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Author(s): M. Hattori [1]; A. Fujiyama [1]; T. D. Taylor [1]; H. Watanabe [1]; T. Yada [1]; H.-S. Park [1]; A. Toyoda [1]; K. Ishii [1]; Y. Totoki [1]; D.-K. [...]

Objectives More than two decades have passed since the first living donor liver transplantation was performed in Japan in 1989. There are many reports about problems in adherence to taking medication and medical follow-ups in children who received liver transplants, because there is no transition strategy for those children and parents or guardians. The objective of this study is to measure the effect of nature and outdoor activity to improve children's medical adherence. Methods We recruited participants from 9-year-old children who are attending the outpatient liver transplant clinic in a stable condition (no event such as rejection or surgical procedure within 6 months). We took participants to a snow camp and measured its effect by using the IKIRU CHIKARA (IKR) tool, which contain 28 items divided into 3 categories: psychosocial ability, moral fitness, and physical ability. Children were tested on three occasions, before, just after, and 1 month after the camp. Results Eight patients participated in the snow camp and 7 patients were eligible for the study. The average age was 12.6 with a range 10 to 17 years. There were 3 girls and 4 boys. The average IKR scores before, just after, and 1 month after the camp were 127.9, 131.5, and 126.6, respectively. Conclusion An outdoor activity such as a snow camp can be safely conducted, and it is an acceptable option to incorporate within a pediatric liver transplant program. There were no significant changes in IKR scores during this short observation. Longer observation is needed to measure the effect of nature and outdoor activities. [ABSTRACT FROM AUTHOR]

Author(s): 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; Y. [...]

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. [ABSTRACT FROM AUTHOR]

Purpose: In Japan, there have been no national surveys on the incidence of de novo malignancy after solid organ transplantation, which is one of the leading causes of death in transplant recipients., Methods: A questionnaire was distributed to institutions that perform solid organ transplantation in Japan, and clinical information was collected from patients who underwent transplantation between 2001 and 2010 and who exhibited de novo malignancies., Results: Nine thousand two hundred ten solid organ transplants (kidney, 49.9%; liver, 45.9%; heart, 0.9%; lung, 1.2%; pancreas, 1.9%; small intestine, 0.2%) were performed. Four hundred seventy-nine (5.2%) cases of de novo malignancy were identified. The transplanted organs of the patients included the kidney (n = 479, 54.8%), liver (n = 186, 38.8%), heart (n = 5, 0.1%), lung (n = 18, 3.8%), pancreas (n = 9, 1.9%), and small intestine (n = 1, 0.02%). The most common malignancies were post-transplant lymphoproliferative disorder (n = 87) and cancers of the kidney (n = 43), stomach (n = 41), large intestine (n = 41), and lung (n = 36)., Conclusions: This is the first national survey of the incidence of de novo malignancy in Japan. Further study is required to identify the risk of de novo malignancy in organ transplant recipients in comparison to the general population, namely the standardized incidence ratio.

Objective: Uterus transplantation (UTx) is a potential option for women with uterine factor infertility to have a child, but there has been no large-scale survey of the views on UTx in women of reproductive age in Japan. The present study was aimed to clarify the views of Japanese women of reproductive age on UTx for uterine factor infertility., Methods: A questionnaire on UTx was conducted by an Internet research company in December 2014 as a cross-sectional study in 3,892 randomly chosen women aged 25 to 39 years old. Responses were analyzed from 3,098 subjects (mean age 32.1±4.2 years old), after exclusion of inappropriate respondents in screening., Results: Of the respondents, 62.1%, 34.7% and 18.1% favored adoption, UTx and gestational surrogacy, respectively. In contrast, 7.0%, 21.9% and 63.3% opposed adoption, UTx and gestational surrogacy, respectively. In choices of candidates for UTx based on highest priority, deceased persons (33.8%) and mothers (19.0%) were favored as donors, and women with congenital absence of the uterus (54.4%) and hysterectomy due to a malignant uterine tumor (20.0%) as recipients. Regarding societal acceptance of UTx, the answer rates were 15.7% for "UTx should be permitted", 77.6% for "UTx should be permitted with discussion", and 6.7% for "UTx should not be permitted, even with discussion". Regarding personal opinions on UTx, 44.2% were in favor, 47.5% had no opinion, and 8.3% were against., Conclusion: Our results suggest that many Japanese women of reproductive age feel that UTx is socially and individually acceptable, but that concerns requiring further discussion remain among these women. There was also a tendency for UTx to be viewed more favorably than gestational surrogacy.

