BIO-MEDICAL SCIENCES Genetics

﹛Constitutional loss or inactivation of one copy of a tumor suppressor gene, as exemplified by hereditary retinoblastoma, increases the propensity for malignancy by reducing the number of events required for the complete loss of the negative regulatory function. An immortalized B-lymphoblastoid cell line, WR10, derived from an obligate heterozygote of hereditary 2,8-dihydroxyadenine urolithiasis, adenine phosphoribosy ltransferase (APRT) deficiency, has enabled us to develop an assay system for dissecting the second step in loss-of-function mutations, i.e. the forward mutation from APRT ≒▼- to APRT -▼- or APRT o▼-, and for determining the potency of physical and chemical agents to produce such mutations. The non-functional APRT allele on chromosome 16 (16q24.3) in WR10 cells bears a nonsense mutation in the exon 3. WR10 was found to be also heterozygous for a SphI RFLP associated with the gene, which allowed the functional and the constitutionally non-functional alleles to be distinguished by Southern blot analysis using an APRT probe.

1.Allelic Losses at the APRT Locus in Human Cells

Kouichi Tatsumi, Akira Tachibana←, Akira Fujimori, Yuko Fujimori-Houki and Masao S. Sasaki← (←Kyoto Univ.)

Keywords: adenine-phosphoribosyltransferase, 2,6-diaminopurine resistance, LOH, RFLP, microsatellite locus, mitotic recombination

﹛The base-line frequency of cells resistant to 100 米M of 2,6-diaminopurine (DAP) was found to be 1.1 x 10-5 with a mutation rate of 1.65 x 10-6 ▼ cell ▼ generation. Recloned DAP r mutants, in general, grew as rapidly as wild-type WR10 cells (population doubling time, 36 h). Exposure of WR10 cells to 污-rays resulted in a dose-dependent increase of DAP污 mutant fraction up to 2.5 x 10-4 at 2 Gy, whereas induced mutant fraction was 4.7 x 10-5 for 6-thioguanine resistance (TG r) with the base-line frequency of 1 x 10-6. A substantial proportion of the spontaneously-arising mutants (22▼26, 85 ⊙) and virtually all of the 污-ray-induced mutants (64▼69, 93 ⊙) lost the functional allele, judging by loss of heterozygosity (LOH). Dosage blotting revealed that about half of the spontaneously-arising and 污-ray-induced mutants with LOH showed a reduction to homozygosity of the mutant allele, implying that the mutated allele was duplicated due to mitotic recombination or gene conversion. Non-disjunction with reduplication of the mutant chromosome 16 was ruled out by the retention of heterozygosity at 16p microsatellite loci in all of the mutants tested with LOH at the APRT locus. About 70 ⊙ of mutants, both those arising spontaneously and those induced by radiation, showed LOH at the D16S265 (16q21) and the D16S308 (16q12.2) loci on the long arm. The distribution of the sites for somatic recombination or for deletion breakpoints in radiation-induced mutants was indistinguishable from that in spontaneously-arising mutants. These results suggest that somatic recombination and▼or deletion occur frequently close to the border between the heterochromatin and the euchromatin regions on the chromosome 16q.

[Publications]

﹛The SCID mutation affects both V(D)J recombination and DNA double- strand break repair. To get an insight as to the relationship between DNA double-strand break repair and mutagenesis, we determined survival and mutation for 6-thioguanine resistance as a function of dose of 污-irradiation in SCA1 cells that were derived from a spontaneous thymic lymphoma in a SCID mouse. SCA1 cells were very sensitive to the cytotoxic effect of 污-rays and D0 value was estimated as 0.25 Gy. DNA dependent protein-kinase activity determined with the crude extract of SCA1 cells was negligibly low and so indicated that this tumor cell line retained the mutated phenotype as SCID. The mutant fraction at the Hprt locus after 污-irradiation was very low, i.e. irradiation of SCA1 cells at the dose as high as that which gave 10⊙ survival yielded only twice as high a mutant frequency as the spontaneous mutant fraction. After exposure to UV-light, SCA1 cells showed an essentially normal profile in the dose survival curve. Mutant fraction following UV irradiation increased linearly against UV dose in SCA1 cells. These results suggest that SCID mutation renders mouse cells hypomutable by 污-rays and that the repair pathway that is defective in SCID appears to be responsible at least in part for the mutagenesis by DNA double-strand breaks.

2.Hypomutability by 污-rays of Thymic Lymphoma Cells Derived from a SCID Mouse

Ikuko Furuno-FukushiI, Kouichi Tatsumi, Ikuko Furuno-Fukushi, Kouichi Tatsumi, Masahiro Muto, Hirobumi Teraoka←, Fumiaki Watanabe and Toshiaki Ogiu (←Tokyo Medical and Dental Univ.)

Keywords : SCID mouse, 污-rays, mutant induction, Hprt, DNA dependent PK

[Publications]

﹛Heritable malformations were examined in the progeny of x-ray irradiated male medaka (Oryzias latipes) by three-generation crosses. Two x-irradiated male fish were pair-mated with nonirradiated females to produce Fㄠ founders, and each Fㄠ fish was pair-mated with a nonirradiated fish to produce Fㄡ progeny. For detection of recessive mutations, pair-matings between Fㄡ siblings were performed for each Fㄠ family. Morphogenesis of the embryos of each generation was observed using a stereomicroscope, throughout the entire period of embryonic development, for identification of anomalies. In the Fㄠ embryos, the frequency of dominant lethals, which were accompanied by various types of malformations, was increased by the X-irradiation. Two out of 30Fㄠ pairs produced a number of malformed and lethal Fㄡ embryos, indicating inheritance of high rates of the dominant lethals in the two Fㄠ families. Moreover, Fㄡ sib-pairs offspring of which exibited high rates of dominant lethals were found in ten out of 28 Fㄠ families. Recessive lethal mutations, which were associated with a particular phenotype, were found in two out of the 28Fㄠ families. These results indicate that the heritable malformations induced by X-irradiation can be studied in the medaka.

