Search

The genetic variations of Lycoris radiata var. radiata, a completely sterile triploid from Japan, were examined by comparing the nucleotide sequences of genomic DNA regions in 11 triploid strains sampled from Japan and four triploid strains sampled from China, and in two diploid strains of Lycoris radiata var. pumila, which is endemic to China and fertile. For this purpose, two genes were analyzed, the lectin gene in the nuclear genome and the maturase gene in the chloroplast genome. A clear genetic constancy was observed in their DNA nucleotide sequences. For both genes, completely identical nucleotide sequences were detected in the 11 Japanese and four Chinese triploid strains and also between the two Chinese diploid strains. However, some genetic variations were observed between the Japanese and Chinese triploid strains, and between the triploid and diploid strains. These results are consistent with the findings obtained from previous chromosome karyotype analyses and allozyme analyses. In addition, in our preliminary FISH analysis of the physical mapping of the rRNA gene family, the 18S-5.8S-26S rRNA and 5S rRNA loci were localized on six and four chromosomes, respectively. Regarding the 18S-5.8S-26S rRNA loci, two were associated with two SAT chromosomes. The remaining four were distinguished by having no secondary constriction. Localization of 5S rRNA loci to chromosome spreads revealed three sites on the proximal part of the long arm of three acrocentric chromosomes and one site on the distal part of the long arm of the SAT chromosome; the latter site was juxtaposed to the 18S-5.8S-26S rRNA loci. These findings indicate that L. radiata var. radiata is not a typical autotriploid. The present paper discusses the possible origin of L. radiata var. radiata from a diploid variety of L. radiata var. pumila, based on the molecular cytogenetic analysis and DNA sequence analysis.

The UTF1 is a transcriptional coactivator expressed mainly in pluripotent embryonic stem cells. Here, we have isolated a genomic DNA fragment carrying the UTF1 gene and found that the gene contains two exons interrupted by a short intron. The gene possesses four GC boxes, but no TATA box in the 5′-flanking region. This is reminiscent of a housekeeping gene type promoter and the functional relevance of these motifs is confirmed by the transient transfection analyses. As to the gene product, our analyses have led to the identification of two different species. One of them corresponds to the full-length protein, while the other is produced by utilizing the second methionine codon for the translation initiation. The oligo-capping analyses reveal multiple transcription start sites. Interestingly, some of them are localized downstream of the first methionine codon, indicating that such transcripts produce a protein starting from the second methionine codon. Chromosomal mapping analyses locate the gene at 7F5, the syntenic region of the human chromosome (10q26) where the human UTF1 gene is located.

A novel DNA-PKcs interacting protein, KIP (kinase interacting protein), was recently isolated using a two-hybrid analysis which showed a significant homology to calcineurin B. We found other ESTs showing significant similarity to KIP gene in the dbEST database and isolated a cDNA clone which encodes a 187 amino acid polypeptide from a human fetal brain cDNA library. This protein (termed KIP2 for kinase interacting protein 2) has sequence homology to KIP (46% identical and 64% similarity). RT-PCR analysis showed that the messenger RNA was ubiquitously expressed in various human tissues. Based on PCR-based analysis with a radiation hybrid cell panel and fluorescence in situ hybridization, the gene was localized to the q24 region of chromosome 15.

Huntington disease (HD) is an inherited neurodegenerative disorder which is associated with CAG expansion in the coding region of the gene for huntingtin protein. Recently, a huntingtin interacting protein, HIP1, was isolated by the yeast two-hybrid system. Here we report the isolation of a cDNA clone for HIP1R (huntingtin interacting protein-1 related), which encodes a predicted protein product sharing a striking homology with HIP1. RT-PCR analysis showed that the messenger RNA was ubiquitously expressed in various human tissues. Based on PCR-assisted analysis of a radiation hybrid panel and fluorescence in situ hybridization, HIP1R was localized to the q24 region of chromosome 12.

The regulator of G-protein signaling (RGS) proteins have recently been identified as signal transduction molecules which have structural homology to SST2 of Saccharomyces cerevisiae and EGL-10 of Caenorhabditis elegans. Multiple genes homologous to SST2 are present in higher eukaryotes, and the group of these genes is termed the RGS family. RGS proteins are involved in the regulation of heterotrimeric G-proteins by acting as GTPase-activators. A putative new member of the RGS family was isolated from a neuroblastoma cDNA library. The amino acid sequence deduced from the cDNA possessed all consensus motifs of the RGS domain and showed closest homology to mouse RGS5 (90% identical), indicating that it was human RGS5 (hRGS5). The messenger RNA of hRGS5 was abundantly expressed in heart, lung, skeletal muscle, and small intestine, and at low levels in brain, placenta, liver, colon, and leukocytes. The chromosome localization of the gene in the 1q23 region was determined by a monochromosomal hybrid panel and a radiation hybrid panel.

