protein. The amino acid sequence -vas highl)' homologoiis, but not identicat, o thut of the hEiman hsp 90ct gene isulated from humun peripheral blood lymphocytes [M, Yamaiaki, K. Akaogi, T. Miwa, T. Imai, E. Soeda and K. Yoko)'ama, iN'uctei(' Acids Re.s.. 17, 71{}8 (1989)], '1'his ct)NA hybridized v'ith RNA species which increased 5te 20-fotd upen heat shock and more than 5-fold in the differentiation siage of humaii Tera 2 cells.

The construction of a cynomolgus macaque (Macaca fascicularis, Mafa) BAC library for genomic comparison between rhesus and cynomolgus macaques is necessary to promote the cynomolgus macaque as one of the important experimental animals for future medical and biological research. In this paper, we constructed a cynomolgus macaque BAC library and a map of the MHC (Mafa) genomic region for comparison of the genomic organization and nucleotide similarities between the human, the chimpanzee, and the rhesus macaque. The BAC library consists of 221,184 clones with an average insert size of 83 kb, providing a sixfold coverage of the haploid genome. A total of 114 BAC clones and 54 PCR primer sets were used to construct a 4.3-Mb contig of the MHC region. Diversity analysis of genomic sequence from selected subregions of the MHC revealed that the cynomolgus sequence varied compared to rhesus macaque, human, and chimpanzee sequences by 0.48, 4.15, and 4.10%, respectively. From these findings, we conclude that the BAC library and Mafa genomic map are useful tools for genome analysis and will have important applications for comparative genomics and identifying regions of consequence in medical research.

A simple strategy for the rapid preparation of an end-specific linking-DNA probe from the YAC-human chromosome 21 DNA recombinant clone and the characterization of this single DNA probe are described. Synthetic oligodeoxynucleotide primers, based on the consensus Alu sequence, and the Sup4 DNA fragment in the YAC arms were used to amplify end-specific DNA sequences by the polymerase chain reaction (PCR) for screening of the linking YAC recombinant clones ("YAC-Alu PCR"). Nucleotide sequencing of the product of PCR from human genomic DNA in a YAC insert confirmed the boundary between the vector and the insert and the presence of the 3'-end Alu-like structure. The probe R1, prepared by "YAC-Alu PCR" amplification, was assigned to chromosome 21 by Southern hybridization of somatic cell hybrid DNAs. In situ hybridization allowed localization of the R1 DNA probe to the human chromosome 21q21-q22.1 region. Thus, this approach has significant advantages not only for isolation of a single DNA probe specific for human chromosome 21 but also for the screening of YAC linking recombinant clones for mapping of the human genome.

Construction of a comprehensive comparative map between swine and human chromosomes is a prerequisite, in order to select candidate swine genes for traits from the human genome database as well as to understand the evolutionary process of the two species. The present study attempted to use 910 sequence-tagged sites (STSs) localized in human chromosome (HSA) 1p36-->p35 (35 Mbp) for radiation hybrid (RH) mapping to swine chromosomes (SSCs). Out of the 910 STSs subjected to amplification of swine orthologues, primer pairs for 13 STSs were found to amplify the respective orthologues and the STSs were assigned to SSCs. Eleven STSs were assigned to SSC6 in the same order as that in HSA1: SSC6cen-(SHGC-150)-(A006H31)-(X82877)-(A007E03)-(IB404)-(stGDB:371372)-(stSG31658)-(A009Q18)-(stSG14201/A009C01)-(H08335)-qter. One of the remaining two STSs, WI-20819, was assigned to SSCX, and the other, R91D18R, was not linked to any first-generation markers of the IMpRH map with a lod score greater than 3.