3.Heritable Malformations in the Progeny of the Male Medaka (Oryzias latipes) Irradiated with X-rays

Yuji Ishikawa and Yasuko Hyodo-Taguchi

Keywords: genetic defects, malformations, x-ray irradiation, development, medaka, fish

[Publications]
Ishikawa, Y. and Hyodo-Taguchi, Y.: Mutation Res. (in press).

﹛Conventionally, in preparing germfree (GF) mice, the uterus is removed from animals immediately before delivery, the fetus is taken out, and nursed by a GF foster mother. With this tehnique, it is difficult to determine the optimum time to remove the uterus. The resuscitation rate of offspring can be reduced if natural delivery occurs earlier than expected or if surgical delivery is too early. Furthermore, foster nursing requires skill. To increase the efficiency of GF mouse production, we examined the embryo transfer technique.

4.Production of Germfree Mouse by Embryo Transfer

Masanori Okamoto and Tsuneya Matsumoto

Keywords: embryo transfer, germfree mouse

﹛Mature female Jcl:MCH (ICR) mice served as donors for embryo collection after they were mated with mature males of the same strain. GF female C3H▼HeMS mice, which had been maintained at our facility, served as recipients after having mated with males of the same strain previously vasectomized. The procedure for producing GF mice, by embryo transfer, is outlined in the flow chart in Fig.1. GF C3H male mice underwent a vasectomy aseptically within a clean bench. Infertile copulation between vasectomized male and female C3H mice was then induced to produce pseudopregnant recipients. Superovulation was induced with 5 IU of PMSG and hCG injected 48 h apart in conventional female ICR mice, which were then mated with males . On Day 4 post-mating, morphologically normal morula and blastocyst embryos were collected, aseptically, from these females in a clean bench. The collected embryos were immediately transferred, aseptically, into the uteri of the recipients, which had been moved, via a sterile lock, into the clean bench, on Day 3 of infertile copulation with sterile males. The recipients were returned to the vinyl isolator and underwent pregnancy, delivery and nursing. A sterility test was performed, according to the methods recommended by the Japan Experimental Animal Research Association using a combination of TGC and CM medium, or GAM medium. To confirm that embryos had been collected aseptically, the embryos collected from a superovulated donor mouse were put into culture medium. One half of the embryo-containing culture medium was then combined with one test medium and the other half was combined with the other test medium. These two test media were then incubated at 20 and 37∵ for 2 weeks. The sterility test of recipient mice was confirmed by checking their feces 4-5 days after embryo transfer and after delivery. The flexible vinyl isolator was also examined for sterility twice, when examining the recipient mice. The newborns were examined for sterility when they were weaned at the age of 3 weeks.

﹛From the 3 donor mice, we collected 16, 13 and 15 morphologically normal morulae and blastocysts, respectively. These embryo-containing culture media were inoculated into each test medium, and incubated for 2 weeks. All culture media containing embryos were found to be sterile. From donor mouse, we collected 12 morphologically normal morulae and blastocysts. All embryos were transferred to recipients and 6 newborns were delivered. The feces samples, collected from the 2 recipients 4-5 days after embryo transfer and after delivery, were sterile. All feces samples from the 6 weaned animals were also sterile. The flexible vinyl isolator was also sterile in both tests.

Hysterectomy has conventionally been used at our facility to produce GF mice. The success rate using this technique was about 20⊙ before 1989 and approximately 45⊙ in 1990 and 1991. The low success rate of this technique is attributable to the difficulty in determining the optimal timing for surgical delivery and in the nursing of newborns by the foster mother. By our new method, the recipient mice, to which the embryos have been transferred, can deliver and nurse the neonates without human intervention. Furthermore, our technique of embryo transfer allows the animals infected by pathogens to be cleansed by transferring their embryos into clean recipient mice.

The present study, indicated that GF mice could be produced by embryo transfer, in addition to the conventional techniques of hysterectomy and cesarotomy. Furthermore, we developed new aseptic techniques for producing vasectomized sterile males from GF mice and for manipulating embryos. We connected the flexible vinyl isolator, which accommodated GF mice, to the clean bench for aseptic surgery. A follow-up study will be performed using a larger number of animals and also to establish a more efficient, more practical technique for producing GF animals. This method of producing GF animals, in combination with reproductive biotechnology such as techniques for storing frozen embryos, should be useful for strain maintenance, transportation of mice, and in vitro fertilization. Fig.24. Experimental procedures for production of germfree mice using an embryo transfer technique. SL: Sterile lock.

[Publications]
Okamoto, M. and Matsumoto, T.: Proceedings of the XIIth International Symposium on Gnotobiology, in press.

﹛Our purpose in the cDNA project using fission yeast Schizosaccharomyces pombe is to compile a comprehensive list of the house-keeping genes of eukaryotes. Since most of the house-keeping genes are conserved throughout eukaryotes and the fission yeast is the simplest eukaryote with a minimum number of essential genes of a eukaryotic cell, we chose it as a model organism to understand the function of house-keeping genes. We have, thus far, sequenced more than 12,000 clones and identified more than 2,500 independent clones.

