COH1 analysis and linkage study in two Japanese families with Cohen syndrome.
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Abnormalities, Multiple↗
Biomedical subjects
S Asakawa
Publications and source records attributed to S Asakawa.
Construction of a bovine bacterial artificial chromosome library from fibroblasts used for cloned cattle.
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Animals↗
Efficient selection of genomic clones from a female chicken bacterial artificial chromosome library by four-dimensional polymerase chain reactions.
A bacterial artificial chromosome (BAC) library consisting of 49,152 genomic clones was constructed from partially HindIII-digested female chicken embryo genomic DNA using the pBAC-Lac vector and maintained in 512 96-well plates. The mean insert size was approximately 150 kb, and the total library was estimated to contain about 3.2 times coverage of the diploid genome. In order to screen this library by the PCR, 296 BAC clone DNA samples were prepared: one sample each from 8 superpools (64 plates per superpool) and 36 samples of four-dimensionally (4-D) mixed clones from each superpool. A BAC clone of interest was selected by two-step PCR. First, 8 DNA samples representing superpools were subjected to PCR with a set of primers to amplify a part of the genomic sequence of interest. Second, 36 4-D DNA samples from the superpool that contained BAC clone(s) of interest were subjected to PCR with the same set of primers. The second step identified a plate and a well containing the BAC clone of interest. Selection of target BAC clone(s) from the whole library with the above procedure can be achieved within 1 to 4 d without using a radioactive probe. This procedure was applied successfully in the selection of BAC clones for Wpkci, chPKCI/HINT, ZOV3, and 17beta-HSD genes.
Amino Acid Sequence↗
Molecular cloning of a member of the facilitative glucose transporter gene family GLUT11 (SLC2A11) and identification of transcription variants.
We isolated a member of the facilitative glucose transporter (GLUT) gene family (GLUT11; SLC2A11 as a HGMW-approved symbol) based on the analysis of a human genomic BAC clone KB1125A3 located on band q11.2 of human chromosome 22. The gene GLUT11/SLC2A11 consists of 12 exons spanning over 29 kb in size and is located between two genes, SMARCB1 and MIF. The deduced amino acid sequence indicated the topological features of transmembrane helices and sequence motifs which are common to the GLUT protein family. The cDNA cloning revealed the presence of three types of variation in its transcripts. The first variation is caused by the existence of three distinct first exons (SLC2A11-a, -b, and -c). PCR analysis of multi-tissue-derived cDNA panels indicated the differential expression of these transcript variants. The second variation is caused by skipping over one exon (exon 6). The third variation is caused by the premature transcription termination at a site between exon 8 and exon 9. Both exon skipping and premature termination caused frameshift, resulting in the production of truncated GLUT11/SLC2A11 transcripts. These results suggested that transcription of GLUT11/SCL2A11 gene is controlled in a complex manner.
Amino Acid Motifs↗
Structure of human holocarboxylase synthetase gene and mutation spectrum of holocarboxylase synthetase deficiency.
Holocarboxylase synthetase (HLCS) is an enzyme that catalyzes the incorporation of biotin into apo-carboxylases, and its deficiency causes biotin-responsive multiple carboxylase deficiency. The reported sequences of cDNA for human HLCS from liver, lymphocyte, and KG-1 myeloid cell lines differ at their 5' regions. To elucidate variations of the human HLCS mRNA and longer 5' cDNA ends, we performed screening of the human liver cDNA library and rapid amplification of the cDNA ends (RACE). Our results suggest the existence of three types of HLCS mRNA that start at different exons. The first type starts at exon 1, and the second type starts at exon 3, and both are found in various human tissues. The third type, corresponding to the cDNA from the KG-1 cell, starts at exon 2 of the HLCS gene. Various splicing patterns from exons 3-6 were also observed. None of the variations of cDNA found created a new initiation codon. Mutation screening from exons 6-14, therefore, was sufficient to detect amino acid changes in HLCS in patients. Our direct sequencing strategy for screening mutations in the HLCS gene revealed mutations in five Japanese patients and seven non-Japanese patients. Our analyses involving 12 Japanese and 13 non-Japanese patients and studies by others indicate that (1) there is no panethnically prevalent mutation; (2) the Arg508Trp, Gly581Ser, and Val550Met mutations are found in both Japanese and non-Japanese populations; (3) the IVS10+5G-->A mutation is predominant and probably a founder mutation in European patients; (4) the 655-656insA, Leu237Pro, and 780delG mutations are unique in Japanese patients; (5) the spectrum of the mutations in the HLCS gene may vary substantially among different ethnic groups.
Base Sequence↗
The genomic structure and promoter region of the human parkin gene.
