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J Oshima

Publications and source records attributed to J Oshima.

At least 37 records · Page 2Linked to original sources
PubMed1999

Werner helicase expression in human fetal and adult aortas.

Werner syndrome is a human progeroid syndrome caused by mutations at the Werner helicase locus (WRN). Progeroid features and diseases associated with aging (including arteriosclerosis) do not become apparent until after puberty. We entertained two alternative hypotheses to explain the post-pubertal onset: 1) WRN expression is induced at the time of puberty, its earlier functions being satisfied by another member of that family of helicases; and 2) it is expressed at all ages, but the phenotype of deficiency becomes apparent only after puberty. We report initial experiments consistent with the second hypothesis. Steady-state levels of WRN mRNA in aortic tissues were determined by semiquantitative reverse transcription-polymerase chain reaction. WRN mRNA was detectable as early as 49 days of gestation (the earliest available material). There was no statistically significant change in these levels between fetal and adult tissues. The presence of the WRN protein in fetal aorta was confirmed by Western analysis. This rules out the possibility that Werner syndrome phenotypes manifest after the puberty because of peripubertal induction of WRN expression.

Adult↗
PubMed1999

The Werner syndrome protein is involved in RNA polymerase II transcription.

Werner syndrome (WS) is a human progeroid syndrome characterized by the early onset of a large number of clinical features associated with the normal aging process. The complex molecular and cellular phenotypes of WS involve characteristic features of genomic instability and accelerated replicative senescence. The gene involved (WRN) was recently cloned, and its gene product (WRNp) was biochemically characterized as a helicase. Helicases play important roles in a variety of DNA transactions, including DNA replication, transcription, repair, and recombination. We have assessed the role of the WRN gene in transcription by analyzing the efficiency of basal transcription in WS lymphoblastoid cell lines that carry homozygous WRN mutations. Transcription was measured in permeabilized cells by [3H]UTP incorporation and in vitro by using a plasmid template containing the RNA polymerase II (RNA pol II)-dependent adenovirus major late promoter. With both of these approaches, we find that the transcription efficiency in different WS cell lines is reduced to 40-60% of the transcription in cells from normal individuals. This defect can be complemented by the addition of normal cell extracts to the chromatin of WS cells. Addition of purified wild-type WRNp but not mutated WRNp to the in vitro transcription assay markedly stimulates RNA pol II-dependent transcription carried out by nuclear extracts. A nonhelicase domain (a direct repeat of 27 amino acids) also appears to have a role in transcription enhancement, as revealed by a yeast hybrid-protein reporter assay. This is further supported by the lack of stimulation of transcription when mutant WRNp lacking this domain was added to the in vitro assay. We have thus used several approaches to show a role for WRNp in RNA pol II transcription, possibly as a transcriptional activator. A deficit in either global or regional transcription in WS cells may be a primary molecular defect responsible for the WS clinical phenotype.

Amino Acid Sequence↗
PubMed1998

Werner syndrome protein. I. DNA helicase and dna exonuclease reside on the same polypeptide.

Werner Syndrome (WS) is a human progeroid disorder characterized by genomic instability. The gene defective in WS encodes a 3' --> 5' DNA helicase (Gray, M. D., Shen, J.-C., Kamath-Loeb, A. S., Blank, A. , Sopher, B. L., Martin, G. M., Oshima, J., and Loeb, L. A.(1997) Nat. Genet. 17, 100-103). Sequence alignment analysis identified an N-terminal motif in WRN that is homologous to several exonucleases. Using combined molecular genetic, biochemical, and immunochemical approaches, we demonstrate that WRN also exhibits an integral DNA exonuclease activity. First, whereas wild-type recombinant WRN possesses both helicase and exonuclease activities, mutant WRN lacking the nuclease domain does not display exonucleolytic activity. In contrast, WRN proteins with defective helicase activity are active in exonucleolytic digestion of DNA. Second, the exonuclease co-purifies with the 160-kDa WRN protein and its associated DNA helicase and ATPase activities through successive steps of ion exchange and affinity chromatography, suggesting that all three activities are physically associated. Lastly, anti-WRN antiserum specifically co-precipitates the WRN helicase and exonuclease activities indicating that both activities reside on the same antigenic WRN polypeptide. The association of an exonuclease with WRN distinguishes it from other RecQ homologs and raises the possibility that the distinct phenotypic characteristics of WS may be due in part to a defective exonuclease.

Chromatography, Affinity↗
PubMed1998

Comparison of methods for identifying transcription units and transcription map of the Werner syndrome gene region.

