cDNA library prepared from a mixture of undifferentiated gonads and mesonephroi of male or female 5-d (stages 26-28) chicken embryos. These two clones were demonstrated to be derived from the mRNA encoding an altered form of PKC inhibitor/interacting protein (PKCI), and its gene was named Wpkci. The Wpkci gene reiterated approximately 40 times tandemly and located at the nonheterochromatic end of the chicken W chromosome. The W linkage and the moderate reiteration of Wpkci were conserved widely in Carinatae birds. The chicken PKCI gene, chPKCI, was shown to be a single-copy gene located near the centromere on the long arm of the Z chromosome. Deduced amino acid sequences of Wpkci and chPKCI showed approximately 65% identity. In the deduced sequence of Wpkci, the HIT motif, which is essential for PKCI function, was absent, but the alpha-helix region, which was conserved among the PKCI family, and a unique Leu- and Arg-rich region, were present. Transcripts from both Wpkci and chPKCI genes were present at significantly higher levels in 3- to 6-d (stages 20-29) embryos. These transcripts were detected in several embryonic tissues, including undifferentiated left and right gonads. When the green fluorescent protein-fused form of Wpkci was expressed in male chicken embryonic fibroblast, it was located almost exclusively in the nucleus. A model is presented suggesting that Wpkci may be involved in triggering the differentiation of ovary by interfering with PKCI function or by exhibiting its unique function in the nuclei of early female embryos.

Amino Acid Sequence↗

PubMed2000

Structure determination and total synthesis of a novel antibacterial substance, AB0022A, produced by a cellular slime mold.

A novel antibacterial substance, AB0022A, was isolated from the cellular slime mold Dictyostelium purpureum K1001. It inhibited the growth of Gram-positive bacteria, and its MICs ranged from 0.39 to 50 microg/ml. Because AB0022A was a highly substituted aromatic compound, we could not determine its structure based on only its physico-chemical and spectral data. We therefore used a dehalogenated derivative from AB0022A and deduced that its structure was 1,9-dihydroxy-3,7-dimethoxy-2-hexanoyl-4,6,8-trichlorodibenzofuran . To confirm this structure, we synthesized the compound having the deduced structure. The synthetic compound was identical to naturally occurring AB0022A.

Animals↗