Parkin has been identified as a causative gene of the autosomal recessive juvenile parkinsonism (AR-JP). In this study, we determined the genomic structure of the Parkin gene and identified a core promoter region based on the DNA sequence of 1.4 Mb. The 5'-flanking region contained no apparent TATA or CAAT box elements but several putative cis-elements for various transcription factors. The GC- and CpG-rich regions were observed not only in the 5'-flanking sequence but also in the 5'-part of the first intron of Parkin. We identified an exact starting point of Parkin transcription. A core promoter region was determined by transfecting a series of deletion constructs with a dual luciferase reporter system into human neuroblastoma cells. Furthermore, we located a neighboring novel gene in a head-to-head direction with Parkin with only a 198-bp interval.
Base Sequence↗
The medaka rs-3 locus required for scale development encodes ectodysplasin-A receptor.
The bodies of most teleost fish species are covered with specialized subepithelial structures known as scales. The scale is an epithelial appendage that differentiates from the dermal mesenchyme. Mammals, on the other hand, have no scales, but instead their bodies are covered with hair. Although their appearances are quite different, scales and hair can be considered structurally similar in that both of them are epithelial appendages distributed over the body surface in an orderly pattern. This analogy suggests that they may have the same evolutionary origin. But, to date, no molecular evidence has been presented that links scales and hair. A mutation at the rs-3 locus of medaka (Oryzias latipes) leads to almost complete loss of scales. We demonstrated that the rs-3 locus encodes ectodysplasin-A receptor (EDAR), which is required for the initiation of hair development in mammals. We identified a novel transposon inserted in the first intron of EDAR, which causes aberrant splicing. This work shows that EDAR is required for scale development in fish and suggests that it is an evolutionarily conserved molecule that is required for the development of epithelial appendages in vertebrates.
Amino Acid Sequence↗
Human biotin-containing subunit of 3-methylcrotonyl-CoA carboxylase gene (MCCA): cDNA sequence, genomic organization, localization to chromosomal band 3q27, and expression.
3-Methylcrotonyl-CoA carboxylase (MCCase; EC 6.4.1.4) is a mitochondrial biotin enzyme and plays an essential role in the catabolism of leucine and isovalerate in animals, bacterial species, and plants. MCCase consists of two subunits, those that are biotin-containing and non-biotin-containing. The genes responsible for these subunits have been isolated in soybean, Arabidopsis thaliana, and tomatoes, but not in mammals. In humans, MCCase deficiency has been thought to be a rare metabolic disease, but the number of patients with MCCase deficiency appears to be increasing with a wide range of clinical presentations, some that result in a lethal condition and others that are asymptomatic. In this report, we have isolated and carried out chromosomal mapping of the gene for the biotin-containing subunit (A subunit) of the human MCCase gene, MCCA. The cDNA predicts an open reading frame coding for a 725-amino-acid protein with mitochondrial signal peptide, biotin carboxylase, and biotin-carrier domains. The gene is composed of at least 19 exons and covers more than 70 kb of sequence on band q27 of chromosome 3. MCCA was abundantly expressed in mitochondria-rich organs, such as the heart, skeletal muscles, kidney, and liver. In exon 13, we observed a His/Pro polymorphism at codon 464 (an A to C transition at nucleotide position 1391 in the cDNA sequence). Then, we determined the DNA sequences of the 5' untranslated region and entire coding regions in two patients with MCCase deficiency, but no sequence substitution was detected, suggesting that the gene mutations might be in the non-biotin-containing subunit (B subunit) gene, MCCB, in these patients.
Amino Acid Sequence↗
A physical map of the human genome.
The human genome is by far the largest genome to be sequenced, and its size and complexity present many challenges for sequence assembly. The International Human Genome Sequencing Consortium constructed a map of the whole genome to enable the selection of clones for sequencing and for the accurate assembly of the genome sequence. Here we report the construction of the whole-genome bacterial artificial chromosome (BAC) map and its integration with previous landmark maps and information from mapping efforts focused on specific chromosomal regions. We also describe the integration of sequence data with the map.
Chromosomes, Artificial, Bacterial↗
Isolation and characterization of the UBASH3A gene on 21q22.3 encoding a potential nuclear protein with a novel combination of domains.
In order to identify candidate genes for Down syndrome phenotypes or monogenic disorders that map to human chromosome 21q22.3, we have used genomic sequence and expressed sequence tags mapping to an autosomal recessive deafness (DFNB10) critical region to isolate a novel 2.5-kb cDNA that maps between TFF1 and D21S49. A semi-quantitative reverse transcription/polymerase chain reaction method revealed that UBASH3A gene expression is limited to only a few tissues, with its highest expression in spleen, peripheral blood leukocytes, and bone marrow. The putative 661-amino-acid protein shows considerable homology to a hypothetical protein from Drosophila melanogaster but only domain homologies to other organisms. Both the human and D. melanogaster proteins contain protein-protein interaction domains, viz., SH3 and ubiquitin-associated (UBA) domains, in addition to a novel domain also containing a nuclear localization signal. This is the first protein described containing both UBA and SH3 domains. The gene, thus called UBASH3A, spans 40 kb and is divided into 15 exons. Mutation analysis excluded UBASH3A as being responsible for DFNB10.