5.Comprehensive Cloning of Schizosaccharomyces pombe Genes Encoding Translation Elongation Factors

Kazuei Mita, Mitsuoki Morimyo, Koichi Ito←, Kimihiko Sugaya, Kanae Ebihara←, Etsuko Hongo, Tomoyasu Higashi, Yasuko Hirayama and Yoshikazu Nakamura← (←Univ. of Tokyo)

Keywords: nucleotide sequence, cDNA catalog, fission yeast, translation elongation factor

﹛In the course of theSchizosaccaromyces pombe cDNA project, we succeeded in cloning all the genes encoding translation elongation factors EF-1a, EF-1b, EF-1g, EF-2 and EF-3. With the exception of the EF-1g gene, the nucleotide sequence of S. pombe elongation factors has not been previously reported. For EF-1a, we found three genes whose amino acid sequences are quite homologous to each other (99.5⊙), but whose 3'UTRs (untranslated region) are completely different. Southern blot indicated that those three EF-1a genes are located at different loci. Northern analysis indicated that one of these EF-1a genes was inducible with UV-irradiation. The amino acid sequence predicted from the nucleotide sequence of the S. pombe EF-1b cDNA clone covered almost all the CDS (coding sequence) of EF-1b except the first methionine which has 55.4⊙ identity with that of S. cerevisiae. We also identified two copies of S. pombe EF-2 genes. Their amino acid sequences deduced from nucleotide sequences are identical (100⊙), but they have different 3'UTRs. The location of these two EF-2 genes in different loci was proved by Southern analysis. The S. pombe EF-3 cDNA clone encoded only a third of the CDS from the C-terminal and its deduced amino acid sequence has a 76⊙ identity with those of other yeasts and fungi. Table 1 summarizes the translation elongation factors of S. pombe identified by the cDNA project.

[Publications]
Mita, K., Morimyo, M., Ito K., Sugaya, K., Ebihara, K., Hongo E., Higashi, T., Hirayama, Y. and Nakamura, Y.: Gene, in press.

﹛The cDNA and genomic clones for the human counterpart of mouse mammary tumor gene int-3 were isolated and sequenced. We designated this human MHC class ╯ gene as NOTCH4 since very recently the complete form of the mouse proto-oncogene int-3 has been clarified by sequencing for cDNA clones and named Notch4. The present human NOTCH4 sequence is the first example of the genomic sequence for the extracellular portion of the mammalian Notch4, and the exon▼intron organization was clarified by comparing with the mouse Notch4 cDNA sequence (Fig.1). The comparison of the predicted amino acid sequence of human NOTCH4 with sequences of other Notch homologues of a wide range of species including mouse Notch4 revealed four subfamilies for mammalian Notch. In the protein coding region of human NOTCH4, we found (CTG)n repeats showing VNTR polymorphism for different HLA haplotypes. We also found ten genes mapped on 6p21.3, including NOTCH4, which have counterparts structurally and functionally similar to those mostly mapped on 9q33-q34, indicating segmental chromosome duplication during the course of evolution.

6.Organization of NOTCH4 and (CTG) n Polymorphism in the Human Counterpart Gene of Mouse Proto-Oncogene int-3

Kimihiko Sugaya, Shun-ichi Sasanuma, Junko Nohata, Terumi Kimura, Tatsuo Fukagawaㄠ, Yasukazu Nakamuraㄠ, Asako Andoㄡ, Hidetoshi Inokoㄡ, Toshimichi Ikemuraㄠ, and Kazuei Mita (ㄠNational Institute of Genetics, ㄡTokai Univ.)

Keywords : gene structure, variable number tandem repeat, phylogenetic relationship, chromosome duplication, major histocompatibility complex

Fig.25. Gene organization of human NOTCH4. (A) Location and structure of NOTCH4 gene. Black boxes on the upper line indicate genes found in the junction area between MHC classes ╯ and III by our group, and two cosmid clones (KS74 and KS71) harboring the NOTCH4 sequence are indicated below the line. The lower line indicates the EcoRI map near and at the NOTCH4 locus. Genomic sequences determined in this study and previously (f1-f7) are indicated by thick horizontal bars below the line; black boxes on the bars show exons. The exon including (CTG)10 repeats and initiator methionine ATG is indicated by an asterisk. A cDNA clone, PB5P4, is shown as a thick horizontal line, CDSs of genomic sequence as thin horizontal line and short vertical bars indicate sites for intron insertion. Regions corresponding to genomic sequences determined are connected by slant lines. (B) A schematic representation of functional domains of the NOTCH4 protein. The CDSs are predicted from open reading frames of genomic sequence and PB5P4 cDNA.

[Publications]

﹛Ataxia telangiectasia (AT) is an autosomal recessive disease of unknown etiology associated with cerebellar ataxia, oculo-cutaneous 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, designed NPAT, from the major AT locus on human chromosome 11q22-23. The gene encoded a 1,427-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 one 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 (Fig.1). The database search with the nucleotide sequence nestled between two genes indicated that this region contained a 256 bp sequence which was previously predicted to be one of the CpG islands isolated by Cross et al. Three degenerative TATA-like boxes were found in the upstream region at 782 bp, 893 bp, and 918 bp from the 5' end of the NPAT cDNA. Also, two GC boxes existed at 179 and 195 bp upstream of the NPAT transcript. Several types of transcriptional regulation motifs including one heat shock responsible element (102 bp apart from the 5' end of the gene) and two sites of the E2F binding domain (69 and 113 bp upstream) could be predicted in the 5' region of the NPAT gene which partly overlapped the first exon and intron of the ATM gene. It is noteworthy that some of the promoter elements and regulatory sequences work in either orientation. Therefore, this NPAT upstream region may regulate transcription of both genes.