To isolate a human disease gene by positional cloning, a critical step is the identification of candidate genes from a targeted genomic region. We used cDNA selection, exon trapping, and genomic sequencing to identify 12 transcription units from a 1.4-Mb genomic region containing the Werner syndrome gene (WRN). This included sequencing of 650 kb in the region of the WRN gene, to date, the most DNA sequenced as part of a positional cloning effort. The result of this combined method was significant overlap among the transcription units identified by each method; yet, no one method identified all of the transcription units. We present here a comparison of the effectiveness and efficiency of these methods and present a transcription map of the Werner syndrome gene region.

Blotting, Northern↗
PubMed1998

Werner helicase is localized to transcriptionally active nucleoli of cycling cells.

Mutations at the Werner helicase locus (WRN) are responsible for the Werner syndrome (WS), a "caricature of aging." We have localized the Werner protein (WRNp) to the nucleoli of replicating mammalian cells, where its appearance is associated with transcriptional activity. A dramatic reduction of the nucleolar signal and of [3H]uridine incorporation occurred when cultures were made quiescent or were exposed to 4-nitroquinoline-1-oxide (4NQO), to which WS cells are particularly susceptible. Total cellular levels of WRNp, however, did not change, and virtually all WRNp was in the nuclear fractions, consistent with translocation to the nucleoplasm and/or masking of the epitopes. The 4NQO-induced altered state of WRNp was prevented by Na3VO4, but not by okadaic acid, suggesting that WRNp localization/function is partially regulated by kinases/phosphatases for Tyr substrates on WRNp or interacting proteins. The repression of rDNA transcription by 4NQO was not reversed by Na3VO4. We suggest that physiological states and genotoxic agents modulate the interaction of WRNp with rDNA, consistent with a role of WRNp in rDNA transcription.

4-Nitroquinoline-1-oxide↗
PubMed1998

Structure and function of the human Werner syndrome gene promoter: evidence for transcriptional modulation.

The Werner syndrome (WS) is an autosomal recessive segmental progeroid syndrome caused by mutations in a novel member ( WRN ) of the RecQ family of helicases. Somatic WS cells are hypermutable and have elongated S phases, suggesting possible defects in DNA replication and/or repair. As an initial approach to the investigation of how this locus might be responsive to DNA damage, we determined the structure of the human WRN promoter. The WRN promoter region has two transcription initiation sites and exhibits several features characteristic of so-called constitutive promoters, including the absence of TATA and CAAT boxes. A luciferase reporter assay revealed that the upstream promoter was used 2-10-fold less frequently than the downstream promoter, the variation being a function of cell type. The activity of the WRN promoter was dramatically reduced in cells from WS patients. The reduction of activity was not seen in three other promoters tested, including one TATA-less promoter and one TATA-containing promoter. This is consistent with the presence of a positive regulatory mechanism of WRN expression.

Base Sequence↗
PubMed1998

Characterization of Werner syndrome protein DNA helicase activity: directionality, substrate dependence and stimulation by replication protein A.

Werner syndrome is an inherited disease characterized by premature aging, genetic instability and a high incidence of cancer. The wild type Werner syndrome protein (WRN) has been demonstrated to exhibit DNA helicase activity in vitro. Here we report further biochemical characterization of the WRN helicase. The enzyme unwinds double-stranded DNA, translocating 3'-->5' on the enzyme-bound strand. Hydrolysis of dATP or ATP, and to a lesser extent hydrolysis of dCTP or CTP, supports WRN-catalyzed strand-displacement. K m values for ATP and dATP are 51 and 119 microM, respectively, and 2.1 and 3.9 mM for CTP and dCTP, respectively. Strand-displacement activity of WRN is stimulated by single-stranded DNA-binding proteins (SSBs). Among the SSBs from Escherichia coli, bacteriophage T4 and human, stimulation by human SSB (human replication protein A, hRPA) is the most extensive and occurs with a stoichiometry which suggests direct interaction with WRN. A deficit in the interaction of WRN with hRPA may be associated with deletion mutations that occur at elevated frequency in Werner syndrome cells.

Adenine Nucleotides↗
PubMed1997

Mismatch repair in extracts of Werner syndrome cell lines.