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.

Sulfur mustard (SM) causes blisters in the skin through a series of cellular changes that we are beginning to identify. We earlier demonstrated that SM toxicity is the result of induction of both death receptor and mitochondrial pathways of apoptosis in human keratinocytes (KC). Because of its importance in apoptosis in the skin, we tested whether calmodulin (CaM) mediates the mitochondrial apoptotic pathway induced by SM. Of the three human CaM genes, the predominant form expressed in KC was CaM1. RT-PCR and immunoblot analysis revealed upregulation of CaM expression following SM treatment. To delineate the potential role of CaM1 in the regulation of SM-induced apoptosis, retroviral vectors expressing CaM1 RNA in the antisense (AS) orientation were used to transduce and derive stable CaM1 AS cells, which were then exposed to SM and subjected to immunoblot analysis for expression of apoptotic markers. Proteolytic activation of executioner caspases-3, -6, -7, and the upstream caspase-9, as well as caspase-mediated PARP cleavage were markedly inhibited by CaM1 AS expression. CaM1 AS depletion attenuated SM-induced, but not Fas-induced, proteolytic processing and activation of caspase-3. Whereas control KC exhibited a marked increase in apoptotic nuclear fragmentation after SM, CaM1 AS cells exhibited normal nuclear morphology up to 48h after SM, indicating that suppression of apoptosis in CaM1 AS cells increases survival and does not shift to a necrotic death. CaM has been shown to activate the phosphatase calcineurin, which can induce apoptosis by Bad dephosphorylation. Interestingly, whereas SM-treated CaM1-depleted KC expressed the phosphorylated non-apoptotic sequestered form of Bad, Bad was present in the hypophosphorylated apoptotic form in SM-exposed control KC. To determine if pharmacological CaM inhibitors could attenuate SM-induced apoptosis via Bad dephosphorylation, KC were pretreated with the CaM-specific antagonist W-13 or its less active structural analogue W-12. Following SM exposure, KC exhibited Bad dephosphorylation, which was inhibited in the presence of W-13, but not with W-12. Consequently, W-13 but not W-12 markedly suppressed SM-induced proteolytic processing and activation of caspase-3, as well as apoptotic nuclear fragmentation. Finally, while the CaM antagonist W-13 and the calcineurin inhibitor cyclosporin A attenuated SM-induced caspase-3 activation, inhibitors for CaM-dependent protein kinase II (KN62 and KN93) did not. These results indicate that CaM, calcineurin, and Bad also play a role in SM-induced apoptosis, and may therefore be targets for therapeutic intervention to reduce SM injury.

HPV16 DNA is often integrated in cancers, disrupting the E1 or E2 genes. E2 can repress the E6/E7 promoter, but other models have been proposed to explain why integration promotes malignant progression. E1 and E2 are required for viral replication, and so genetic analysis of their role in transcriptional regulation is complex. Therefore, we developed an extrachromosomal vector containing HPV16 to undertake a genetic analysis of the E1 and E2 genes. We demonstrate that the E2 protein is primarily a transcriptional repressor when expressed from the virus. Furthermore, repression requires both the transactivation function of E2 and specific binding of E2 to the LCR. We find no evidence that the E1 protein directly modulates HPV16 gene expression. However, certain E1 mutations modulated transcription indirectly by altering splicing of E2 mRNA species. These data provide important insight into which E1 and E2 functions are optimal targets for anti-viral therapies.

Transplantation in Japan, as well as the environment in which nurses work, has been slowly changed. Consequently, those nurses started working as recipient coordinators to meet the needs of patients, physicians and nurses who work at the patients' bedside. In the future, recipient coordinators can be a valuable asset to each transplant program because of their education, commitment to personalized care to recipients and their families. This article describes a variety of roles of recipient coordinators in Japan.

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., (Copyright 2002 S. Karger AG, Basel)

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 approximately 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-beta) of the mouseKif1B-beta gene in this region. The KIF1B-beta 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-beta gene in 23 cell lines using 46 sets of primers and also an allelic imbalance (AI) analysis of KIF1B-beta 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-beta 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-beta gene revealed obvious expression in all NB cell lines except NB-1, although decreased expression of the KIF1B-beta gene was found in a subset of early- and advanced-stage NBs. These results suggest that the KIF1B-beta gene may not be a candidate for tumor suppressor gene of NB.

