Eiichi Soeda

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.

Genomic analysis of DUSP6, a dual specificity MAP kinase phosphatase, in pancreatic cancer

DUSP6 (alias PYST1), one of the dual-specificity tyrosine phosphatases, is localized on 12q21, one of the regions of frequent allelic loss in pancreatic cancer. This gene is composed of three exons, and two forms of alternatively spliced transcripts are ubiquitously expressed. Although no mutations were observed in 26 pancreatic cancer cell lines, reduced expressions of the full-length transcripts were observed in some cell lines, which may suggest some role for DUSP6 in pancreatic carcinogenesis.

Identification and characterization of a 500-kb homozygously deleted region at 1p36.2-p36.3 in a neuroblastoma cell line

Loss of heterozygosity of the distal region of chromosome 1p where tumor suppressor gene(s) might harbor is frequently observed in many human cancers including neuroblastoma (NBL) with MYCN amplification and poor prognosis. We have identified for the first time a homozygously deleted region at the marker D1S244 within the smallest region of overlap at 1p36.2-p36.3 in two NBL cell lines, NB-1 and NB-C201 (MASS-NB-SCH1), although our genotyping has suggested the possibility that both lines are derived from the same origin. The 800-kb PAC contig covering the entire region of homozygous deletion was made and partially sequenced (about 60%). The estimated length of the deleted region was 500 kb. We have, thus far, identified six genes within the region which include three known genes (DFF45, PGD, and CORT) as well as three other genes which have been reported during processing our present project for the last 3½ years (HDNB1/UFD2, KIAA0591F/KIF1B-β, and PEX14). They include the genes related to apoptosis, glucose metabolism, ubiquitin-proteasome pathway, a neuronal microtubule-associated motor molecule and biogenesis of peroxisome. At least three genes (HDNB1/UFD2, KIAA0591F/KIF1B-β, and PEX14) were differentially expressed at high levels in favorable and at low levels in unfavorable subsets of primary neuroblastoma. Since the 1p distal region is reported to be imprinted, those differentially expressed genes could be the new members of the candidate NBL suppressor, although RT-PCR-SSCP analysis has demonstrated infrequent mutation of the genes so far identified. Full-sequencing and gene prediction for the region of homozygous deletion would elucidate more detailed structure of this region and might lead to discovery of additional candidate genes.

Cosmid assembly and anchoring to human chromosome 21

A human chromosome 21-specific cosmid library from the Lawrence Livermore National Laboratory has been analyzed by two complementary methods, fingerprinting and hybridization; 40% coverage of the entire chromosome 21 has been achieved. To prepare a contig pool, approximately 9300 cosmid clones randomly selected from the library were fingerprinted and automatically assembled into 467 overlapping sets by the fluorescence-tagged restriction fragment method. The average size of the overlapping sets was 9.5 cosmids with minimal tiling paths consisting of 5.4 cosmids with a 10-kb extension each. However, as many as 10% of overlaps within members were estimated to be false. For regional localization, we hybridized gridded arrays of cosmids with inter-Alu-PCR probes obtained from YAC clones and somatic cell hybrids and assigned 592 cosmids to 26 subregions of 21q. Of these, 371 clones were incorporated into 139 contigs, anchoring the total 1864 cosmids to the subregion. The remaining 221 clones were mapped as orphans. To correlate the cytogenetic, YAC, and cosmid maps on 21q, the translocation breakpoints of the chromosomes contained in the somatic cell hybrids were mapped with respect to the STS content of the YACs. From the gene cluster regions, 176 ribosomal and 25 alphoid clones were isolated by hybridization. Together, these sets of anchored contigs and cosmids will provide a valuable resource for construction of a high-resolution map and for isolation of genes of interest from chromosome 21.

Cloning of a new kinesin-related gene located at the centromeric end of the human MHC region

We previously reported the presence of a new gene (HSET) with an unknown function, in the centromeric side of the class II gene region of the human major histocompatibility complex (MHC). cDNA clones corresponding to the HSET gene were isolated from a human testis cDNA library. A 2.4 kilobase transcript from the HSET gene was abundantly expressed in testis, B-cell, T-cell, and ovary cell lines but was not detected in lung or stomach. Analysis of the nucleotide sequence of the HSET cDNA clones revealed significant similarity to kinesin-related proteins in yeast, Drosophila, and human. Its predicted amino acid sequence contains a domain with strong sequence similarity to the ATP-binding and motor domains of a plus end-directed microtuble motor protein, kinesin, which might be involved in mitotic chromosome segregation, suggesting that the HSET gene encodes a novel kinesin-related protein.