Animal GeneticsVolume 33, Issue 5 p. 379-381 Construction of a bovine bacterial artificial chromosome library from fibroblasts used for cloned cattle S. Fujisaki, S. Fujisaki Genome Research Department, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-0901, Japan,Search for more papers by this authorY. Mizoguchi, Y. Mizoguchi Shirakawa Institute of Animal Genetics, Odakura Nishigo, Nishishirakawa, Fukushima 961-8061, Japan,Search for more papers by this authorS. Takahashi, S. Takahashi Department of Animal Breeding and Reproduction, National Institute of Livestock and Grassland,Search for more papers by this authorY. Z. Chen, Y. Z. Chen Advanced Genotechs Co., Tsukuba, Ibaraki 305-0074, Japan,Search for more papers by this authorK. Suzuki, K. Suzuki Animal Genome Program Team, STAFF Institute, Tsukuba, Ibaraki 305-0854, JapanSearch for more papers by this authorS. Asakawa, S. Asakawa Department of Molecular Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-0016, Japan,Search for more papers by this authorE. Soeda, E. Soeda Advanced Genotechs Co., Tsukuba, Ibaraki 305-0074, Japan,Search for more papers by this authorN. Shimizu, N. Shimizu Department of Molecular Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-0016, Japan,Search for more papers by this authorY. Sugimoto, Y. Sugimoto Shirakawa Institute of Animal Genetics, Odakura Nishigo, Nishishirakawa, Fukushima 961-8061, Japan,Search for more papers by this authorH. Yasue, H. Yasue Genome Research Department, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-0901, Japan,Search for more papers by this author S. Fujisaki, S. Fujisaki Genome Research Department, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-0901, Japan,Search for more papers by this authorY. Mizoguchi, Y. Mizoguchi Shirakawa Institute of Animal Genetics, Odakura Nishigo, Nishishirakawa, Fukushima 961-8061, Japan,Search for more papers by this authorS. Takahashi, S. Takahashi Department of Animal Breeding and Reproduction, National Institute of Livestock and Grassland,Search for more papers by this authorY. Z. Chen, Y. Z. Chen Advanced Genotechs Co., Tsukuba, Ibaraki 305-0074, Japan,Search for more papers by this authorK. Suzuki, K. Suzuki Animal Genome Program Team, STAFF Institute, Tsukuba, Ibaraki 305-0854, JapanSearch for more papers by this authorS. Asakawa, S. Asakawa Department of Molecular Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-0016, Japan,Search for more papers by this authorE. Soeda, E. Soeda Advanced Genotechs Co., Tsukuba, Ibaraki 305-0074, Japan,Search for more papers by this authorN. Shimizu, N. Shimizu Department of Molecular Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-0016, Japan,Search for more papers by this authorY. Sugimoto, Y. Sugimoto Shirakawa Institute of Animal Genetics, Odakura Nishigo, Nishishirakawa, Fukushima 961-8061, Japan,Search for more papers by this authorH. Yasue, H. Yasue Genome Research Department, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-0901, Japan,Search for more papers by this author First published: 30 September 2002 https://doi.org/10.1046/j.1365-2052.2002.00896_3.xCitations: 7Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume33, Issue5October 2002Pages 379-381 RelatedInformation

We have devised a mapping method for rapid assembly and ordering of bacterial artificial chromosome (BAC) clones on a radiation hybrid (RH) panel, using sequence-tagged sites (STSs) and PCR. The protocol consists of two rounds of two-dimensional screening from a limited number of BACs to correspond each to an STS. In the first round, STSs are assembled in the RH bins and ordered according to PCR signals derived from 384-well microtiter plates (MTPs) in which BAC clones have been arrayed. In the second round, individual BAC clones are isolated from the MTPs to build a contig. We applied this method to a 35-Mb region spanning human chromosome 1p35–p36 and assembled 1366 BACs in 11 contigs, the longest being about 20 Mb. The working draft sequences of the human genome have been integrated into the contigs to validate the accuracy.

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. © 2001 Wiley‐Liss, Inc.

Recently, loss of heterozygosity (LOH) studies suggest that more than two tumor suppressor genes lie on the short arm of chromosome 1 (1p) in neuroblastoma (NB). To identify candidate tumor suppressor genes in NB, we searched for homozygous deletions in 20 NB cell lines using a high-density STS map spanning chromosome 1 p36, a common LOH region in NB. We found that the 45-kDa subunit of the DNA fragmentation factor (DFF45) gene was homozygously deleted in an NB cell line, NB-1. DFF45 is the chaperon of DFF40, and both molecules are necessary for caspase 3 to induce apoptosis. DFF35, a splicing variant of DFF45, is an inhibitor of DFF40. We examined 20 NB cell lines for expression and mutation of DFF45 gene by reverse transcription (RT)-polymerase chain reaction (PCR) and RT-PCR-single-strand conformation polymorphism. Some novel variant transcripts of the DFF45 gene were found in NB cell lines, but not in normal adrenal gland and peripheral blood. These variants may not serve as chaperons of DFF40, but as inhibitors like DFF35, thus disrupting the balance between DFF45 and DFF40. No mutations of the DFF45 gene were found in any NB cell line, suggesting that the DFF45 is not a tumor suppressor gene for NB. However, homozygous deletion of the DFF45 gene in the NB-1 cell line may imply the presence of unknown tumor suppressor genes in this region.