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., (Copyright 2001 Wiley-Liss, Inc.)

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., (Copyright 2001 Academic Press.)

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.

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 1p36, 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.

Background: We 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., Procedure: The 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., Results: Currently, 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., Conclusions: Full 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., (Copyright 2000 Wiley-Liss, Inc.)

Homeodomain transcription factors play important roles in directing cellular proliferation and differentiation. A TALE-superclass homeodomain protein, multifunctional repressor of TGFbeta-induced transcription. Here we report identification of TGIF2, a novel TALE-superclass homeodomain protein that shows distinct homology with TGIF, especially in its DNA-binding domain. TGIF2 is expressed ubiquitously in human tissues, with the highest levels being found in heart, kidney, and testis. The TGIF2 product contains a putative nuclear localization signal; translocation of the protein to the nucleus was confirmed by transfection of epitope-tagged cDNA. TGIF2 lies on chromosome 20q11.2-12. Since amplification of 20q is often observed among ovarian cancers, we determined the status of DNA copy-number and expression of TGIF2 in 14 ovarian-cancer cell lines. This gene was over-expressed in all lines that showed amplification by FISH analysis. The results suggested that TGIF2 may play an important role in the development and/or progression of some ovarian tumors through a mechanism of gene amplification., (Copyright 2000 Academic Press.)

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 3(1/2) years (HDNB1/UFD2, KIAA0591F/KIF1B-beta, 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-beta, 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. Oncogene (2000) 19, 4302 - 4307

Deletion of chromosome 10 is one of the most common chromosomal alterations in glioma. At 10p15, the telomeric region of the short arm of chromosome 10, loss of heterozygosity (LOH) has been frequently observed by microsatellite analysis, suggesting the presence of a tumor suppressor gene. We examined LOH in 34 gliomas on chromosome 10, and frequent LOH on 10p was detected on 10p15, in agreement with deletion mapping studies on chromosome 10. We then constructed a bacterial artificial chromosome (BAC) clone contig covering the critical region, which spanned the interval between D10S249 and D10S533 on 10p15. The map contained 68 BAC clones connected by 74 sequenced tag sites (STSs) and covered approximately 2.7 Mb, with one gap. A total of 74 STSs, including 6 microsatellite markers, 29 expressed sequenced tags (ESTs), and 39 BAC end STSs, were physically arranged. Twenty-eight ESTs were mapped in the interval between D10S249 and D10S559 (approximately 1200 kb), and another EST was mapped in the interval between D10S559 and D10S533 (approximately 1300 kb). This sequence-ready BAC clone contig map will be a basic resource for high-quality sequencing and positional cloning of the putative tumor suppressor gene at 10p15 in glioma., (Copyright 2000 Academic Press.)

MEST/PEG1, a gene expressed paternally in mesodermal derivatives in early embryonic stages, is the first imprinted gene mapped to chromosome 7q32. Since imprinted genes are clustered in general at a chromosomal region, we speculated that a similar imprinted-gene cluster may exist at chromosome region 7q32 and that the functions of some such genes may contribute to the phenotype of Silver-Russell syndrome including maternal uniparental disomy for chromosome 7 (maternal UPD7). As an initial step toward the isolation of imprinted genes at 7q32, we adopted an integrated approach involving the construction of a PAC contig and ESTs mapping in the vicinity of MEST. Here, we have constructed a complete contig of PAC and BAC clones and a transcript map spanning the entire approximately 1-Mb region between D7S530 and D7S649. We developed 60 novel STSs and precisely mapped 47 genes/ESTs. This map contains a putative autistic disorder locus that has been suggested to be localized near markers D7S530 and D7S684. This integrated physical and transcript map provides a valuable resource for identification of an imprinted gene(s) in this region as well as a candidate gene(s) for autistic disorder., (Copyright 2000 Academic Press.)