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.

Genomic structure and mutational analysis of the human KIF1B gene which is homozygously deleted in neuroblastoma at chromosome 1p36.2.

In order to clone candidate tumor suppressor genes whose loss contributes to the pathogenesis of neuroblastoma (NB), we performed polymerase chain reaction (PCR) screening using a high-density sequence tagged site-content map within a commonly deleted region (chromosome band 1p36) in 24 NB cell lines. We found a ∼480 kb homozygously deleted region at chromosome band 1p36.2 in one of the 24 NB cell lines, NB-1, and cloned the human homologue (KIF1B-β) of the mouseKif1B-β gene in this region. The KIF1B-β gene had at least 47 exons, all of which had a classic exon–intron boundary structure. Mouse Kif1B is a microtubule-based putative anterograde motor protein for the transport of mitochondria in neural cells. We performed mutational analysis of the KIF1B-β gene in 23 cell lines using 46 sets of primers and also an allelic imbalance (AI) analysis of KIF1B-β in 50 fresh NB samples. A missense mutation at codon 1554, GTG (Gly) to ATG (Met), silent mutations at codon 409 (ACG to ACA) and codon 1721 (ACC to ACT), and polymorphisms at codon 170, GAT (Asp) to GAA (Glu), and at codon 1087, TAT (Tyr), to TGT (Cys), were all identified, although their functional significances remain to be determined. The AI for KIF1B-β was slightly higher (38%) than those for the other two markers (D1S244, D1S1350) (35 and 32%) within the commonly deleted region (1p36). Reverse transcriptase-PCR analysis of the KIF1B-β gene revealed obvious expression in all NB cell lines except NB-1, although decreased expression of the KIF1B-β gene was found in a subset of early- and advanced-stage NBs. These results suggest that the KIF1B-β gene may not be a candidate for tumor suppressor gene of NB.

Homozygous deletion in a neuroblastoma cell line defined by a high-density STS map spanning human chromosome band 1p36

Recent molecular studies have shown a relatively high rate of loss of heterozygosity (LOH) in neuroblastoma (NB) as well as other types of tumors in human chromosome band 1p36. To identify candidate tumor suppressor genes in NB, we searched for homozygous deletions in NB cell lines with PCR according to a high‐density sequence tagged site (STS)‐content map spanning 1p35–36. Among 25 NB cell lines examined, only one cell line, NB‐1, showed no signal with 27 STSs in a 480 kb region in 1p36.2. The sequence analysis has revealed that the defective region included seven known genes (E4, KIF1B, SCYA5, PGD, Cortistatin, DFF45, and PEX14), nine expressed sequence tags (ESTs), and two microsatellite markers. These genes are related to apoptosis, an ubiquitin‐proteasome pathway, a neuronal microtubule‐associated motor molecule, and components of a common translocation machinery. The region between the DFF45 and KIF1B genes was defined as homozygous deletion by Southern blotting. The search in LOH regions with high‐density STSs may be useful for the isolation and identification of tumor suppressor genes in other tumors as well as NBs.

Analysis of YAC clones by pulsed-field gel electrophoresis: Physical mapping of Cu/Zn superoxide dismutase gene locus

Procedures for constructing a physical map of yeast artificial chromosome (YAC) clones by pulsed-field gel electrophoresis are described. Included are practical protocols for preparation of yeast chromosomal DNA containing YAC in agarose plugs, restriction enzyme digestion of DNA within the plugs, and running conditions for pulsed-field gel electrophoresis and Southern hybridization. As an illustration, we have applied these methods to two independent YAC clones encoding the Cu/Zn superoxide dismutase gene (SOD-1) and constructed from these two overlapping clones a restriction enzyme map spanning approximately 600 kb. The map of chromosome 21q22 may prove useful in investigating the genetic basis of Down syndrome.

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.