Werner syndrome (WS) is an autosomal recessive disease, the phenotype of which is a caricature of premature aging. WS cells and cell lines display several types of genetic instability, and WS patients have an increased risk of developing cancer. The WS locus (WRN) encodes a protein that shows significant sequence homology to the RecQ family of DNA helicases. Because a DNA helicase may function in DNA mismatch repair, we examined extracts of WS cell lines for mismatch repair activity. Extracts from four different WS lymphoblastoid cell lines containing different WRN mutations and from three within-pedigree control cell lines were all proficient in mismatch repair. In marked contrast, extracts from three independent WS fibroblastoid cell lines were deficient in repair of base-base and insertion/deletion mismatches. Extracts of one of these lines restored activity to extracts of mismatch repair-deficient tumor cells with defined mutations in hMSH2, hMSH3, hMSH6, hMLH1, or hPMS2. This suggests that the WRN mutation in this fibroblast line is not a dominant negative inhibitor of mismatch repair activity and that the repair defect does not reside in these five known mismatch repair genes. Defective mismatch repair in fibroblastoid but not lymphoblastoid cells is consistent with the possibility that WRN protein could have a cell type- and/or tissue-specific role in mismatch repair. Alternatively, a mutation in WRN could predispose cells to mutations in other genes required for mismatch repair activity, at least one of which could be an unknown gene.

Cell Line↗
PubMed1997

Association of a polymorphic variant of the Werner helicase gene with myocardial infarction in a Japanese population.

The Werner syndrome (WS) is a rare autosomal recessive progeroid syndrome characterized by the premature onset of multiple age-related disorders, including atherosclerosis, cancer, non-insulin-dependent diabetes mellitus (NIDDM), ocular cataracts and osteoporosis [Epstein et al., 1966]. The major cause of death (at a median age of 47) is myocardial infarction (MI) [Epstein et al., 1966]. The WS mutation involves a member (WRN) of the RecQ family of helicases and may perturb DNA replication, repair, recombination, transcription, or chromosomal segregation [Yu et al., 1996]. We now report data on 149 MI cases and age-matched controls suggesting that a polymorphic WRN variant is associated with increased risk for MI. Based on our data, homozygosity for a cysteine at amino acid 1367 (the most prevalent genotype) predicts a 2.78 times greater risk of MI (95% confidence intervals: 1.23 to 6.86). The variant was not significantly associated with NIDDM. The two alleles (cysteine vs. arginine) could influence helicase activity, turnover, macromolecular interactions or, alternatively, could be markers for haplotypes influencing WRN regulation or reflecting gene action at linked loci. However, given the caveats implicit in genetic association studies, it is imperative that the present results be replicated in independent populations.

Adult↗
PubMed1997

An apoptosis-inducing genotoxin differentiates heterozygotic carriers for Werner helicase mutations from wild-type and homozygous mutants.

Immortalized B lymphocytes from Werner syndrome subjects are shown to be hypersensitive to 4-nitroquinoline-1-oxide (4NQO), supporting earlier work on T lymphocytes. We also show that B cell lines from clinically normal heterozygous carriers exhibit sensitivities to this genotoxic agent, which are intermediate to those of wild-type and homozygous mutants. 4NQO is shown to induce an apoptotic response. These data encourage research on DNA repair with such cell lines and raise the question of an enhanced sensitivity of the relatively prevalent heterozygous carriers to certain environmental genotoxic agents.

4-Nitroquinoline-1-oxide↗
PubMed1997

The Werner syndrome protein is a DNA helicase.

Werner syndrome (WS) is an uncommon autosomal recessive disorder characterized by premature aging. The clinical manifestations of WS, including atherosclerosis and osteoporosis, appear early in adulthood, and death in the fourth to sixth decade commonly ensues from myocardial infarction or cancer. In accord with the aging phenotype, cells from WS patients have a reduced replicative life span in culture. Genomic instability is observed at the cytogenetic level in the form of chromosome breaks and translocations and at the molecular level by multiple large deletions. The Werner syndrome gene (WRN) has recently been cloned. The predicted product is a 1,432-amino-acid protein whose central domain is homologous to members of the RecQ family of DNA helicases. Such homology does not necessarily mean that WRN encodes an active helicase. For example, the Saccharomyces cerevisiae RAD26 gene protein and the human transcription-repair coupling factor CSB (Cockayne syndrome 8) are highly homologous to known helicases, yet neither encodes an active helicase. Moreover, the Bloom's syndrome gene (BLM), discovered before WRN, is also homologous to the RecQ family of DNA helicases, though we still await demonstration that it encodes an active helicase. Here we report that the WS protein does indeed catalyze DNA unwinding.

Adult↗
PubMed1997

Mutations in the consensus helicase domains of the Werner syndrome gene. Werner's Syndrome Collaborative Group.