Human chromosome 11q23.2 has been proposed to contain a tumor suppressor gene(s) whose deletion has been associated with cancer of the lung and breast and with neuroblastoma. To analyze the genomic structure and to isolate a candidate tumor suppressor gene from this region, we constructed a 2-Mb sequence-ready contig map using bacteriophage P1 (P1), bacterial artificial chromosome (BAC), and P1-derived artificial chromosome (PAC). The map comprises a contig of 24 overlapping P1, BAC, and PAC clones. To isolate gene fragments from the region, we performed direct cDNA library screening, exon trapping, EST mapping, and genomic sequencing using the P1, BAC, and PAC clones. Sequence analysis of 5 clones, which spans 23% (458,738 bp) of the region, and extensive gene scanning along the entire region revealed that the region is extraordinarily scarce in genes, but we identified one ubiquitously expressed novel gene and one testis-specific gene fragment. The novel gene, which we call IGSF4 (immunoglobulin superfamily 4), is transcribed into a 1.6- or 4.4-kb RNA encoding a 442-amino-acid protein. It shares strong homology with mouse IGSF-B12 and cell adhesion molecules NCAM1 and NCAM2 within their Ig-like C2-type domains. The IGSF4 gene, a novel gene that is shown to be located in the common loss of heterozygosity region, possesses a number of interesting features and may be good candidate for a tumor suppressor gene., (Copyright 1999 Academic Press.)

The intensely studied MHC has become the paradigm for understanding the architectural evolution of vertebrate multigene families. The 4-Mb human MHC (also known as the HLA complex) encodes genes critically involved in the immune response, graft rejection, and disease susceptibility. Here we report the continuous 1,796,938-bp genomic sequence of the HLA class I region, linking genes between MICB and HLA-F. A total of 127 genes or potentially coding sequences were recognized within the analyzed sequence, establishing a high gene density of one per every 14.1 kb. The identification of 758 microsatellite provides tools for high-resolution mapping of HLA class I-associated disease genes. Most importantly, we establish that the repeated duplication and subsequent diversification of a minimal building block, MIC-HCGIX-3.8-1-P5-HCGIV-HLA class I-HCGII, engendered the present-day MHC. That the currently nonessential HLA-F and MICE genes have acted as progenitors to today's immune-competent HLA-ABC and MICA/B genes provides experimental evidence for evolution by "birth and death," which has general relevance to our understanding of the evolutionary forces driving vertebrate multigene families.

From a human fetal brain cDNA library, we isolated two transcripts (ZIS-1 and ZIS-2) corresponding to the human ZIS gene, an ortholog of the rat Zis (zinc finger, splicing). A comparison of base sequences of the cDNA and its corresponding genomic DNA (a P1-derived artificial chromosome clone) revealed that both transcripts have an ORF of 1011bp and encodes 337 amino acids, but ZIS-1 has 10 exons and ZIS-2 contains 11 exons. Although both transcripts share the first nine exons, exon 10 of ZIS-2 is lacking in ZIS-1, and instead, exon 11 (10th exon) of ZIS-1 is larger in size, leading to the longer 3'-UTR. Thus, the two transcripts result from differential splicing. A Northern blot analysis on various adult and fetal tissues revealed that 5.2- and 3.2-kb transcripts were ubiquitously expressed, and 3.9- and 1.9-kb transcripts were highly expressed in the fetal brain and kidney, respectively. There were several other transcripts that may be alternatively processed forms of the human ZIS. Considering the ZIS gene size, the 3.2-kb transcripts most likely corresponds to ZIS-1 and may act as a major transcript of ZIS. The human ZIS has a high homology to the rat Zis for the coding DNA sequence with 91% identity and for the amino acid sequence with 87% identity. ZIS and Zis contain the same numbers of exons and introns. Both genes have unusually long 3'-UTR, and their encoding proteins contain similar components, i.e. a zinc finger domain, a nuclear localization signal, an Asp-Glu region, and a Ser-Arg-rich region. Furthermore, the expression patterns of the two genes in tissues are similar each other. Thus, the human ZIS may act as a transcriptional factor to regulate transcription and/or splicing, as does the rat Zis.

In human endometrial cancer, we have previously identified a 790-kb region of common allelic loss in chromosome bands 10q25-q26, flanked by D10S587 and D10S1723. We constructed a contig covering the entire deleted region using YACs, PACs, and BACs. Five overlapping cosmid clones derived from YAC clones completely covered the entire deleted region: its size was estimated to be no larger than 200 kb. We further performed two-color fluorescence in situ hybridization (FISH) analysis to confirm the deletion and narrowed down the deleted region to 100 kb or less; it was covered by three overlapping cosmid clones that were included in one BAC clone. Restriction endonuclease mapping identified a region in which NotI, SalI, SmaI, and Xhol were clustered, suggesting the possible existence of a CpG island.