Progression through the eukaryotic cell cycle requires the activation of cyclin-dependent kinases (CDKs). The activities of these kinases are tightly regulated by both positive and negative factors in normal cells, and perturbation of CDK activities can result in tumorigenesis (Hunter and Pines 1994; Sherr 1996). Although the regulation of CDK activation has been studied intensively in recent years, the mechanisms by which these CDKs regulate cell cycle progression are not fully understood partly because few substrates of these kinases have been identified. Previous studies have shown that some protein kinases, including CDKs, form stable complexes with their respective substrates in vivo (Kato et al. 1993; Dynlacht et al. 1994, 1997; Krek et al. 1994; Karin and Hunter 1995; Zhu et al. 1995; Adams et al. 1996; Kallunki et al. 1996). This physical interaction has been suggested as a mechanism by which the specificity and efficiency of the kinase reactions are achieved. Thus, a possible approach for identifying substrates of a protein kinase is to search for proteins that interact physically with the kinase. We have developed a method to screen expression libraries for potential CDK substrates and identified a novel substrate of cyclin E–CDK2, a kinase complex that regulates the G1/S-phase transition and is required for the initiation of DNA replication in higher eukaryotes (Fang and Newport 1991; Ohtsubo and Roberts 1993; Tsai et al. 1993; Resnitzky et al. 1994).

Hereditary multiple exostoses (EXT) is an autosomal dominant disorder that is characterized by the appearance of multiple outgrowths of the long bones (exostoses) at their epiphyses. Genetical heterogeneities have segregated at least on chromosome 8, 11, and 19 and been designated EXT1, EXT2, and EXT3, respectively. Recently, the responsible genes for EXT1 and EXT2 have been isolated and appeared to define a structurally related gene family. In the present study, we have identified novel genes which share significant sequence homologies with the EXT genes. The predicted protein products of the novel EXT-related genes, EXTR1 and EXTR2 (forEXT-related genes 1 and 2), consist of 919 and 330 amino acid residues, respectively. These genes were transcribed ubiquitously in various tissues. Based on PCR-assisted analyses of both a human/rodent mono-chromosomal hybrid cell panel and a radiation hybrid mapping panel, EXTR1 was localized to the chromosome 8p21 region, where loss of heterozygosity has been frequently observed in various tumors, and EXTR2 was assigned to the chromosome 1p21 region, where osteopetrosis, a dominant hereditary disease of bone, has been mapped by genetic linkage analysis, implying that the protein products of these two EXT-related genes, as well as of the EXT genes, have potential tumor suppressor activity.

Placental protein 5 (PP5)/tissue factor pathway inhibitor-2 (TFPI-2) is a new homologue of TFPI, which contains three tandemly repeated Kunitz-type proteinase inhibitory (KPI) domains and potently inhibits the extrinsic blood coagulation cascade. In this study, mouse PP5/TFPI-2 cDNA was cloned using a human PP5/TFPI2 cDNA fragment as a probe. The characteristic three KPI domains with short spacer sequences and a basic amino acid stretch in the carboxyl-terminal region present in human PP5/TFPI-2 were well conserved in mouse PP5/TFPI-2. In general, the P1 reactive site residues of active KPI domains are basic amino acids. However, the putative P1 residues of the first, second, and third KPI domains were glutamine, aspartic acid, and serine, respectively. Mouse PP5/TFPI-2 mRNA was highly expressed in developing placenta as in humans. Adult liver and kidney also contained a significant amount of its transcripts. The mouse PP5/TFPI-2 gene was found to be located in the R-positive A2 band by the direct R-banding FISH and identified at 2.7 cM proximal to D6Mit 1 by interspecific backcross analysis.

To assess the relative contributions of trans-acting factors (replication and repair functions) and cis-acting elements (repeat and flanking DNA composition) to the mechanism of trinucleotide repeat sequence mutation we have analysed the distribution of copy number polymorphisms at 12 loci associated with dynamic mutations in 15 populations of different ethnic origins. Genome wide instability of repeats in a particular population would be evidence of trans-acting factor instigation of the mutation process, whereas instability at a particular locus (perhaps even in several populations) would be evidence that the composition of the particular locus was the most significant factor contributing to mutation. The FRA16A locus is highly polymorphic in only the European population. Some other loci exhibit distinct distributions of alleles between different populations. Therefore sequences in the vicinity of the repeat -- the cis component of a particular locus -- appear(s) to be more important in the mutation mechanism than sporadic genome-wide instability induced by trans-acting factors such as the DNA mismatch repair enzymes.

Chromosomal locations of the Atm (ataxia-telangiectasia (AT)-mutated) and Acat1 (mitochondrial acetoacetyl-CoA thiolase) genes in mouse, rat, and Syrian hamster were determined by direct R-banding FISH. Both genes were colocalized to the C-D band of mouse chromosome 9, the proximal end of q24.1 of rat chromosome 8, and qa4-qa5 of Syrian hamster chromosome 12. The regions in the mouse and rat were homologous to human chromosome 11q. Fine genetic linkage mapping of the mouse AT region was performed using the interspecific backcross mice. Atm, Acat1, and Npat, which is a new gene isolated from the AT region, and 12 flanking microsatellite DNA markers were examined. No recombinations were found among the Atm, Npat, Acat1, and D9Mit6 loci, and these loci were mapped 2.0 cM distal to D9Mit99 and 1.3 cM proximal to D9Mit102. Comparison of the linkage map of mouse chromosome 9 (MMU9) and that of human chromosome 11 (HSA11) indicates that there is a chromosomal rearrangement due to an inversion between Ets1 and Atm-Npat-Acat1 and that the inversion of MMU9 originated from the chromosomal breakage at the boundary between Gria4 and Atm-Npat-Acat1 on HSA11. This type of inversion appeared to be conserved in the three rodent species, mouse, rat, and Syrian hamster, using additional comparative mapping data with the Rck gene.