7.Identification and Characterization of a New Gene Physically Linked to the ATM Gene

Takashi Imai, Masatake Yamauchi, Naohiko Seki←, Takehiko Sugawara, Toshiyuki Saito, Yoichi Matsuda, Hiroko Ito, Takahiro Nagase←, Nobuo Nomura← and Tada-aki Hori (←Kazusa DNA Res. Inst.)

Keywords: ataxia telangiectasia, ATM, NPAT, chromosome 11q22-23

These properties of the NPAT product and the chromosomal position suggest that NPAT, together with ATM, may be included in complex AT phenotypes.

[Publications]
1)Imai, T., Seki, N., Saito, T., Yamauchi, M., Matsuda, Y., Ito, H., Ogiwara, A., Nomura, N., and Hori, T.: DNA Res.,  2, 113-121, 1995.
2)Imai, T., Yamauchi, M., Seki, N., Sugawara, T., Saito, T., Matsuda, Y., Ito, H., Nagase, T., Nomura, N. and Hori, T.: Genome Res. 6, 439-447, 1996.

Fig.1 Schematic diagram of the upstream region nestled between ATM and NPAT genes. The 5' untranslated region in the first exon of both genes is denoted by the white boxes. The first intron of both genes is represented by shaded bars.  Possible E2F binding domain, SP1 binding sites, the heat shock responsible element or TATA-like motifs are marked by ellipses.

﹛Ataxia telangiectasia (AT) is a hereditary disorder characterized by cerebellar ataxia, telangiectases, immune defects, a predisposition to malignancy and chromosomal aberration. These features are considered to be due to a defect in the Gㄠ-S checkpoint in AT cells after suffering DNA damage. Savitsky et al. (Science 268, 1749, 1995) reported a gene termed ATM from 11q22-23, the major AT locus, as a candidate gene for AT. The predicted protein product of ATM shared a homology with a signal transduction mediator, phosphatidylinositol 3-kinase of Saccaromyces cerevisiae. The described sequence of ATM cDNA in the report was, however, approximately a half of the transcript, implying the deduced amino acid sequence might be incomplete. We attempted to determine the whole structure of the gene product to investigate ATM function. A RACE (rapid amplification of cDNA ends) method was applied to isolate unknown 5' portion of the transcript and five types, at least, of ATM cDNAs were identified (Fig.1A). One type of them had the same structure of the reported in Science. Another of the other forms contained the largest open reading frame that might code for a relatively large, 351 kilodalton, protein consists of 3,057 amino acid residues (Fig.1B). The predicted protein contained a leucine zipper motif that suggests ATM protein may bind another protein (including ATM protein) and function in a protein complex form. Artificial expression of an anti-sense strand of the 5'-sequence of this ORF gave hyper sensitivity to X-ray irradiation to HeLa cells, indicating the protein encoded by the ORF would play a role in the responsive process to radiation-induced DNA damage of cells. The other three types of ATM cDNAs may derive from splicing-intermediate molecules or alternatively processed transcripts, requiring further study on heterogeneity of ATM transcripts. Biochemical analysis of ATM protein is now under way.

8.Isolation of 5' Portion cDNAs of ATM Gene for Ataxia Telangiectasia: A Large Open Reading Frame and Structural Heterogeneity of ATM Transcripts

Toshiyuki Saito, Masaki Kato, Masatake Yamauchi, Yoichi Matsuda, Takashi Imai, Takehiko Sugawara, Naohiko Seki← and Tada-aki Hori (←Kazusa DNA Research Institute)

Keywords: ataxia telangiectasia, ATM gene, ATM transcripts

Fig.1 Structure of ATM gene product. A) Schematic representation of various ATM transcripts: Each bar represents the deduced protein from independently-isolated cDNAs. The top is from the report by Savitsky et al. White boxes represent the PI3 kinase domain. B) A putative large ATM protein: This protein is coded in the open reading frame of the third cDNA clone in A. The amino acid sequence written in bold letters is the newly isolated portion.

[Publications]

﹛Ataxia telangiectasia (AT) is an autosomal recessive genetic disease characterized by pleomorphic clinical manifestations. At the cellular level, the AT cells exhibit a hypersensitivity to the killing effect of ionizing radiation and various abnormalities consistent with a defect involving DNA metabolism and▼or maintenance of genomic stability. Recently, a candidate gene ATM was cloned and found to be mutated in AT patients from all complimentation groups, suggesting the possibility that the ATM gene is the sole gene responsible for AT phenotypes. To investigate the molecular pathology of this disorder, it is important to identify the mouse counterpart of the human AT gene. In vivo functions of the AT gene can be examined in a mouse model in which the mouse AT gene is targeted or knocked out.

