Toshihiko Eki

Analysis of the nucleotide sequence of chromosome VI from Saccharomyces cerevisiae.

The complete nucleotide sequence of Saccharomyces cerevisiae chromosome VI (270 kb) has revealed that it contains 129 predicted or known genes (300 bp or longer). Thirty–seven (28%) of which have been identified previously. Among the 92 novel genes, 39 are highly homologous to previously identified genes. Local sequence motifs were compared to active ARS regions and inactive loci with perfect ARS core sequences to examine the relationship between these motifs and ARS activity. Additional ARS sequences were predominantly observed in 3′ flanking sequences of active ARS loci.

Systematic identification, classification, and characterization of the open reading frames which encode novel helicase‐related proteins in Saccharomyces cerevisiae by gene disruption and Northern analysis

Helicase‐related proteins play important roles in various cellular processes incuding DNA replication, DNA repair, RNA processing and so on. It has been well known that the amino acid sequences of these proteins contain several conserved motifs, and that the open reading frames (ORFs) which encode helicase‐related proteins make up several gene families. In this study, we have identified 134 ORFs that encode helicase‐like proteins in the Saccharomyces genome, based on similarity with the ORFs of authentic helicase and helicase‐related proteins. Multiple alignment of the ORF sequences resulted in the 134 ORFs being classified to 11 clusters. Seven out of 21 previously uncharacterized ORFs (YDL031w, YDL070w, YDL084w, YGL150c, YKL078w, YLR276c, and YMR128w) were identified by systematic gene disruption, to be essential for vegetative growth. Three (YDR332w, YGL064c, and YOL095c) out of the remaining 14 dispensable ORFs exhibited the slow‐growth phenotype at 30°C and 37°C. Furthermore, the expression profiles of transcripts from 43 ORFs were examined under seven different growth conditions by Northern analysis and reverse transcription‐polymerase chain reaction, indicating that all of the 43 tested ORFs were transcribed. Interestingly, we found that the level of transcript from 34 helicase‐like genes was markedly increased by heat shock. This suggests that helicase‐like genes may be involved in the biosynthesis of nucleic acids and proteins, and that the genes can be transcriptionally activated by heat shock to compensate for the repressed synthesis of mRNA and protein. Copyright

Genomic structure, chromosomal localization and identification of mutations in the xeroderma pigmentosum variant (XPV) gene

The xeroderma pigmentosum variant (XP-V) is one of the most common forms of this cancer-prone syndrome. XP groups A through G are characterized by defective nucleotide excision repair, whereas the XP-V phenotype is proficient in this pathway. The XPV gene encodes DNA polymerase η, which catalyzes an accurate translesion synthesis, indicating that the XPV gene contributes tumor suppression in normal individuals. Here we describe the genomic structure and chromosomal localization of the XPV gene, which includes 11 exons covering the entire coding sequence, lacks a TATA sequence in the upstream region of the transcription-initiation, and is located at the chromosome band 6p21.1-6p12. Analyses of patient-derived XP-V cell lines strongly suggested that three of four cell lines carried homozygous mutations in the XPV gene. The fourth cell line, XP1RO, carried heterozygous point mutations in the XPV gene, one of which was located at the splice acceptor site of exon 2, resulting in the omission of exon 2 from the mature mRNA. These findings provide a basis for diagnosis and therapy of XP-V patients.

Dicer-related drh-3 gene functions in germ-line development by maintenance of chromosomal integrity in Caenorhabditis elegans.

In the course of systematic RNA interference (RNAi)‐based screens with helicase‐like genes in Caenorhabditis elegans, we have identified the drh‐3(D2005.5) gene as a candidate gene for protection against X‐ray irradiation. This gene encodes a novel RNA helicase‐like protein that is similar to two nematode Dicer‐related helicases (DRH). Here, we have showed the increased expression of drh‐3 transcripts during maturation of larvae to adults, and characterized the phenotype of drh‐3‐interferred nematodes using feeding RNAi method. RNAi‐mediated depletion of the drh‐3 transcripts caused embryonic lethality of F1 progeny and temperature‐sensitive reproductive capacity but did not affect the nematode life span. F1 progeny from drh‐3(RNAi) animals exhibited increased lethality after X‐ray irradiation or exposure to camptothecin. In drh‐3(RNAi) worms, aggregated chromosomes were observed in diakinesis oocyte nuclei. In developing early zygotic embryos from drh‐3(RNAi) worms, abnormally segregated chromosomes were observed and embryonic development was largely arrested at the mid‐stages of embryogenesis. Finally, examination of checkpoint responses in mitotic germ cells with regards to replication arrest by hydroxyurea and X‐ray‐induced DNA damage suggested that both checkpoints function normally under these genotoxic stress conditions. Taken together, these results indicate that the drh‐3 gene is essential for the development of germ‐lines by maintaining chromosomal integrity in C. elegans.

Expression Profiles of Transcripts from 126 Open Reading Frames in the Entire Chromosome VI of Saccharomyces cerevisiae by Systematic Northern Analyses

Chromosome VI of Saccharomyces cerevisiae contains 126 open reading frames (ORFs), and the functions of proteins encoded by 80 ORFs are still unknown. In this report, we have systematically examined the expression profiles of all 126 ORFs on chromosome VI under five kinds of growth conditions by quantitative Northern hybridization. A series of Northern analyses and reverse transcription polymerase chain reactions have revealed that more than 64 novel ORFs are transcribed. Two ORFs (YFL059w and YFR011c) are specifically expressed in the presence of galactose. Two ORFs (YFL012w and YFR032c) are specifically transcribed in sporulation. Six ORFs (YFL049w, YFL035c, YFL010c, YFR006w, YFR010w and YFR017c) are abundantly expressed in many growth conditions.