Werner syndrome (WS) is an autosomal recessive disease with a complex phenotype that is suggestive of accelerated aging. WS is caused by mutations in a gene, WRN, that encodes a predicted 1,432-amino-acid protein with homology to DNA and RNA helicases. Previous work identified four WS mutations in the 3' end of the gene, which resulted in predicted truncated protein products of 1,060-1,247 amino acids but did not disrupt the helicase domain region (amino acids 569-859). Here, additional WS subjects were screened for mutations, and the intron-exon structure of the gene was determined. A total of 35 exons were defined, with the coding sequences beginning in the second exon. Five new WS mutations were identified: two nonsense mutations at codons 369 and 889; a mutation at a splice-junction site, resulting in a predicted truncated protein of 760 amino acids; a 1-bp deletion causing a frameshift; and a predicted truncated protein of 391 amino acids. Another deletion is >15 kb of genomic DNA, including exons 19-23; the predicted protein is 1,186 amino acids long. Four of these new mutations either partially disrupt the helicase domain region or result in predicted protein products completely missing the helicase region. These results confirm that mutations in the WRN gene are responsible for WS. Also, the location of the mutations indicates that the presence or absence of the helicase domain does not influence the WS phenotype and suggests that WS is the result of complete loss of function of the WRN gene product.

Asian People↗
PubMed1996

Narrowing the position of the Werner syndrome locus by homozygosity analysis-extension of homozygosity analysis.

Werner syndrome (WS) is an autosomal recessive disorder characterized by the premature occurrence of many age-related features. Previously, the WS gene (WRN) was mapped between D8S131 and D8S87, in an 8.3-cM interval. In this study, regions of homozygosity in 36 WS patients from inbred families were searched for by genotyping for 35 dinucleotide repeat polymorphic markers to narrow down the WRN critical region. The region most consistently homozygous in these patients was between the D8S1219/D8S1220 cluster and D8S278, within a 4.4-cM interval. For 16 markers mapped in this interval, 24 WS patients (22 Japanese patients and 2 Caucasian patients) in whom consanguinity failed to be proved were also genotyped, under the assumption that some of these patients might still be from consanguineous marriages. The data were analyzed by Fisher's exact test with a 2 x 2 contingency table for the 22 Japanese patients, excluding the 2 Caucasian patients. The frequencies of homozygosity in the 22 patients at 10 of 16 markers tested were significantly higher than those detected in the general population. Analysis of homozygosity patterns indicated that the region most consistently homozygous was between D8S1445 and D8S278. Thus the WRN locus is most likely between the two markers D8S1445 and D8S278, in a 1.6-cM interval.

Asian People↗
PubMed1996

A YAC, P1, and cosmid contig and 17 new polymorphic markers for the Werner syndrome region at 8p12-p21.

A yeast artificial chromosome (YAC), P1, and cosmid clone contig was constructed for the Werner syndrome (WRN) region of chromosome 8p12-p21 and used to clone a candidate gene for WRN. This region also possibly contains a familial breast cancer locus. The contig was initiated by isolating YACs for the glutathione reductase (GSR) gene and extended in either direction by walking techniques. Sequence-tagged site (STS) markers were generated from subclones of 2 GSR YACs and used to identify P1 and cosmid clones. Additional STSs were generated from P1 and cosmid clones and from potential expressed sequences identified by cDNA selection and exon amplification methods. The final contig was assembled by typing 17 YACs, 20 P1 clones, and 109 cosmids for 54 STS markers. The WRN region could be spanned by 2 nonchimeric YACs covering approximately 1.4 Mb. A P1/cosmid contig was established covering the core 700-800 kb of the WRN region. Fifteen new short tandem repeat polymorphisms and 2 biallelic polymorphic markers were identified and included as STSs in the contig. Analysis of these markers in Werner syndrome subjects demonstrates that the candidate WRN gene is in a region of linkage disequilibrium.

Base Sequence↗
PubMed1996

Genomic structure and expression of STM2, the chromosome 1 familial Alzheimer disease gene.

Mutations in the gene STM2 result in autosomal dominant familial Alzheimer disease. To screen for mutations and to identify regulatory elements for this gene, the genomic DNA sequence and intron-exon structure were determined. Twelve exons including 10 coding exons were identified in a genomic region spanning 23,737 bp. The first 2 exons encode the 5'-untranslated region. Expression analysis of STM2 indicates that two transcripts of 2.4 and 2.8 kb are found in skeletal muscle, pancreas, and heart. In addition, a splice variant of the 2.4-kb transcript was identified that is the result of the use of an alternative splice acceptor site located in exon 10. The use of this site results in a transcript lacking a single glutamate. The promotor for this gene and the alternatively spliced exons leading to the 2.8-kb form of the gene remain to be identified. Expression of STM2 was high in skeletal muscle and pancreas, with comparatively low levels observed in brain. This expression pattern is intriguing since in Alzheimer disease, pathology and degeneration are observed only in the central nervous system.

Alternative Splicing↗