To find the genes contributing to Down syndrome, we constructed a 4-Mb sequence-ready map spanning chromosome 21q22.2 with megabase-sized cosmid/P1-derived artificial chromosome (PAC) contigs. The restriction map with rare cutting enzymes, followed by sequencing from the clustering sites, has defined CpG islands and revealed the genes associated with CpG islands (Accession No. D85771). Of these, two human carbonyl reductases (CBR; EC1.1.1.184) were found in a PAC 25P16 clone. CBR catalyzes the reduction of a large number of biologically and pharmacologically active carbonyl compounds to their corresponding alcohols and has been mapped in 21q22.1. To confirm these results, we sequenced the PAC clone in shotgun strategies and identified a novel carbonyl reductase, designated CBR3, 62 kb downstream from the original CBR. In addition, three ribosomal pseudogenes, L23a, S9, and L3, and some cDNAs with ESTs were mapped in the sequence. In conclusion, the sequence analysis for CpG islands predicted from the megabase-sized contigs will reveal and identify the genes involved in Down syndrome., (Copyright 1998 Academic Press.)

We previously assigned a 14q13 breakpoint of t(2; 14) in a patient with mirror-image polydactyly to a segment between two loci, AFM200ZH4 and D14S306, within a genetic distance of 0.6 cM. In the present study, we constructed a 1.2-Mb high-resolution physical map with a contig composed of 16 bacterial artificial chromosomes (BACs) and 6 P1-derived artificial chromosomes (PACs) at a region around the breakpoint, extending from D14S75 to D14S728 loci. Thirty-four novel sequence-tagged sites (STSs) were also characterized at this region. Of nine ESTs that had been mapped between D14S75 and D14S288, T99065 was confirmed to be in two BAC clones, B102 and B319. This BAC/PAC contig with STSs is useful for further genomic sequencing, for construction of a transcription map, and for the isolation of the putative gene for mirror-image polydactyly.

Despite the presence of several human disease genes on chromosome 11q13, few of them have been molecularly cloned. Here, we report the construction of a contig map encompassing 11q13.1-q13.3 using bacteriophage P1 (P1), bacterial artificial chromosome (BAC), and P1-derived artificial chromosome (PAC). The contig map comprises 32 P1 clones, 27 BAC clones, 6 PAC clones, and 1 YAC clone and spans a 3-Mb region from D11S480 to D11S913. The map encompasses all the candidate loci of Bardet-Biedle syndrome type I (BBS1) and spinocerebellar ataxia type 5 (SCA5), one-third of the distal region for hereditary paraganglioma 2 (PGL2), and one-third of the central region for insulin-dependent diabetes mellitus 4 (IDDM4). In the process of map construction, 61 new sequence-tagged site (STS) markers were developed from the Not I linking clones and the termini of clone inserts. We have also mapped 30 ESTs on this map. This contig map will facilitate the isolation of polymorphic markers for a more refined analysis of the disease gene region and identification of candidate genes by direct cDNA selection, as well as prediction of gene function from sequence information of these bacterial clones.

To help in isolating the genes involved in Down syndrome, we sought CpG islands in 4 Mb cosmid/PAC contigs spanning most of the 21q.22.2 band using seven rare cutting enzymes. A striking feature was observed upstream of hSIM2 where at least 41 rare-cutting sites were clustered within a 20-kb region. To investigate the structure of the cluster, a cosmid containing hSIM2 was submitted to shotgun sequencing. Sequence analysis revealed that the cluster was a long CpG island extending 19, 128 nucleotides which includes in the first and second exons of hSIM2. Taken together with our observation in which the CpG islands were concentrated within 1.2 Mb around hSIM2, we propose that this region functions as an R-band, and the cluster provides a unique element for marking of DNA for the spatial and temporal expression of the hSIM2 locus.

The major phenotypic features of Down syndrome have been correlated with partial trisomies of chromosome 21, allowing us to define the candidate gene region to a 4-Mb segment on the 21q22.2 band. We present here a high-resolution physical map with megabase-sized cosmid/PAC contigs. This ordered clone library has provided unique material for the integration of a variety of mappable objects, including exons, cDNAs, restriction sites, etc. Furthermore, our results have exemplified a strategy for the completion of the chromosome 21 map to sequencing.