Mapping of the gene family for human heat-shock protein 90α to chromosomes 1, 4, 11, and 14

Abstract The HSP90 family of heat-shock proteins (encoded by genes for HSP90α and β) constitutes one of the major groups of proteins that are synthesized at increased rates in response to heat and other forms of stress. We previously isolated two distinct cDNA clones for HSP90α from human peripheral blood lymphocytes and from HeLa cells transfected with the adenovirus E1A gene, respectively. To determine the organization of this complex multigene family in the human genome, we used three complementary approaches: Southern analysis of a panel of human/hamster somatic cell hybrids, molecular cloning of the cosmid HSP90α clones from libraries prepared with DNAs from human lymphoblastoid cells, and in situ hybridization to human chromosomes. We demonstrate here that nucleotide sequences that encode HSP90α map to human chromosomes 1q21.2–q22, 4q35, 11p14.1–p14.2, and 14q32.3. The chromosomal mapping of the loci, HSPCAL1, HSPCAL2, HSPCAL3, HSPCAL4, and the characterization of the respective genes should facilitate clarification of the organization of this gene family and lead to a better understanding of the biological functions of the gene product.

Assignment of the 36.5-kDa (RFC5), 37-kDa (RFC4), 38-kDa (RFC3), and 40-kDa (RFC2) subunit genes of human replication factor C to chromosome bands 12q24.2–q24.3, 3q27, 13q12.3–q13, and 7q11.23

Replication factor C is a multimeric primer-recognition protein consisting of five subunits (p145, p40, p38, p37, and p36.5) and is essential for the processive elongation of DNA chains catalyzed by DNA polymerase delta or epsilon in human cells. We have mapped the locations on human chromosomes of the genes coding for the four smaller subunits [p36.5 (RFC5), p37 (RFC4), p38 (RFC3), and p40 (RFC2)] using both PCR amplification from DNAs of a panel of somatic hybrids and fluorescence in situ hybridization to bands 12q24.2-q24.3, 3q27, 13q12.3-q13, and 7q11.23, respectively.

Assignment of STK6 to human chromosome 20q13.2→q13.3 and a pseudogene STK6P to 1q41→q42

Fluorescence in situ hybridization analysis of human STK6 encoding a mitotic centrosomal protein kinase, Aik, revealed two signals in chromosome bands 20q13.2-->q13.3 and 1q41-->q42. Somatic cell hybrid panel analyses showed the existence of an identical sequence to STK6 cDNA on chromosome 20, and a processed pseudogene on chromosome 1. These results suggest that STK6 is localized at 20q 13.2-->q13.3 and a pseudogene STK6P at 1q41-->q42.

Profiling Nematode Communities in Unmanaged Flowerbed and Agricultural Field Soils in Japan by DNA Barcode Sequencing

Soil nematodes play crucial roles in the soil food web and are a suitable indicator for assessing soil environments and ecosystems. Previous nematode community analyses based on nematode morphology classification have been shown to be useful for assessing various soil environments. Here we have conducted DNA barcode analysis for soil nematode community analyses in Japanese soils. We isolated nematodes from two different environmental soils of an unmanaged flowerbed and an agricultural field using the improved flotation-sieving method. Small subunit (SSU) rDNA fragments were directly amplified from each of 68 (flowerbed samples) and 48 (field samples) isolated nematodes to determine the nucleotide sequence. Sixteen and thirteen operational taxonomic units (OTUs) were obtained by multiple sequence alignment from the flowerbed and agricultural field nematodes, respectively. All 29 SSU rDNA-derived OTUs (rOTUs) were further mapped onto a phylogenetic tree with 107 known nematode species. Interestingly, the two nematode communities examined were clearly distinct from each other in terms of trophic groups: Animal predators and plant feeders were markedly abundant in the flowerbed soils, in contrast, bacterial feeders were dominantly observed in the agricultural field soils. The data from the flowerbed nematodes suggests a possible food web among two different trophic nematode groups and plants (weeds) in the closed soil environment. Finally, DNA sequences derived from the mitochondrial cytochrome oxidase c subunit 1 (COI) gene were determined as a DNA barcode from 43 agricultural field soil nematodes. These nematodes were assigned to 13 rDNA-derived OTUs, but in the COI gene analysis were assigned to 23 COI gene-derived OTUs (cOTUs), indicating that COI gene-based barcoding may provide higher taxonomic resolution than conventional SSU rDNA-barcoding in soil nematode community analysis.

Mapping of the humanGSPT1 gene, a human homolog of the yeastGST1 gene, to chromosomal band 16p13.1

TheGSPT1 gene, a human homolog of the yeastGST1 gene (formerly namedGST1-Hs), was mapped on human chromosome 16p13.1 by a combination of nonradioactive in situ hybridization and Giemsa staining. Southern blot hybridization with a panel of human-rodent somatic cells confirmed the location of theGSPT1 gene on chromosome 16 and also showed the existence of a homologous gene on the X chromosome. A breakpoint for nonrandom chromosome rearrangements has been found in the region ofGSPT1 in patients with acute nonlymphocytic leukemia.