9.Comparative Genome Mapping of the Ataxia Telangiectasia Region in Mouse, Rat, and Syrian Hamster

Yoichi Matsuda, Takashi Imai, Tadahiro Shiomi, Toshiyuki Saito, Masatake Yamauchi, Hiroko Ito and Tada-aki Hori

Keywords: ataxia telangiectasia, comparative genome mapping, mouse

﹛In the present study, the chromosomal locations of the Atm, Acat1, and Rck genes in the mouse, rat, and Syrian hamster were determined by direct R-banding FISH using a rat cDNA fragment of Acat1 and mouse cDNA fragments of Atm and Rck as probes. Both Atm and Acat1 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. These regions on the mouse and rat chromosomes have been identified as homologous to human chromosome 11q. To determine the order of genes in this region, fine genetic linkage mapping of the mouse AT region was performed using the interspecific backcross mice between (C57BL▼6 x M. spretus) Fㄠ females and M. spretus males. The Atm, Acat1, and Npat which is new gene physically linked to the ATM gene in human, and 12 flanking microsatellite DNA markers were examined. Fig.1 shows the gene order and recombination frequencies of each pair of loci examined in 150 backcross mice. No recombinations were found among the Atm, 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 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 three rodent species, mouse, rat, and Syrian hamster, using additional comparative mapping data with Rck gene. The present mapping information on MMU9 will facilitate consideration of the evolution of MMU9 and HSA11 and also be useful for construction of AT-model mice to investigate its gene functions in vivo.

[Publications]
Matsuda, Y., Imai, T., Shiomi, T., Saito, T., Yamauchi, M., Fukao, T., Akao, Y., Seki, N., Ito, H. and Hori, T.: Genomics, 34,347-352,1996.

Fig.1‘ Comparison of locations of homologous gene typed on MMU9 and HSA11. 
The recombination distances between loci are shown in centimorgans (cM).

﹛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 have been cloned and characterized. The molecular basis of these fragile site mutations was shown to be a new class of mutation,called dynamic mutation, due to 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 played a mechanistic role in breakage and recombination, and may also be integration and modification sites of foreign viral genome. A distamycin A-inducible fragile site FRA8E was mapped to 8q24.1 in which various loci implicated in genomic instability are located. This region is involved in various chromosomal rearrangements associated with genetic diseases and cancers. Furthermore, this region was shown to be the most common integration site of human papillomavirus, especially HPV16 and HPV18 in cervix cancers. In the present study aimed at positional cloning of the FRA8E locus and integration site of HPV16 DNA sequence (Fig.1), we have constructed a YAC contig at 8q24.1 region by using published integrated physical mapping data and by two-color fluorescence in situ hybridization (FISH) method. To identify YAC clones covering the FRA8E region,FISH analysis was carried out using chromosome spreads prepared from a heterozygous carrier of FRA8E. We identified a YAC clone which produced split signals at the expressed site of fra(8)(q24.1). This YAC clone was found to contain both FRA8E locus and breakpoints involved in chromosomal rearrangements associated with Langer-Giedion syndrome (LGS) and hereditary multiple exostoses (EXT1). Using P1 phage clones, the FRA8E locus was further localized to a genomic region (ca. 300kb) of the EXT1 gene (data not shown). The integration and amplification site of HPV16 DNA sequence in cervic cancers was shown to be involved in an extensively broad region including c-myc oncogene, but it did not coincide with the FRA8E locus. The identifications of YAC clones and P1 phage clones presented here will facilitate the cloning and characterization of DNA sequences associated with FRA8E locus and HPV16 DNA integration site. Fig.1. An integrated physical map of YAC clones at 8q24.1, and locations of the FRA8E locus and HPV16 DNA-integrated site.

10.A Rare Fragile Site, FRA8E, Is Localized to Hereditary Multiple Exostosis Gene

Tada-aki Hori, Naohiko Seki←, Toshiyuki Saito, Masatake Yamauchi, Hiroko Ito and Takashi Imai (←Kazusa DNA Res. Inst.)

Keywords : chromosomal fragile site, FRA8E, 8q24.1, EXT1 gene

[Publications]

﹛Ataxia telangiectasia (AT) is a human genetic disease that is genetically recessive and manifests itself as cerebellar ataxia and dilation of blood vessels. AT patients also show immunodeficiency, genomic instability, and high incidence of tumors, as well as hypersensitivity to ionizing radiations. To investigate the function of the gene product which defect is responsible for AT phenotypes, we covered the entire region of the major AT locus at 11q22-24, where the localization of the responsible gene(s) was suggested by genetic linkage analyses, using yeast artificial chromosome (YAC) clones. Cosmid contig covering the AT locus was constructed using YAC clones, and the cosmid clones were used to search for the DNA regions that can be transcribed. Four independent transcripts were identified by using the CpG island rescue method. ﹛To know the exact positions and directions of the transcribable units (﹥genomic genes), we constructed a restriction map of the overlapping cosmid clones covering the major AT locus (Fig.1). Three genes, ATM, NPAT, and ACAT, were mapped within ℅300 kb of the genomic DNA, although the exact position of another gene, T451, has not been determined on the cosmid contig yet. ATM gene is possibly a candidate gene that is responsible for AT phenotype, since mutations were identified in ATM gene of AT patients. The functional basis of ATM gene has not been established. Genomic DNA fragments covering ATM gene were extremely unstable during proliferation in the cosmid vector, and a bacterial artificial chromosome (BAC) clone was employed to cover the region. NPAT gene was mapped very close to ATM gene. The first exons of the two genes, ATM and NPAT were ℅500 bp apart and orientation of the two genes was in a head-to-head manner. This suggested that ATM and NPAT share the same promotor sequence, and that their transcription were possibly regulated by the same factors. This implies that NPAT can be another possible candidate gene for AT phenotype. ACAT was originally reported as a genomic gene coding for mitochondrial acetoacetyl-coenzyme A thiolase by another group, and mapped at the junction of 11q22.3 and 11q23.1 by us using FISH method. ACAT gene was found to be in a intron of NPAT gene. The restriction map of the cosmid contig constructed here will provide useful informations for further analyese of the genes identified, including gene transfer experiments for functional complementation of the AT phenotypes using each of genomic genes as well as mutation analyses of AT patients and heterozygotes. Fig.1 YAC(yeast artificial chromosome, represented by solid bars) contig was constructed to cover the entire genomic region between S1818 and S1819, wher the existence of the responsible gene(s) for the radio-sensitive AT (ataxia telangiectasia) phenotype was suggested by the genetic linkage analysis. The small open circles on solid bars of a YAC clone represent STS markers detected. Cosmid contig (gray bars) was then constructed by using human genomic DNA prepared from the YAC clones. BAC (bacterial artificial chromosome, shaded bar) clone was also employed to cover the region where cosmid clones were highly unstable. Two genes, ATM and NPAT, were newly identified in the contig region by using the CpG island rescue procedure, and their positions and transcriptional-directions were determined (represented by arrows). Another gene, ACAT (gray box), was also identified and mapped in as intron of NPAT gene. It has already been reported by another research group as a genomic gene coding mitochondrial acetoacetyl-coenzyme A thiolase.