GTP cyclohydrolase I is the first and rate-limiting enzyme for the biosynthesis of tetrahydrobiopterin in mammals. Previously, we reported three species of human GTP cyclohydrolase I cDNA in a human liver cDNA library (Togari, A., Ichinose, H., Matsumoto, S., Fujita, K., and Nagatsu, T. (1992) Biochem. Biophys. Res. Commun. 187, 359-365). Furthermore, very recently, we found that the GTP cyclohydrolase I gene is causative for hereditary progressive dystonia with marked diurnal fluctuation, also known as DOPA-responsive dystonia (Ichinose, H., Ohye, T., Takahashi, E., Seki, N., Hori, T., Segawa, M., Nomura, Y., Endo, K., Tanaka, H., Tsuji, S., Fujita, K., and Nagatsu, T. (1994) Nature Genetics 8, 236-242). To clarify the mechanisms that regulate transcription of the GTP cyclohydrolase I gene and to generate multiple species of mRNA, we isolated genomic DNA clones for the human and mouse GTP cyclohydrolase I genes. Structural analysis of the isolated clones revealed that the GTP cyclohydrolase I gene is encoded by a single copy gene and is composed of six exons spanning ∼30 kilobases. We sequenced all exon/intron boundaries of the human and mouse genes. Structural analysis also demonstrated that the heterogeneity of GTP cyclohydrolase I mRNA is caused by an alternative usage of the splicing acceptor site at the sixth exon. The transcription start site of the mouse GTP cyclohydrolase I gene and the 5′-flanking sequences of the mouse and human genes were determined. We performed regional mapping of the mouse gene by fluorescence in situ hybridization, and the mouse GTP cyclohydrolase I gene was assigned to region C2-3 of mouse chromosome 14. We identified missense mutations in patients with GTP cyclohydrolase I deficiency and expressed mutated enzymes in Escherichia coli to confirm alterations in the enzyme activity.

A human chromosomal region 11q23-specific DNA library has been constructed by means of microdissection-microcloning method with polymerase chain reaction (PCR) technique (Seki et al., Genomics 16: 1993). DNA sequences were determined for 25 microclones that contained approximately 300-500 bp insert and gave a unique (single copy) signal in Southern blot analysis. The sequence tagged site (STS) was designed and appropriate condition for PCR was determined for each unique microclone. Twelve STSs were established and used for PCR-screening of human genomic libraries constructed with yeast artificial chromosome (YAC). Thirteen YAC clones have been isolated from eight STSs. These chromosomal region-specific STSs and YAC clones will be useful in the positional cloning of disease-related genes localized to the q23 region of chromosome 11.

Interferon regulatory factor 1 (IRF-1) and IRF-2 are structurally similar DNA-binding factors which were originally identified as regulators of the type I interferon (IFN) system; the former functions as a transcriptional activator, and the latter represses IRF-1 function by competing for the same cis elements. More recent studies have revealed new roles of the two factors in the regulation of cell growth; IRF-1 and IRF-2 manifest antioncogenic and oncogenic activities, respectively. In this study, we determined the structures and chromosomal locations of the human IRF-1 and IRF-2 genes and further characterized the promoters of the respective genes. Comparison of exon-intron organization of the two genes revealed a common evolutionary structure, notably within the exons encoding the N-terminal portions of the two factors. We confirmed the chromosomal mapping of the human IRF-1 gene to 5q31.1 and newly assigned the IRF-2 gene to 4q35.1, using fluorescence in situ hybridization. The 5{prime} regulatory regions of both genes contain highly GC-rich sequences and consensus binding sequences for several known transcription factors, including NF-{kappa}B. Interestingly, one IRF binding site was found within the IRF-2 promoter, and expression of the IRF-2 gene was affected by both transient and stable IRF-1 expression. In addition, one potential IFN-{gamma}-activatedmore » sequence was found within the IRF-1 promoter. Thus, these results may shed light on the complex gene network involved in regulation of the IFN system. 61 refs., 7 figs.« less

Fragile X syndrome is the most common familial form of mental retardation and known to be associated with the fragile site at Xq27.3 (FRAXA). The syndrome has recently been characterized by a unique genetic mechanism which involves dynamic mutation due to a heritable unstable DNA sequence, p(CCG)n repeat, in the FRAXA locus. We were asked to make a genetic diagnosis on the case of a normal male who has two brothers and a maternal uncle with mental retardation. We performed the pedigree analysis of the fragile X syndrome using both cytogenetic and molecular techniques. The affected two brothers and the uncle showed cytogenetic expression of the fra (X)(q27.3) and carried hypermethylated full mutation in the FRAXA locus. The phenotypically normal mother also exhibited fragile X expression and was found to be a carrier of premutation. Via female transmission, the premutation converted to full mutation and exhibited somatic heterogeneity and hypermethylation. However, both cytogenetic and molecular data did not show any evidence of fragile X mutation in the normal male client and, thus, excluded the possibility of his being a carrier.