Adaptor Proteins, Signal Transducing↗
Insertion of beta-satellite repeats identifies a transmembrane protease causing both congenital and childhood onset autosomal recessive deafness.
Approximately 50% of childhood deafness is caused by mutations in specific genes. Autosomal recessive loci account for approximately 80% of nonsyndromic genetic deafness. Here we report the identification of a new transmembrane serine protease (TMPRSS3; also known as ECHOS1) expressed in many tissues, including fetal cochlea, which is mutated in the families used to describe both the DFNB10 and DFNB8 loci. An 8-bp deletion and insertion of 18 monomeric (approximately 68-bp) beta-satellite repeat units, normally present in tandem arrays of up to several hundred kilobases on the short arms of acrocentric chromosomes, causes congenital deafness (DFNB10). A mutation in a splice-acceptor site, resulting in a 4-bp insertion in the mRNA and a frameshift, was detected in childhood onset deafness (DFNB8). This is the first description of beta-satellite insertion into an active gene resulting in a pathogenic state, and the first description of a protease involved in hearing loss.
Adult↗
Construction and characterization of BAC libraries for three fish species; rainbow trout, carp and tilapia.
Bacterial artificial chromosome (BAC) libraries are important tools for genomic research. We have constructed seven genomic BAC libraries from three fish species, rainbow trout (Oncorhynchus mykiss), carp (Cyprinus carpio) and tilapia (Oreochromis niloticus). The two rainbow trout BAC libraries have average insert sizes of 58 and 110 kb. The average size of inserts in the carp BAC library is 160 kb. The average insert sizes of the four tilapia BAC libraries are 65, 105, 145 and 194 kb, respectively. These libraries represent good coverage of each genome (2-64 x coverage). The libraries can be screened by conventional colony hybridization and provide a starting point for the construction of high-density filtres or polymerase chain reaction (PCR) screening approaches. These BAC libraries will facilitate the positional cloning of quantitative trait loci (QTLs) for a variety of economically important traits in these species.
Animals↗
Parkin is associated with cellular vesicles.
We recently identified a novel gene, parkin, as a pathogenic gene for autosomal recessive juvenile parkinsonism. Parkin encodes a 52-kDa protein with a ubiquitin-like domain and two RING-finger motifs. To provide a insight into the function of parkin, we have examined its intracellular distribution in cultured cells. We found that parkin was localized in the trans-Golgi network and the secretory vesicles in U-373MG or SH-SY5Y cells by immunocytochemical analyses. In the subsequent subcellular fractionation studies of rat brain, we showed that parkin was copurified with the synaptic vesicles (SVs) when we used low ionic conditions throughout the procedure. An immunoelectromicroscopic analysis indicated that parkin was present on the SV membrane. Parkin was readily released from SVs into the soluble phase by increasing ionic strength at neutral pH, but not by a non-ionic detergent. To elucidate its responsible region for membrane association, we transfected with green fluorescent protein-tagged deletion mutants of parkin into COS-1 cells followed by subcellular fractionation. We demonstrated the ability of parkin to bind to the membranes through a broad region except for the ubiquitin-like domain. The significance of SV localization of parkin is discussed.
Animals↗
Developmental changes in the expression of parkin and UbcR7, a parkin-interacting and ubiquitin-conjugating enzyme, in rat brain.
Parkin is a product of the Park2 gene the mutation of which causes autosomal recessive juvenile parkinsonism (AR-JP) characterized by selective dopaminergic neuronal death and absence of Lewy bodies. Recently we found that parkin is directly linked to the ubiquitin (Ub)-proteasome pathway as a Ub-protein ligase (E3) collaborating with a Ub-conjugating enzyme (E2) UbcH7. Here we analysed by in situ hybridization the expression of mRNAs for parkin and UbcR7 (rat orthologue of human UbcH7) in the developing rat brain. Parkin mRNA increased in parallel with neuronal maturation, but was unevenly distributed in various brain regions after four postnatal days. The expression pattern of the UbcR7 mRNA was almost identical to that of the parkin mRNA in all cases examined. Both parkin and UbcR7 mRNAs were distributed in neurones but not glial cells. Our findings indicate that parkin is expressed not only in the substantia nigra, but also uniformly in various brain regions in a development-dependent manner. Co-expression of UbcR7 with parkin suggests that UbcR7 may interact with parkin in vivo for ubiquitination of yet unidentified target protein(s).
