関 直彦

p73, a novel p53 family member, is a recently identified candidate neuroblastoma (NBL) suppressor gene mapped at chromosome 1p36.33 and was found to inhibit growth and induce apoptosis in cell lines. To test the hypothesis that p73 is a NBL suppressor gene, we analysed the p73 gene in primary human NBLs, Loss of heterozygosity (LOH) for p73 was observed in 19% (28/151) of informative cases which included 92 mass-screening (MS) tumors. The high frequency of p73 LOH was significantly associated with sporadic NBLs (9% vs 34%, P < 0.001), N-myc amplification (10% vs 71%, P < 0.001), and advanced stage (14% vs 28%, P < 0.05), Both p73 alpha and p73 beta transcripts were detectable in only 46 of 134 (34%) NBLs at low levels by RT-PCR methods, while they were easily detectable in most breast cancers and colorectal cancers under the same conditions. They found no correlation between p73 LOH and its expression levels (P > 0.1), We found two mutations out of 140 NBLs, one somatic and one germline, which result in amino acid substitutions in the C-terminal region of p73 which may affect transactivation functions, though, in the same tumor samples, no mutation of the p53 gene was observed as reported previously. These results suggest that allelic loss of the p73 gene may be a later event in NBL tumorigenesis. However, p73 is infrequently mutated in primary NBLs and may hardly function as a tumor suppressor in a classic Knudson's manner.

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. (C) 1999 Elsevier Science B.V. All rights reserved.

A novel gene, termed p73, encodes a protein with a significant homology to p53 and has been mapped at chromosome 1p36.3, which is a locus of multiple suppressor genes for tumors including neuroblastoma and other cancers. Since the 1p36 locus is reported to be deleted and p53 is frequently mutated in esophageal carcinomas, we examined loss of heterozygosity (LOH) and mutation of the p73 gene in 48 untreated esophageal tumors, as well as mRNA expression in 8 tumors. We screened the pi genomic library to obtain a pi clone containing the p73 gene and found a polymorphic short tandem CT repeat site at intron 9, Intragenic sequences for 14 PCR primer sets and a primer pair flanking the repeat were also determined for the analysis of PCR single-strand conformation polymorphism (SSCP) and LOH studies, respectively, Expression of p73 mRNA was detectable but at low levels in all 8 tumor tissues by reverse transcriptase PCR, We did not find any type of mutation other than polymorphisms in the 48 esophageal carcinomas, though aberration of the p53 gene on the PCR-SSCP gels was observed in 15 of 38 (39%) tumors of the same set. In addition, LOH for p73 was found in only 2 of 25 (8%) tumors. These results suggest that, at least in esophageal carcinomas, allelic loss or mutation of p73 may not be a main genetic event for the tumorigenesis as it is with P53. (C) 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 nerv class of mutation, called dynamic mutation. resulting from an allelic expansion of (CCG)n repeats. The mechanism responsible for other types of rare fragile sires, i.e., distamycin A-inducible and BrdU-requiring, is unknown, although cytogenetic studies suggested that these fragile sires 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 ar as further localized to a 400-kb region including the EXT1 gene. Furthermore, the integration and amplification site of human papillomavirus 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. (C) Elsevier Science Inc., 1998.

Clinical interest in the genes on human chromosome 21, especially with respect to Down syndrome (DS), has provided a strong impetus for the creation of a transcript map of this chromosome. In an effort to identify new human genes on the basis of cDNA analysis, we found several cDNA clones that corresponded to chromosome 21-specific transcripts. One of these, ha2303, showed strong similarity to the murine transcription factor Bach1. We subsequently determined the entire nucleotide sequence of this cDNA clone and found it to contain the whole coding sequence. The gene, termed BACH1, encodes a 736-amino-acid polypeptide with 80.3% identity to the murine Bach1 protein and contains a Cap'n'collar (CNC)-type basic leucine zipper (bZip) domain and a protein interaction motif, the BTB domain. Northern blot analysis revealed that BACH1 was expressed in all tissues examined. Mapping using the NotI restriction map and the YAC contig map showed that the BACH1 gene is located at 21q22.1 between the NotI sites LA329 (D21S338) and LL60 (D21S389) and within approximately 400 kb of LA329. Both the prospective function and the chromosomal location suggest that this gene may be a DS candidate gene, contributing to certain DS phenotypes, and is possibly involved in certain features of monosomy 21. (C) 1998 Academic Press.