A human genomic DNA library was constructed by using a microdissection-microcloning procedure with polymerase chain reaction (PCR) techniques on DNA from the chromosome 11q23 region. A total of 450 recombinant pUC clones were isolated from the library. Their insert sizes ranged from 150 to 850 bp with a mean of 320 bp. Fifty pUC clones were randomly selected and analyzed in detail. Southern blot analyses showed that 21 (42%) clones were unique DNA sequences, 20 (40%) clones were repetitive sequences, and 9 (18%) clones had no detectable hybridization. The unique sequences were used further in a secondary screening of a partially digested human genomic DNA library constructed in phage vector, and 4 clones were isolated. The chromosomal locations of these phage clones were confirmed to be in the q23 region of chromosome 11 by fluorescence in situ suppression hybridization. These pUC microclones isolated from the chromosomal region-specific genomic DNA library will be useful in the construction of physical contig maps with yeast artificial chromosome and/or cosmid clones and in the positional cloning of disease-associated genes localized to the q23 region of chromosome 11.

The fragile X syndrome is a common familial form of mental retardation and is associated with a rare fragile site at Xq27.3 (FRAXA). This disorder has recently been reported to correlate with length variations of restriction genomic DNA fragments which may due to the amplification of (CCG)n trinucleotide repeats located at the FRAXA locus. We described here a rapid preparation method of diagnostic DNA probes for the fragile X syndrome by direct enzymatic amplification of human chromosomal DNA. The PstI-assay, which is Southern blot analysis of DNA samples probed by PCR products, was shown to be sensitive method for diagnostic purposes to detect the size variations specific in the fragile X syndrome.

We examined the expression of a fragile site-like lesion and induction of sister chromatid exchanges (SCEs) at 1 lq23.1 in EBV-transformed lymphoblastoid cell lines derived from carriers of distamycin A-inducible fragile sites and ataxia telangiectasia patients. The fragile site-like lesion at llq23.1 was found to be BrdU-enhanceable in all cell lines examined, and the expression frequencies increased linearly with the rates of BrdU substitution in replicated DNA. In addition, an increased frequency of SCEs was observed at 1 lq23.1 on the expressed chromosome. Thus, the BrdU-enhanceable fragile site-like lesion at 1 lq23.1 is a “hot spot” for the formation of SCEs, as has been reported for other rare and common fragile sites.

RNF16 (ring finger protein 16; alias terf), a member of the RING finger family, has been shown to be exclusively expressed in the testis. Human RNF16 is located at 1q42 based on PCR-assisted analysis of both a human/rodent mono-chromosomal hybrid cell panel and a radiation hybrid-mapping panel. On the other hand, chromosomal mapping of the RNF16 gene by fluorescence in situ hybridization reveals that mouse Rnf16 is located at 11B1.2–B1.3 and rat Rnf16 at 10q22. These results provide additional evidence that the mouse 11B region displays conserved linkage homology with the rat 10q22 region, whereas in the case of RNF16, this homology is only conserved among rodents, distinct from the 1q42 region of the human genome.

We have isolated and mapped 75 new DNA markers including 52 restriction fragment length polymorphism (RFLP) markers on human chromosome 3. Clones were mapped by nonisotopic in situ hybridization, in which discrete fluorescent signals can be detected on prometaphase R-banded chromosomes. Thirty-seven markers were mapped to each arm of chromosome 3, and one was localized to the centromere. Five markers defined variable number of tandem repeat (VNTR) loci. Although the 75 clones were scattered throughout the chromosome, they were concentrated in the R-positive bands. This physical map of chromosome 3 will contribute to the characterization of the chromosomal and molecular aberrations involved in renal cell carcinoma, small-cell lung cancer, and other malignancies and in single-gene disorders such as von Hippel-Lindau disease and autosomal dominant retinitis pigmentosa.

A population survey of a common folate-sensitive fragile site, fra(3)(p14), was carried out on PHA-stimulated peripheral lymphocytes of 1,078 healthy subjects. Fra(3)(p14) was expressed more frequently in the younger than in the older, and in males than in females. It also showed some seasonal variation. The age difference of the expression frequency was also observed by aphidicolin treatment. A positive correlation of the expression frequency was found, though not so strongly, between both culture conditions of folate deprivation and aphidicolin treatment, These findings suggest that the inter-individual variation in the expressivity of common fragile sites is not only ascribable to chance, but to some physiological conditions of blood donors such as response rate of lymphocyte to PHA stimulation and blood concentration of folic acid.

The recently discovered regulators of G-protein signaling proteins, termed the RGS family, have been shown to modulate the functioning of G-proteins by activating the intrinsic guanosine triphosphatase (GTPase) activity of the alpha subunits. Here, we report the chromosomal location and tissue expression of the human regulator of RGS6 gene. The messenger RNA was ubiquitously expressed in various tissues. Polymerase chain reaction (PCR)-based analysis with a human/rodent monochromosomal hybrid panel and a radiation hybrid panel indicated that the gene was mapped between genetic markers WI-5202 and D14S277 on chromosome 14q24.3 region.

A cDNA for a putative member of the serine/threonine kinase family was cloned from an adult human testis cDNA library. The predicted translation product was identical to ZIP kinase, which has been suggested to play an important role in the induction of apoptosis. The messenger RNA was ubiquitously expressed in various tissues. The chromosomal location of the gene was determined by fluorescence in-situ hybridization and polymerase chain reaction-based analyses with both a human/rodent monochromosomal hybrid cell panel and a radiation hybrid mapping panel. This gene was mapped on the q13.3 region of chromosome 19.