Animals↗
Methanoculleus chikugoensis sp. nov., a novel methanogenic archaeon isolated from paddy field soil in Japan, and DNA-DNA hybridization among Methanoculleus species.
A strictly anaerobic, irregularly coccoid, methanogenic archaeon, strain MG62T (= JCM 10825T = DSM 13459T), was isolated from paddy field soil in Chikugo, Fukuoka, Japan. The cells stained gram-negative, were 1.0-2.0 microm in diameter, were lysed by SDS and hypotonic solutions and were flagellated. Motility was not observed. The strain was able to use H2/CO2, 2-propanol/CO2, formate, 2-butanol/CO2 and cyclopentanol/CO2 as substrates for methanogenesis, but did not utilize acetate, ethanol, methanol or methylamines. The optimum temperature and pH were 25-30 degrees C and 6.7-7.2. Analysis of lipid component parts (core lipids, phospholipid polar head groups and glycolipid sugar moieties) showed the characteristic pattern of members of the family Methanomicrobiaceae except for the absence of glucose as a glycolipid sugar moiety. The G+C content of the DNA was 62.2 mol %. Sequence analysis of the 16S rDNA revealed that the strain belonged to the genus Methanoculleus. The strain had DNA-DNA hybridization values of less than 50% with type strains of Methanoculleus species. On the basis of phenotypic, genotypic and phylogenetic characteristics, the name Methanoculleus chikugoensis sp. nov. is proposed for strain MG62T (= JCM 10825T = DSM 13459T). The DNA hybridization study also revealed the close relationships of three species, Methanoculleus olentangyi, Methanoculleus bourgensis and Methanoculleus oldenburgensis, among Methanoculleus species.
Base Composition↗
Construction of a BAC library derived from the inbred Hd-rR strain of the teleost fish, Oryzias latipes.
A large insert genomic bacterial artificial chromosome (BAC) library was constructed from the inbred Hd-rR strain of the medaka, Oryzias latipes. Approximately 92,000 clones were gridded on high-density replica filters. Insert analysis of randomly selected clones indicated a mean insert size of 210 kb and predicted a 24 times coverage of the medaka genome. The library was hybridized with a single locus DNA fragment, and the resulting positive clones were characterized and shown to be compatible with a 24-fold redundant library. This first large insert genomic library of the medaka should increase the speed of genomic analyses for this fish species.
Animals↗
Evolutionary dynamics of the human immunoglobulin kappa locus and the germline repertoire of the Vkappa genes.
We have determined the entire nucleotide sequence of the human immunoglobulin kappa locus, comprising a total of 1,010,706 nucleotides. The 76 Vkappa genes found by a hybridization-based approach and their classification in 7 families were confirmed. A Vkappa orphon located near the locus was also sequenced. In addition, we identified 55 novel Vkappa relics and truncated pseudogenes, which establish 5 new families. Among these 132 Vkappa genes, 46 have open reading frames. According to the databases and the literature, 32 unique Vkappa genes and 5 identical gene pairs form VJ-joints, 27 unique genes and 4 gene pairs are transcribed, and 25 unique genes and 4 gene pairs produce functional proteins. The Vkappa gene locus contains a 360-kb inverted duplication, which harbors 118 Vkappa genes. A comparison of the duplicated Vkappa genes suggests positive selection on the complementarity-determining regions of the duplicated genes by point mutations. The entire duplication unit was divided into 13 blocks, each of which has its distinct nucleotide sequence identity to its duplication counterpart (98.1 - 99.9 %). An inversion-mediated mechanism is suggested to generate the high-homology blocks. Based on the homology blocks and the mutation rates, the inverted duplication is assumed to have taken place approximately 5 million years ago. An orphon Vkappa gene near the kappa locus and a cluster of five Vkappa orphons on chromosome 22 have no counterparts within the kappa locus. This suggests possible mechanisms of the transposition of orphon Vkappa genes.
Base Composition↗
Familial Parkinson's disease. Alpha-synuclein and parkin.
We have reviewed recent progress in establishing the function of alpha-synuclein and parkin in relation to nigral degeneration in autosomal dominant and autosomal recessive PD. Mutations of alpha-synuclein (Ala53Thr and Ala30Pro) cause a form of autosomal dominant PD with early onset. Parkin is a novel protein expressed in the cytoplasm, including the terminal regions and Golgi apparatus. Mutations of parkin cause a form of autosomal recessive young-onset PD (ARJP). Both proteins appear to be associated with fast axonal transport. In addition, in sporadic PD, normal alpha-synuclein shows an increased tendency to self-aggregate. Thus, altered axonal transport of presynaptic proteins appears to play a crucial role in neurodegeneration in PD.
Humans↗