11.Construction of Physical Map Covering the Maijor AT Locus

Masatake Yamauchi, Takashi Imai, Toshiyuki Saito, Tada-aki Hori, Satsuki Tsuji, and Tetsuya Saeki

Keywords: genomic instability, ataxia telangiectasia, radiosensitive, genome analysis

[Publications]

﹛Despite recent achievements in analyses of the primary structure of the human genome, its functional aspect has yet to be analyzed. Our three year project aims to examine the possibility of establishing the first model system to analyze the functional organization of the DNA precursor metabolic pathway (DPM pathway) at the molecular level, by combining information obtained from biochemical analyses, and mutations, and primary structures of the genes involved in the pathway.

12.Molecular Analysis of the Relationship between Radiologically Induced Mutations and Genes Involved in the DNA Precursor Metabolism

Masatake Yamauchi, Satsuki Tsuji, Toshiyuki Saito, Hideo Tsuji, Tetsuya Saeki, Etsuko Hongo, Mitsuoki Morimyo, Kazuei Mita, Masahiko Takahagi, Koh-ichi Tatsumi and Tada-aki Hori

Keywords: purine metabolism, genome analysis, mutagenesis, genetic variation

﹛The metabolic pathway of the DNA precursor compounds is one of the most strictly regulated pathways in all organisms, including humans. Defects in the key regulatory enzymes in the pathway result in nucleotide pool imbalances that eventually lead to occurrence of mutations in genes. Genetic variations in the DPM pathway among the human population may possibly play an important role in the development of various diseases including neoplastic transformation induced by ionizing radiation and chemical compounds in the environment. The existence of genetic variation in the DPM pathway among human population is evident, being supported by the population who tend to manifest gout. Gout is a disease that is often caused by genetic defects in the DNA precursor metabolism. The number of gout patients in the Japanese population is estimated to be nearly one million, although the proportion having genetic gout is not known.

﹛To investigate the relationship between the DNA precursor metabolism and the mutagenesis caused by the envirobnmental mutagens at the molecular level, we have started to isolate genes involved in the DNA precursor metabolism of various organisms, human, mouse, and fission yeast. Fission yeast is thought to provide an ideal system to investigate the function of the mammalian genes isolated, since the procedures for homologous recombination to disrupt the target gene and its function are established.

﹛We isolated seven human cDNA clones and nine yeast cDNA ones in the first budget year. In the second budget year, we isolated eight more human cDNA clones, three rodent ones, and three yeast ones. Their nucleotide sequences were determined in parallel with isolation, completely for a full-length cDNA clone of the human purH gene, and partially for other incomplete-length ones. Human purH cDNA was composed of 2068 nucleotides, and a single open reading frame encoding 592 amino acids was identified. Biological function of the human purH gene was confirmed by functional complementation achieved by introducing the cDNA clone in the expression vector to the mouse purH-negative mutant cells.

[Publications]
Yamauchi, M., Seki, N., Mita, K., Saito, T., Tsuji, S., Hongo, E., Morimyo, M., Shiomi, T., Koyama, H., Ayusawa, D., and Hori, T.,: DNA Research, 2:269-275,1995.

﹛Some of the fragile sites on human chromosomes were recently isolated, which showed the involvement of (CGG) n repeat expansion and the association of genetic diseases and chromosomal instability. The heritable fragile site FRA16q22.1 (FRA16B) has been suspected as being predisposed to the chromosome 16 inversion found in patients with acute myeloid leukemia M4Eo, because some of the patients expressed the FRA16B in normal peripheral lymphocytes. We examined the involvement of FRA16B with the breakpoint of chromosome 16 inversion by fluorescence in situ hybridization (FISH) using the genomic clones as a probe, which contained the CBFB gene located in the breakpoint. FISH analysis with chromosomes expressing FRA16B revealed that the CBFB gene was located distal to the fragile site, indicating the dissociation of the FRA16B to the cancer breakpoint. The isolation of genomic DNA including FRA16B is needed to clarify the genomic structure and the biological significance of FRA16B. To identify the chromosomal localization of FRA16B, yeast artificial chromosome (YAC) clones containing human genomic DNA located in 16q22.1 were isolated and chromosomal localization was examined by FISH. The DNA of the clone 821G9 carrying the STS D16S3238 and D16S3021, and the clone 768F12 with the STS D16S3021 and D16S186 crossed the FRA16B. The clones possessing the STS D16S3238 had the FISH signal proximal to the fragile site, while the location of the clones containing D16S2629 was distal. The detailed analysis of the STS content data clarified the order of the STS, D16S3238, D16S3021, D16S186, and D16S2629 from the centromere to the telomere. Thus the of FRA16B was placed between D16S3021 and D16S186.