The genus Lycoris of Amaryllidaceae comprises approximately 20 species that are distributed only in the moist warm temperate woodlands of eastern Asia. The objectives of this study were: (1) to clarify the phylogeny of the Lycoris species by using the definitive DNA sequencing method and (2) to examine the possible maternal donor of the hybrid origin Lycoris species and the Japanese triploid strains of Lycoris radiata var. radiata. The nucleotide sequence of the maturase K (matK) gene and the noncoding intergenic spacer (IGS) between the atpB and rbcL genes in the chloroplast genome were determined in a total of 27 strains of 11 species of the genus Lycoris. Variation among taxa was mainly due to nucleotide substitution, although deletions and an insertion were found in the IGS. For two chloroplast regions, the phylogenetic trees showed essentially similar topology, indicating the existence of four clades, I, II, III, and IV. For all the species except L. radiata, intraspecific variation was smaller than interspecific variation. For L. radiata, triploid strains were divided into clades I and II, and diploid strains were divided into clades I and IV. This implies that the diploid species of L. radiata var. pumila is a probable ancestral species. The clustering indicated that the chloroplast genome has not evolved in parallel with the karyotype in genus Lycoris. Regarding the hybrid origin species, the maternal parents of L. squamigara, L. albiflora and L. rosea were revealed to be L. longituba, L. radiata and L. radiata var pumila, respectively. We also suggest that a diploid strain of L. radiata var. pumila in clade I might be a candidate of the maternal donor of the Japanese triploid strains. A possible model of the maternal donor of Lycoris species is proposed.

From a newly determined cDNA sequence of the human ERK gene, a highly hydrophobic portion was identified upstream of the putative tyrosine kinase domain. This is the first evidence that the ERK protein possesses a receptor-like membrane-spanning structure. Fluorescence in situ hybridization analysis of R-banded metaphase chromosomes revealed that the ERK gene is located in chromosome region 1p36.1. This locus is near the frequent translocation breakpoint or deletion region of neuroblastoma and some other cancers. A comparative mapping study of the mouse and rat homologues indicated that each counterpart maps to the mouse chromosome 4D2.2-D3 and rat chromosome 5q36.13 regions, both of which have conserved linkage homology to human chromosome 1p.

The distal region of a short arm of chromosome 1p is frequently deleted in many human cancers including neuroblastoma (NBL), in which it has been narrowed down to the smallest region of overlap between D1S244 and D1S214 (approximately 7 cM). During the search for the candidate tumor suppressor genes mapped within the region, we found the KIAA0591 gene which encoded a new human kinesin-related protein with a homology to human axonal transporter of synaptic vesicles (ATSV). The kinesin is an intracellular motor protein and often associated with neuronal differentiation and survival. Here we identified a complete open reading frame of the KIAA0591 gene by screening a cDNA library derived from human substantia nigra. The KIAA0591 protein contains a possible pleckstrin homology (PH) domain at its carboxy-terminus. However, it did not possess a force-generating motor domain which is well conserved among kinesin superfamily members (KIFs). Northern blot analysis demonstrated that KIAA0591 mRNA was preferentially expressed in both adult and fetal brains, kidney, skeletal muscle and pancreas. KIAA0591 was expressed in favorable NBLs at higher levels than in unfavorable NBLs, although RT-PCR SSCP analysis showed no mutation within the coding region of the KIAA0591 gene, when 8 neuroblastoma tissues and 15 neuroblastoma-derived cell lines were examined. Thus, the full-length KIAA0591 gene may be a novel member of human KIF superfamily which lacks motor domain and might function as a tumor suppressor in an epigenetic but not a classic Knudson's manner.

Here, we report on the sequence features and chromosomal location of a novel human gene which shares significant homology with the bovine brain-specific protein p25. Based on polymerase chain reaction analysis with a human/rodent monochromosomal hybrid cell panel and a radiation hybrid panel, the gene was mapped on to p15.3 region of chromosome 5.

Here, we report on the chromosomal location of the human apoptosis-associated tyrosine kinase gene. Based on polymerase chain reaction analysis with a human/rodent monochromosomal hybrid cell panel and fluorescence in situ hybridization, the gene was mapped on 25.3 region of chromosome 17.

We identified a novel gene encoding a RING finger (C3HC4-type zinc finger) protein from a human neuroblastoma full-length enriched cDNA library. This cDNA clone consists of 1919 nucleotides with an open reading frame of a 485-amino acid protein. From reverse transcription (RT)-polymerase chain reaction (PCR) analysis, the messenger RNA was ubiquitously expressed in various human adult tissues. The chromosomal location of the gene was determined on the chromosome 6p21.3 region by PCR-based analyses with both a human/rodent monochromosomal hybrid cell panel and a radiation hybrid mapping panel.

We report the chromosomal location of the gene for DNA-PKcs interacting protein KIP. Based on fluorescence in situ hybridization and polymerase chain reaction (PCR)-based analyses with both a human/rodent monochromosomal hybrid cell panel and a radiation hybrid mapping panel, this gene was mapped to q25.3-q26.1 region on chromosome 15.