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.

As a part of our project for accumulating sequence information of the coding regions of unidentified human genes, we herein report the sequence features of 78 new cDNA clones isolated from human brain cDNA libraries as those which may code for large proteins. The sequence data showed that the average size of the cDNA inserts and their open reading frames was 6.0 kb and 2.8 kb (925 amino acid residues), respectively, and these clones produced the corresponding sizes of protein products in an in vitro transcription/translation system. Homology search against the public databases indicated that the predicted coding sequences of 68 genes contained sequences similar to known genes, 69% of which (47 genes) were related to cell signaling/communication, nucleic acid management, and cell structure/motility. The expression profiles of these genes in 14 different tissues have been analyzed by the reverse transcription-coupled polymerase chain reaction method, and 8 genes were found to be predominantly expressed in the brain.

To evaluate the size-fractionated cDNA libraries of human brain previously constructed (O. Ohara et al. DNA Research, 4, 53-59, 1997), the occurrence of chimeric clones and the content of clones with coding potentiality were analyzed using the randomly sampled clones with insert sizes of 5 to 7 kb. When the chromosomal location of 30 clones was determined by the radiation-hybrid mapping method, the map positions assigned from the 3′- and 5′-end sequences separately were coincident for 29 clones, suggesting that the occurrence of chimeric clones is at most 1/30. Using 91 clones mapped to chromosome 1, the content of clones that have the potentiality coding for proteins larger than 100 amino acid residues was estimated to be approximately 50% (46 out of 91 clones) on the basis of nucleotide sequence analysis and coding potentiality assay in vitro. No significant open reading frames were detected in the remaining clones. Although the clones coding for short peptides may not have been included in the above estimation, the libraries constructed from the whole brain mRNA fraction appear to contain a considerable amount of clones corresponding to the 5′-truncated transcripts in an unprocessed form and/or those with long 3′-untranslated regions.

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.

In this series of projects of sequencing human cDNA clones which correspond to relatively long transcripts, we newly determined the entire sequences of 100 cDNA clones which were screened on the basis of the potentiality of coding for large proteins in vitro. The cDNA libraries used were the fractions with average insert sizes from 5.3 to 7.0 kb of the size-fractionated cDNA libraries from human brain. The randomly sampled clones were single-pass sequenced from both the ends to select clones that are not registered in the public database. Then their protein-coding potentialities were examined by an in vitro transcription/translation system, and the clones that generated proteins larger than 60 kDa were entirely sequenced. Each clone gave a distinct open reading frame (ORF), and the length of the ORF was roughly coincident with the approximate molecular mass of the in vitro product estimated from its mobility on SDS-polyacrylamide gel electrophoresis. The average size of the cDNA clones sequenced was 6.1 kb, and that of the ORFs corresponded to 1200 amino acid residues. By computer-assisted analysis of the sequences with DNA and protein-motif databases (GenBank and PROSITE databases), the functions of at least 73% of the gene products could be anticipated, and 88% of them (the products of 64 clones) were assigned to the functional categories of proteins relating to cell signaling/communication, nucleic acid managing, and cell structure/motility. The expression profiles in a variety of tissues and chromosomal locations of the sequenced clones have been determined. According to the expression spectra, approximately 11 genes appeared to be predominantly expressed in brain. Most of the remaining genes were categorized into one of the following classes: either the expression occurs in a limited number of tissues (31 genes) or the expression occurs ubiquitously in all but a few tissues (47 genes).

Analysis of proteins registered in the PIR protein database implied that most of relatively large proteins are related to important functions in higher multicellular organisms, but not many large proteins have been registered to date. To establish a protocol for efficient analysis of cDNA clones coding for large proteins, we constructed a series of strictly size-fractionated cDNA libraries of human brain, where the average insert sizes of cDNA clones ranged from 3.3 kb to 10 kb. As judged by hybridization analysis with probes derived from mRNAs of known sizes, the libraries with insert sizes up to 7 kb, at least, contained the clones corresponding to full-length transcripts in addition to truncated products of longer transcripts, but few chimeric clones. Using one of the fractionated libraries with an average insert size of 7 kb, the single-pass sequences from both the ends of randomly sampled clones were determined and searched against DNA databases. Approximately 90% of the clones were found to be new with respect to their 5′-sequences, while their 3′-sequences were frequently similar to the registered expression sequence tags. Examination of the protein-coding capacity in an in vitro transcription/translation system showed that about 20% of the clones direct the synthesis of proteins with apparent molecular masses larger than 50 kDa. The set of libraries constructed here should be very useful for the accumulation of sequence data on large proteins in the human brain.