Transient abnormal myelopoiesis (TAM) is a leukemoid reaction occurring occasionally on Down syndrome (DS) newborn infants. It has been hypothesized that "disomic homozygosity" in 21-trisomic cells plays an important role in the genesis of TAM, and the putative TAM gene was suggested to be mapped at a 21q11 region. We encountered a DS-associated TAM infant with a 47,XY,inv(21)(q11.1q22.13),+inv(21)(q11.1q22.13) karyotype. On the basis of another presumption that in this patient the putative TAM gene is disrupted by the break, we tried to isolate a breakpoint DNA. FISH analysis with cosmid clones corresponding to various sequence-tagged-site (STS) markers mapped at around 21q11.1-q11.2, we confirmed that the proximal breakpoint of the inv(21) was located between two STSs, G51E07 and D21S215, the latter locus being consistent with the previous tentative mapping. After construction of a cosmid contig encompassing between the two markers, we have isolated a cosmid clone corresponding to the proximal breakpoint of the inversion. This breakpoint was located near a previously identified duplicated region that is homologous to the sequence at 21q22.1. The isolated cosmid clone is useful for analysis of other TAM patients and for a search for a transcript at or flanking the breakpoint.

We previously isolated the AML1 gene, which is rearranged by the t(8;21) translocation in acute myeloid leukemia. The AML1 gene is highly homologous to the Drosophila segmentation gene runt and the mouse transcription factor PEBP2 alpha subunit gene. This region of homology, called the Runt domain, is responsible for DNA-binding and protein--protein interaction. In this study, we isolated and characterized various forms of AML1 cDNAs which reflect a complex pattern of mRNA species. Analysis of these cDNAs has led to the identification of two distinct AML1 proteins, designated AML1b (453 amino acids) and AML1c (480 amino acids), which differ markedly from the previously reported AML1a (250 amino acids) with regard to their C-terminal regions, although all three contain the Runt domain. The large C-terminal region common to AML1b and AML1c is suggested to be a transcriptional activation domain. AML1c differs from AML1b by only 32 amino acids in the N-terminal. Characterization of the genomic structure revealed that the AML1 gene consists of nine exons and spans > 150 kb of genomic DNA. Northern blot analysis demonstrated the presence of six major transcripts, encoding AML1b or AML1c, which can all be explained by the existence of two promoters, alternative splicing and differential usage of three polyadenylation sites. A minor transcript encoding AML1a which results from alternative splicing of a separate exon can be detected only by reverse transcription-polymerase chain reaction amplification. The distinct proteins encoded by the AML1 gene may have different functions, which could contribute to regulating cell growth and/or differentiation through transcriptional regulation of a specific subset of target genes.

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.

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.

A cosmid library of 3 x 10(5) clones has been constructed from a human x hamster hybrid cell line, 153E9a3, which contains human chromosome 21 (HC21) as the only human chromosome. From 56,500 clones of this library, 229 HC21-specific cosmids have been isolated by their hybridization to total human DNA and by their failure to hybridize to total Chinese hamster DNA. The cosmids isolated were then characterized, of these, 28 cosmids (12.2% of those tested) contained Not1 site(s), and 41 cosmids were localized on the eight subregions of HC21 by differential hybridization with Alu-PCR products obtained from a hybrid mapping panel. The cosmids localized were further integrated into the existing contigs using the end-specific probes of the clone insert. Therefore, they provided useful anchor points for contig mapping and walking.

Chymotrypsinogen is widely present in various animal pancreases. To study evolutionary relationship of chymotrypsinogen gene in species, we used a cDNA probe of human prechymotrypsinogen to investigate the species distribution of chymotrypsinogen gene, and designed oligodeoxynucleotide primers to investigate the genomic organization in the three domains of active sites. The genomic analyses showed that chymotrypsinogen gene is evolutionary conserved in species. On the basis of the deduced amino acid residues, a three-dimensional model for human chymotrypsinogen was further built by computer graphics. The model showed high similarity to the X-ray crystal structure of bovine chymotrypsinogen A, thus, demonstrated that the three-dimensional structure is more conserved in evolution than protein sequences.

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 microtubule motor protein, kinesin, which might be involved in mitotic chromosome segregation, suggesting that the HSET gene encodes a novel kinesin-related protein.