13.Identification of Chromosomal Localization of FRA16q22.1

Hideo Tsuji, Ei-ichi Takahashiㄠ, Motoi Murataㄡ and Kazuei Mita (ㄠOhtsuka Pharmaceutical Co.;ㄡChiba Cancer Center Res. Inst.)

Keywords: fragile site, FRA16B, YAC, FISH

[Publications]

﹛Cells from patients suffering from xeroderma pigmentosum (XP) or Cockayne syndrome (CS) are hypersensitive to UV-irradiation and are known to be defective in some steps of the nucleotide excision repair. Using a cell fusion technique, excision repair-defective XP cells have been divided into seven genetic complementation groups (A to G), whereas in CS two complementation groups (A and B) have been identified. In addition to these human mutant cells, large numbers of UV-sensitive repair deficient mutants have been isolated from established rodent (Chinese hamster or mouse) cells as another source of UV-sensitive mutants and classified into at least 11 genetic complementation groups.

14.ERCC8 Involved in Cockayne Syndrome Group A Is Mapped to Human Chromosome 5p12-p13.1

Tadahiro Shiomi, Naoko Shiomi, Yoshinobu Harada, Manabu Koike, Ei-ichi Takahashiㄠ,Toshiki Itoㄡ and Masaru Yamaizumiㄡ (ㄠOtsuka Pharmaceutical Co.; ㄡKumamoto Univ.)

Keywords : ERCC8, group A Cockayne syndrome, irradiation hybrid, complementation test, gene mapping

﹛To assign a human gene complementing the defect of the group 8 rodent mutant (ERCC8), the mutant (6L1030) cells were fused with the UV-resistant hybrid (6L1030 X human fibroblast) cells irradiated with X-rays, followed by four cycles of UV-selection (irradiation hybrid formation). To obtain the human sequences which are retained in the fourth irradiation hybrid, 6LH4R1, a lambda phage library was constructed with genomic DNA of 6LH4R1 and screened with total human DNA as a probe. Five independent phage clones (L6LH4R-1-5) which showed positive signals were isolated. The lengths of human DNAs containing in these clones ranged from 13 kb to 20 kb. A direct mapping, which is based on FISH combined with replicated prometa-phase R-bands, was carried out, with DNAs of the lamda phage clones as probes. We examined 50 typical R-banded prometa phase chromosome for each probe. The fluorescent signals for each probe were observed on the short arm of chromosome 5 at p12-13.1 band, strongly suggesting that ERCC8 gene is assigned to the same band of chromosome 5.

﹛Cell fusion complementation tests measuring unscheduled DNA synthesis (UDS) as a cellular marker are often difficult with UV-sensitive rodent cell lines, because a high proportion of cells are in the S phase of the cell cycle, which hampers discrimination of grains due to UDS from those due to regular DNA synthesis. To overcome this difficulty, we adopted recovery of RNA synthesis (RRS) instead of UDS as a marker for complementation tests, since RRS after UV irradiation in 6L1030 cells is severely depressed. Among the nine excision repair defective complementation groups in humans (seven groups for XP and two groups for CS), only three genes corresponding to XP group E, XP group F, and CS group A cells have not been identified. Since the characteristics of XP group E cells are quite different from those of 6L1030 cells, cell fusion was performed with XP group F and CS group A cells. These two cells also have impaired RRS after UV irradiation. Since the fused cells were placed between 6L1030 cells and reference cells on the same coverslip in the complementaion tests, minor changes in grain numbers of the fused cells were recognized easily by comparison with the grain numbers of the two parental cells plated on each side of the fused cells. An increased RRS was observed in heterokaryons of 6L1030 cells with XP group F (Nps1) or CS group B (CS1MO), but not with CS group A (Mps1) . To confirm the fidelity of the inspection method, we counted the grains of the fused cells. Although the grain number in UV-irradiated mononuclear 6L1030 cells varied in different experiments, possibly due to differences in culture conditions, the grain number in heterokaryons was higher than in the parental cells in all combinations except 6L1030 cells with CS group A (CS2SE, Mps1) cells. These results indicate that 6L1030 cells belong to CS group A.

[Publications]
Itoh, T., Shiomi, T., Shiomi, N., Harada, Y., Wakasugi, M., Matunaga, T., Nikaido, O., Friedberg, E. C. and Yamaizumi, Y.: Mutat. Res., 362, 167-174, 1996.

﹛Xeroderma pigmentosum (XP) is a rare inheritable disorder which is characterized by a clinical and cellular hypersensitivity to the ultraviolet (UV) component of the sunlight spectrum. In eight complementation groups of XPs, XP-G is one of the most rare and phenotypically heterogeneous groups. Although the XPG gene that complements defects of the XP-G cells has been shown to function as a DNA endonuclease, it was suggested to have an additional function essential for viability. To investigate further unknown functions of the XPG gene, mice with non-functional xpg (the mouse counterpart of the human XPG gene) alleles were generated using gene targeting methods and embryonic stem cell technology.