DAN gene is shown to be localized at human chromosome 1p36.11-p36.13, which resides within the consensus deletion observed in neuroblastoma. In the present study, we have isolated the genomic DNA containing the entire human DAN gene and determined its nucleotide sequence. Structural analysis revealed that DAN gene is composed of four exons and spans approximately 15 kb. Its overall structure was basically the same as that of rat DAN gene. Two distinct transcription initiation sites (major and minor) were identified by the primer extension experiment. Putative TATA and CAAT-like elements are present 38 and 366 bp upstream from the major transcription start site, respectively, however, no apparent TATA-like sequence was found in the upstream region of the minor transcription start site. The 400-bp region immediately upstream from the major transcription initiation site was strongly GC-rich (79% GC). Genomic Southern experiments demonstrated that the allelic loss of DAN gene might occur in neuroblastoma. Interestingly, there exist two dinucleotide repeats, (CA)7 and (CA)8, in the first intron of DAN gene, raising the possibility to distinguish two alleles of DAN gene in some of the cancer cells.

We identified a novel phosphatidylinositol (PI) 3-kinase by screening human brain cDNA libraries with probes designed from the conserved kinase-domain sequence. Analysis of cDNAs indicated that two different forms of transcripts are present: one is the full-length form composed of 1,044 amino acid residues and the other is the short form that the N-terminal 216 amino acid residues including a putative p85 binding domain has been truncated (828 amino acid residues). Database search revealed the sequence of the full-length form to be identical to that recently registered by D. Chantry et al. (Accession No. U86453 in GenBank release, August 1997). Northern blot analysis showed this mRNA to be ubiquitously expressed in various tissues, with relatively higher expression was observed in spleen, thymus and leukocytes. Based on fluorescence in situ hybridization and PCR-based analyses with both human/rodent mono-chromosomal hybrid cell panels and radiation hybrid mapping panels, this gene was localized to chromosome region 1p36.2. This region is frequently lost in a variety of human malignancies, including neuroblastoma. The novel PI3K could be a candidate target of the 1p36 alteration that occurs in neuroendocrine tumors.

The Down syndrome (DS) region on chromosome 21, which is responsible for the DS main features, has been defined by analysis of DS patients with partial trisomy 21. Within the DS region, we constructed a 1.6-Mb P1 contig map previously. To isolate gene fragments from the 1.6-Mb region, we performed direct cDNA library screening and exon trapping using the PI clones and a human fetal brain cDNA library, and obtained 67 cDNA fragments and 52 possible exons. Among them, 23 cDNA fragments and 4 exons were interpreted to be derived from a single gene by localization on PI clones and by Northern analysis. To obtain the full-length cDNA sequence, longer cDNA clones were further screened from another human cDNA library which was enriched with longer cDNA species. These clones were sequenced and assembled to a sequence of 9045 bp. This transcribed sequence encodes a novel 2025 amino-acid protein containing tetratricopeptide repeat (TPR) motifs and therefore the gene was designated as TPRD (a gene containing the TPR motifs on the Down syndrome region). The TPR domain has been found in a certain protein phosphatase and in other proteins involved in the regulation of RNA synthesis or mitosis. The TPRD gene, the novel gene which was proved to be in the 1.6-Mb region and to have the interesting features described above, is a candidate for genes responsible for the DS phenotypes.

A subtraction procedure was developed for identification and isolation of a human gene transcribed in mouse transformant cells. The procedure was based on subtractive enrichment of the products that were amplified by the combination of reverse transcription and polymerase chain reaction from the 3′-untranslated region (3′-UTR) of human poly(A)+ RNA expressed in the mouse transformant cells. To assess the ability and usefulness of the procedure, we attempted to recover the human purH gene from a mouse transformant cell line, which was originally established by functional complementation using the human metaphase chromosome-mediated gene transfer technique from a mouse purH-negative mutant cell line. Using our procedure, a part of the human transcript in the transformant cells was successfully identified and isolated. The full-length cDNA was isolated using the 3′-UTR clone as a probe, and its biological activity was confirmed by introducing it into the mouse purH-negative mutant cells.

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.

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. © 1993 Academic Press, Inc.