We have recently cloned a cDNA encoding an embryonic stem cell transcriptional coactivator termed UTF1 from the mouse F9 teratocarcinoma cell line (Okuda, A., Fukushima, A., Nishimoto, M., Orimo, A., Yamagishi, T., Nabeshima, Y., Kuro-o, M., Nabeshima, Y., Boon, K., Keaveney, M., Stunnenberg, H.G., and Muramatsu, M. (1998) EMBO J. 17, 2019–2032). Here we have cloned a cDNA for human UTF1 and identified two highly conserved domains termed conserved domain (CD)1 and CD2. Human UTF1, like that of mouse, binds to ATF-2 and the mutagenesis analyses reveal that the leucine zipper motif within the CD2 of the UTF1 and metal binding motif of ATF-2 are involved in this interaction. The factor also binds to TATA-binding protein containing complex. By means of immunoprecipitation analysis, we mapped two domains which are independently able to bind to the complex. Importantly, both domains are located within the conserved domains (one in CD1 and the other in CD2). Furthermore, transient transfection analyses point out the importance of these domains for activating ATF-2. Thus, these results suggest that these two conserved domains identified here play important roles in activating specific transcription at least in part by supporting physical interaction between the upstream factor, ATF-2, and basal transcription machinery.

ATM has been identified as a gene that is responsible for ataxia telangiectasia (AT), a pleiotropic disorder of autosomal recessive inheritance. While many mutations of this gene in AT patients of various ethnicities have been reported, data on Japanese patients are scarce. In this report, we present the results of a thorough survey of ATM mutations in 14 unrelated AT patients, with an emphasis on Japanese subjects. We used a hierarchical strategy in which we extensively analyzed the entire coding region of the cDNA. In the first stage, point mutations were sought by PCR-SSCP in short patches. In the second and third stages, the products of medium- and long-patch PCR, each covering the entire region, were examined by agarose gel electrophoresis to search for length changes. We found a total of 15 mutations (including 12 new) and 4 polymorphisms. Abnormal splicing of ATM was frequent among Japanese, and no hotspot was obvious, suggesting no strong founder effects in this ethnic group. Eleven patients carried either one homozygous or two compound heterozygous mutations, one patient carried only one detectable heterozygous mutation, and no mutation was found in two patients. Overall, mutations were found in at least 75% of the different ATM alleles examined. Possible reasons for the inability to detect mutations in some patients are discussed. Hum Mutat 12:186–195, 1998. © 1998 Wiley-Liss, Inc.

The rare fragile site is a specific point on a chromosome that is expressed as an isochromatid gap or break under certain conditions of cell culture and is inherited in a Mendelian codominant fashion. Five folate-sensitive fragile sites were cloned, and the molecular basis of fragile site mutation was shown to be a new class of mutation, called dynamic mutation, resulting from an allelic expansion of (CCG)n repeats. The mechanism responsible for other types of rare fragile sites, i.e., distamycin A-inducible and BrdU-requiring, is unknown, although cytogenetic studies suggested that these fragile sites play a mechanistic role in breakage and recombination and may also be integration and modification sites of foreign viral DNA genomes. A distamycin A-inducible fragile site, FRA8E, is mapped to 8q24.1 in which various loci implicated in genomic instability are located. Here we identified a YAC clone spanning both FRA8E and the hereditary multiple exostosis (EXT1) gene, using fluorescence in situ hybridization (FISH) analysis of a yeast artificial chromosome (YAC) contig. By using P1 clones as probes, the FRA8E locus was further localized to a 400-kb region including the EXT1 gene. Furthermore, the integration and amplification site of human papillomovirus 16 DNA in the ASCC (argyrophil small cell carcinoma) cells were shown not to coincide with FRA8E, but to be involved in an extensively broad genomic region of 8q24.1, including the c-myc gene.

The chromosomal locations of the human TEP1 (telomerase protein component 1) and mouse Tep1 genes, which were originally named TLP1 (telomerase protein 1) or TP1 (telomerase-associated protein 1), were determined by direct R-banding FISH and a molecular linkage analysis with interspecific backcross mice. The human TEP1 and mouse Tep1 genes were mapped by FISH to human chromosome 14q11.2 and to the C2-D1 band of mouse chromosome 14, respectively. By means of genetic linkage mapping, the mouse gene was further localized as being 2.7 cM distal toD14Mit18andD14Mit134and 2.0 cM proximal toD14Mit5on mouse chromosome 14, where conserved linkage homology with human chromosome 14q11–q12 has been identified.

A cDNA for a putative new member for phosphatidylinositol kinase family was cloned from an adult human whole brain cDNA library. The predicted translation product was composed of 961 amino acid residues and contained a sequence feature characteristic for lipid/protein kinases. The messenger RNA was ubiquitously expressed in various tissues, while relatively higher expression was observed in heart, skeletal muscle and testis. The chromosomal location of the gene was determined by fluorescence in situ hybridization and PCR-based analyses with both a human/rodent monochromosomal hybrid cell panel and a radiation hybrid mapping panel.

Ataxia telangiectasia (AT) is an autosomal recessive gene disorder, and ATM, a housekeeping gene, has been identified as the gene responsible for AT. Recently we found that another housekeeping gene, NPAT, is located upstream of ATM on human chromosome 11. The two housekeeping genes are transcribed in opposite directions and share a 0.5-kb 5' flanking sequence. The structure and organization of NPAT were determined by direct sequencing of cosmid clones carrying the gene and by application of the long and accurate (LA)-PCR method to amplify regions encompassing the exon/intron boundaries and all of the exons. The gene spans at least 44 kb and consists of 18 exons and 17 introns. It has been suggested that AT heterozygotes have an increased risk of developing cancer, especially breast cancer in women. Frequently, loss of heterozygosity at loci on 11q22-q24 has been observed in DNA isolated from tumors of the breast, uterine cervix, and colon, perhaps suggesting the location of a tumor suppressor gene in 11q22-q24. For investigation of the role of NPAT in AT and these tumors with allelic loss of 11q22-q24, appropriate primer sequences and PCR conditions for amplification of all the NPAT exons from genomic DNA were determined. We previously reported that no recombinations are found among Atm, Npat, and Acat1 (acetoacetyl-CoA thiolase) loci as determined by fine genetic linkage mapping of the mouse AT region. The results of the LA-PCR analysis using NPAT- and ACAT-specific primers and human genomic DNA allowed us to map ACAT 12 kb centromeric to NPAT.