15.Generation of Mouse Strain with Knockout Mutations in the DNA Excision Repair Gene (xpg)

Yoshi-nobu Harada, Naoko Shiomi, Manabu Koike and Tadahiro Shiomi

Keywords: xpg, mouse model, gene targeting, group G xeroderma pigmentosum, excision repair

﹛To generate a mutant allele of xpg, the pMER5▼TV2 targeting vector was designed to generate an insertional mutation in one exon of the mouse xpg gene. Targeted ES cells were injected into C57BL▼6 blastocysts to generate chimeric mice which transmitted the mutant allele to F1 offspring. The heterozygous F1 mice were interbred in an attempt to obtain mutant homozygous mice. Of 163 mice born, 35 (21.5⊙) were severely runted and died between 0 and 23 days post partum. DNAs were successfully collected from 31 of 35 dead mice. Southern blot and PCR analyses of these DNAs revealed that all 31 mice were homozygous for the targeted xpg gene. The 128 survivors were either wild-type (39, 23.9⊙) or heterozygotes (89, 54.6⊙). Although the size of the mutant homozygotes at birth seemed to be almost the same as those of wild-type and heterozygous mice, growth of the xpg mutant homozygotes was severely inhibited thereafter. Fig.1 shows a photograph of three littermates with different genotypes at 16 days old, demonstrating the severe inhibition of growth associated with xpg deficiency.

﹛To examine the effects of the insertional mutation on expression of the xpg gene, total RNA from newborn mice was analyzed by Northern blotting. No stable (intact or truncated) xpg transcript was detected in the homozygous mice using the xpg cDNA as a probe. In heterozygous mice, the xpg mRNA content was approximately half of that in wild-type mice. These findings indicate that disruption of the xpg gene by our method was effective. Primary fibroblasts were isolated from newborn mice of all three xpg genotypes and their susceptibilities to UV were examined by measuring colony forming ability after exposure to various doses of UV. The fibroblasts from xpg mutant homozygotes proliferated in culture as rapidly as in wild-type cells, and plating efficiencies were almost the same among these fibroblasts. The fibroblasts from mutant homozygous mice were hypersensitive to UV (254 nm) irradiation. Cells from heterozygotes were as resistant to UV irradiation as wild-type cells. The UV survival curve for mutant homozygous fibroblasts was almost the same as that for cells from a severe XP-G patient.

Fig.1. Three littermates with severe growth inhibition associated with xpg deficiency.

[Publications]

16.Chromosomal Localization of the Mouse and Rat DNA Double-Strand Break Repair Genes Ku p70 and Ku p80▼XRCC5, and their mRNA Expression in Various Mouse Tissues

Manabu Koike, Yoichi Matsuda, Tsuneyo Mimori←, Yosh-nobu Harada, Naoko Shiomi and Tadahiro Shiomi (←Keio Univ.)

Keywords: Ku p70, Ku p80▼XRCC5, repair gene, Northern analysis, gene mapping

﹛DNA damages induced by ionizing radiation result in measurable endpoints such as cell death, mutation and cell transformation. In particular, double-strand breaks in DNA due to radiation could induce the lethal effect. In yeast, double-strand break repair occurs mainly by homologous recombination. In mammalian cells, however, double-strand break repair seems to occur by a different pathway. In the case of rodent cells, ionizing-radiation sensitive mutants have been isolated and divided into at least eight X-ray repair cross complementation (XRCC) groups. Out of these, XRCC4, XRCC5, and XRCC7 mutants have been demonstrated to be defective in the double-strand break repair systems. Recently, the XRCC7 gene has been shown to be defective in scid mouse and to encode the p350 protein.

﹛The DNA-dependent protein kinase (DNA-PK) complex is a nuclear serine▼threonine protein kinase composed of a catalytic subunit p350 (DNA-PKcs) and a DNA binding component named Ku. The Ku autoimmune antigen, abundant in the nuclei and capable of binding to DNA, is a complex composed of two subunits of 70 and 80 kDa, which are designated as Ku p70 and Ku p80, respectively. The antigen is recognized by autoantibodies in sera of certain patients with systemic lupus erythematosus, Graves disease and scleroderma-polymyositis overlap syndrome. The Ku p70 and Ku p80▼XRCC5 genes are involved in DNA double-strand break repair and V(D)J recombination, and their gene products are the components of the DNA-PK.

﹛We have determined chromosomal location of the mouse Ku p70 and Ku p80▼XRCC5 genes by both in situ hybridization and molecular linkage analysis: the Ku p70 gene was localized to mouse chromosome 15 and rat chromosome 7; and the Ku p80▼XRCC5 gene was localized to mouse chromosome 1 and rat chromosome 9. Both genes were mapped in a region of conserved linkage homology among the three species, i.e. the mouse, rat and human. Molecular linkage analysis using interspecific backcross mice revealed that the murine Ku p70 locus was localized at 0.7 cM terminal to D15Mit1, and that the murine Ku p80▼XRCC5 locus was at 0.7 cM proximal to D1Mit46 . These results suggest that these genes originate from a common genetic linkage in mammalian evolution. To determine the size and tissue transcription specificity of the mouse Ku p70 and Ku p80▼XRCC5 mRNA, Northern blot analysis was carried out with six mouse tissues. Each tissue expressed one species of the Ku p70 gene transcript with 2.4 kb and one species of the Ku p80▼XRCC5 gene transcript with 2.6 kb. In the latter case, however, the brain showed two sizes of transcript, 2.6 and 2.9 kb.

[Publications]
Koike, M., Matsuda, Y., Mimori, T., Harada, Y.-N., Shiomi, N. and Shiomi, T.: Genomics, 38, 38-44, 1996.