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.

Human chromosomes 1q21-q25, 6p21.3-22.2, 9q33-q34, and 19p13.1-p13.4 carry clusters of paralogous loci, to date best defined by the flagship 6p MHC region. They have presumably been created by two rounds of large-scale genomic duplications around the time of vertebrate emergence. Phylogenetically, the 1q21-25 region seems most closely related to the 6p21.3 MHC region, as it is only the MHC paralogous region that includes bona fide MHC class I genes, the CD1 and MR1 loci. Here, to clarify the genomic structure of this model MHC paralogous region as well as to gain insight into the evolutionary dynamics of the entire quadriplication process, a detailed analysis of a critical 1.7 megabase (Mb) region was performed. To this end, a composite, deep, YAC, BAC, and PAC contig encompassing all five CD1 genes and linking the centromeric +P5 locus to the telomeric KRTC7 locus was constructed. Within this contig a 1.1-Mb BAC and PAC core segment joining CD1D to FCER1A was fully sequenced and thoroughly analyzed. This led to the mapping of a total of 41 genes (12 expressed genes, 12 possibly expressed genes, and 17 pseudogenes), among which 31 were novel. The latter include 20 olfactory receptor (OR) genes, 9 of which are potentially expressed. Importantly, CD1, SPTA1, OR, and FCERIA belong to multigene families, which have paralogues in the other three regions. Furthermore, it is noteworthy that 12 of the 13 expressed genes in the 1q21-q22 region around the CD1 loci are immunologically relevant. In addition to CD1A-E, these include SPTA1, MNDA, IFI-16, AIM2, BL1A, FY and FCERIA. This functional convergence of structurally unrelated genes is reminiscent of the 6p MHC region, and perhaps represents the emergence of yet another antigen presentation gene cluster, in this case dedicated to lipid/glycolipid antigens rather than antigen-derived peptides.

BACKGROUNDWe have identified for the first time a homozygously deleted region within the smallest region of overlap at 1p36.2-3 in two neuroblastoma cell lines.PROCEDUREThe 800-kb PAC contig covering the entire homozygously deleted region was made and sequenced. To date, approximately 70% of sequencing has been accomplished, and the estimated length of the deleted region was 500 kb.RESULTSCurrently, we have found six genes within the region, which include three known genes as well as three other genes that have been reported during processing of our present project for the last 3(1/2) years. We report here the results of expression and mutation analyses of those genes.CONCLUSIONSFull sequencing for the region of homozygous deletion as well as further analyses of the genes mapped within the region may reveal whether or not there is a neuroblastoma suppressor gene as proposed by the Knudson's two-hit hypothesis.

The chromosome 21 mapping and sequencing consortium M. Hattori*, A. Fujiyama*, T. D. Taylor*, H. Watanabe*, T. Yada*, H.-S. Park*, A. Toyoda*, K. Ishii*, Y. Totoki*, D.-K. Choi*, E. Soedat, M. Ohki:, T. Takagi§, Y. Sakaki*§; S. Taudienl, K. Blechschmidtl, A. Polleyl, U. Menzell, J. Delabar�, K. Kumpfl, R. Lehmannl, D. Patterson#, K. Reichwaldl, A. Rumpl, M. Schillhabell, A. Schudyl, W. Zimmermannl, A. Rosenthall; J. Kudoh, K. Shibuya, K. Kawasaki, S. Asakawa, A. Shintani, T. Sasaki, K. Nagamine, S. Mitsuyama, S. E. Antonarakis**, S. Minoshima, N. Shimizu, G. Nordsiektt, K. Hornischertt, P. Brandttt, M. Scharfett, O. Schointt, A. Desario::, J. Reichelttt, G. Kauertt, H. Bloickertt; J. Ramser§§, A. Beck§§, S. Klages§§, S. Hennig§§, L. Riesselmann§§, E. Dagand§§, T. Haaf§§, S. Wehrmeyer§§, K. Borzym§§, K. Gardiner#, D. Nizeticll, F. Francis§§, H. Lehrach§§, R. Reinhardt§§ & M.-L. Yaspo§§

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.