Ataxia telangiectasia [AT} is an autosomal recessive disease of unknown etiology associated with cerebellar ataxia, oculocutaneous telangiectasia, immunodeficiency, and hypersensitivity to ionizing radiation. Although AT has been divided into four complementation groups by its radioresistant-DNA synthesis phenotype, the ATM gene has been isolated as the candidate gene responsible for all AT groups. We identified a new gene, designated NPAT, from the major AT locus on human chromosome Ilq22-q23. The gene encoded a 1421-amino-acid protein containing nuclear localization signals and phosphorylation target sites by cyclin-dependent protein kinases associated with E2F. The messenger RNA of NPAT was detected in all human tissues examined, and its genomic sequence was strongly conserved through eukaryotes, suggesting that the NPAT gene may be essential for cell maintenance and may be a member of the housekeeping genes. Analysis of the genomic region of NPAT surprisingly revealed that the gene existed only 0.5 kb apart from the 5' end of the ATM transcript with opposite transcriptional direction, it may be possible to propose the idea that the promoter region could be shared by both housekeeping genes and that each gene could influence the expression of the other. Ataxia telangiectasia (AT) is an autosomal recessive gene disorder defined by a wide spectrum of defects involving a severe neurological disorder attributable to progressive degeneration of the cerebellum, particularly the Purkinje cells, dila

The bfp, a member of the RING finger family, has been shown to be predominantly expressed in brain and up-regulated in neural differentiation of P19 embryonic carcinoma cells. Chromosome mapping of the bfp gene by fluorescence in situ hybridization reveals that human BFP (ZNF179) is located at 17p11.2, mouse Bfp at 11B1.3, and rat BFP at 10q22. These results provide additional evidence that the mouse 11B region displays conserved linkage homology with the 17p11.2 region of the human genome and the 10q22 region of the rate genome.

Ataxia telangiectasia (AT) is an autosomal recessive disease of unknown etiology associated with cerebel- lar ataxia, telangiectasia, immune dysfunction, higher cancer risk, genomic instability and hypersensitivity to ionizing radiation. The major AT loci, AT-A and AT-C, are shown to be closely linked at chromosome Ilq22-q23. The most recent genetic linkage mapping and linkage disequilibrium analysis have localized the major AT loci to a sequence of approximately 850 kb between the markers D11S1819 and D11S1818. The isolation of yeast artificial chromosomes spanning the A T region is an essential step to identify the gene or genes responsible for the mutation(s). We isolated a total of 20 YAC clones from three independent YAC libraries, using sequence tagged sites mapped in the AT region as primers for PCR-based YAC screening. The PCR assay for the presence or absence of 16 different DNA markers allowed us to construct and to order four YAC contigs at the AT" region. One of the contigs which consists of the 10 YAC clones, covers about 2 Mb of DNA at the boundary between Giemsa-positive band Ilq22.3 and Giemsa-negative band Ilq23.1 and includes the entire region of the major AT locus between D11S1819 and D11S1818. Thus, the YAC contigs will facilitate the positional cloning approach for searching transcribed sequences from the defined genomic region.

Rare, folate-sensitive fragile sites are the result of the unstable expansion of trinucleotide p(CCG)n repeats, which are normally polymorphic in copy number. Differences in the number and frequency of alleles of the fragile site FRA16A p(CCG)n repeat were observed between different ethnic populations suggesting that certain alleles might be predisposed to instability. Sequence analysis demonstrated that the longer and more variable alleles were associated with loss of repeat interruption. Perfect repeat configuration therefore appears to be a necessary precondition for the instability associated with fragile site genesis.

We have previously identified an estrogen-responsive gene, efp (estrogen-responsive finger protein), that encodes a putative zinc finger protein (Proc. Natl. Acad. Sci. USA 90: 11117-11121, 1993). The efp protein has a RING finger, a variant type of zinc finger motif, B1 box, and B2 box, each having a pair of zinc fingers, present in a family of apparent DNA-binding proteins. Some members of this family have transformation capabilities when found in chromosomal translocations. Chromosome mapping of the efp gene by fluorescence in situ hybridization reveals that human EFP (ZNF147) is located at 17q23.1 and that mouse Efp is located at 11C. These results provide additional evidence that the mouse 11C region displays conserved synteny with the 17q23.1 region of the human genome.

The complete gene for human thrombopoietin (TPO) has been cloned by screening a human genomic library using human TPO cDNA as a probe. This gene is 6.2 kb in length and contains six exons and five introns. It is shown that the human genome contains a single copy of the human TPO gene according to Southern blotting analysis. The transcription initiation site was determined by S1 nuclease mapping. The human TPO gene expressed TPO activity when transfected into COS-1 cells. The human TPO gene has been mapped to chromosome 3q27 by in situ hybridization using a biotin-labeled probe.

Fragile X syndrome, one of the most common human genetic diseases, is characterized by a unique genetic mechanism which involves dynamic mutation in a heritable unstable DNA sequence, a p(CCG)n repeat, in the FRAXA locus. It has recently been suggested that a few founder chromosomes are responsible for most fragile X mutations in the Caucasian population. In order to investigate the origin of the fragile X mutations in the Japanese population, we analyzed haplotypes of the FRAXA locus in 40 unrelated fragile X chromosomes and 142 normal X chromosomes in Japanese males, by using two polymorphic AC repeats, FRAXAC1 and FRAXAC2, which flank the fragile site. This analysis provided evidence for founder fragile X chromosomes in the Japanese population, similar to that in Caucasians, although different haplotypes are involved. The distribution of normal allele size of the p(CCG)n repeat among the X chromosomes in the Japanese population is very similar to that reported for Caucasians, except that the most frequent copy number (n = 28) is one copy less than that in Caucasians and that there is an additional peak at 35 copies. There is significant correlation between FRAXAC alleles and the p(CCG)n repeat copy number in non-fragile X chromosomes, however, alleles with more than 31 copies of the p(CCG)n repeat do not segregate with either of the fragile X common FRAXAC haplotypes. © 1994 Wiley-Liss, Inc.

We mapped the human (RECA) and mouse (Reca) homologs of the yeast RAD51 and Escherichia coli recA genes to human and mouse chromosomes by direct R-banding fluorescence in situ hybridization. This gene was assigned to human chromosome 15q15.1 and to mouse chromosome 2F1, respectively. This is the first report on the precise localization of this gene to human and mouse chromosomes. This gene was mapped to a region on human chromosome 15q15.1 and mouse 2F1 that is believed to be a conserved syntenic group.

The fragile X syndrome is the most common familial form of mental retardation. The mutation causing the syndrome is dynamic mutation due to an unstable DNA (CCG)n repeat localized at Xq27.3. We have previously reported a PCR procedure to prepare a diagnostic probe, pPCRfx1, which can be used to determine the genotype of fragile X mutation individuals by Southern blot analysis. In the present study, pPCRfx1 was applied to the prenatal diagnosis, using chorionic villus cells, of a fetus which was at risk of having fragile X syndrome. In the PstI assay, the Southern blot showed the typical pattern of a female carrier with the full mutation. Analysis of the DNA methylation patterns by EcoRI + EagI assay showed that the EagI restriction site was not methylated on the mutated X chromosome of chorionic villi, but the sites were totally methylated in the brain and other tissues of the fetus. Thus the fetus was diagnosed to be a heterozygous female carrier of the dynamic mutation involved in the fragile X syndrome.

An interstitial deletion, del(11)(q14q22), found in a female infant was examined by fluorescencein situ hybridization with cosmid DNA markers mapped on the long arm of chromosome 11. Three cosmids mapped on 11q14.1-11q22.1 region were not hybridized to the del(11) chromosome, while all the other DNA markers mapped on 11cen-11q14.1 and 11q23.1-11 qter region gave hybridization signals on the del(11) chromosome. Cytogenetic analysis after R-banding confirmed an apparent deletion of 11q14-q22, but containing a small R-negative band, a part of 11q22.3 and/or 11q14.1, in the middle part of del(11) chromosome. The karyotype thus was determined to be 46, XX, del(11)(q14.1q22.3).

Fragile X syndrome, associated with the fragile site at Xq27.3 (FRAXA), is the most common form of familial mental retardation. The fragile X mutation has recently been characterized as a heritable unstable DNA sequence, p(CCG)n/p(CGG)n, in the FRAXA locus. In the present study, a correlation between fragile X-genotypes in the FRAXA locus and hypermethylation of an adjacent CpG island was examined in four Japanese families with fragile X syndrome. We show here that the heritable unstable DNA sequences in the fragile X chromosome usually increase in size when transmitted by female carriers, and that the degree of methylation in the CpG island correlated with the increased sizes of the unstable DNA sequences. When a hypermethylated full mutation was transmitted by a male to his daughters, both the size of the unstable DNA sequence and the degree of the methylation reduced to the premutation range. Our observations suggest that female meiosis has a greater potential for amplifying unstable DNA sequences and that amplified DNA sequences can be transmitted through germ cells, while male germ cells seem not to be able to tolerate highly amplified unstable DNA sequences.

We have examined constitutional chromosome abnormalities and fragile sites in 40 patients with azoospermia. Chromosome abnormalities were found in four cases. Three cases showed a deletion of the long arm of the Y chromosome 46,X,del(Yq) and the other case had a ring of G group chromosome 46,XY,r(G). In a rare fragile sites test, four fragile site carriers were detected and three rare autosomal fragile sites were identified; fra(8)(q24.1), fra(11)(p15.1), and fra(17)(p12). The expression of these fragile sites were induced specifically by AT-specific DNA ligands, such as distamycin A and Hoechst 33258. In addition, one patient was found to be the case of double ascertainment of fragile sites, fra(8)(q24.1) and fra(17)(p12). The overall frequency of distamycin A-inducible fragile sites in azoospermia patients appeared to be higher than those reported for Japanese healthy subjects and cancer patients. However, no significant relation among fragile sites, clinical and histological findings has been detected so far.