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M Oshimura

Publications and source records attributed to M Oshimura.

At least 127 records · Page 7Linked to original sources
PubMed1995

Construction of 110 cosmid markers and a 4.5-Mb YAC contig on human chromosome 8p12-q11.

Microcell hybrids containing various regions of human chromosome 8 were formed by microcell-mediated transfer of neo-tagged chromosome 8 into the cells derived from severe combined immunodeficiency (SCID) mouse. Thus, 110 cosmid markers were isolated from SV40-transformed SCID fibroblast cell line (SCVA) containing a p12-q11.1 region of human chromosome 8 and were assigned to eight regions in 8p12-q11.1, using a microcell-hybrid panel. For positional cloning of a human gene that restores the DNA-repair defect in a mouse with SCID on 8p11.1-q11.1 (SCID region), we constructed a yeast artificial chromosome (YAC) contig of about 4.5 Mb. Overlapping YACs were further aligned by restriction mapping, using rare-cutting restriction endonucleases. The cosmids and YAC contig should facilitate isolation of the SCID gene and other genes, such as the Werner syndrome-responsible gene in or near this region.

Animals↗
PubMed1995

Isolation and mapping of 186 new DNA markers on human chromosome 1.

To obtain DNA markers on human chromosome 1, we first isolated 500 cosmid clones from mouse A9 cells containing a human chromosome 1 tagged with pSV2neo. Of these, 186 were localized on each band of human chromosome 1 by R-banding fluorescence in situ hybridization; 118 and 68 were on the short and long arms, respectively. We performed restriction fragment length polymorphism (RFLP) analysis of these cosmid clones, and polymorphism was recognized with one or more enzyme in 43 of them. Two markers proved to have variable numbers of tandem repeats. Since several tumor suppressor genes, as well as genes responsible for hereditary disorders, may be located on this human chromosome, the DNA markers will be useful for RFLP analysis or the isolation of new genes related to various disorders.

Chromosome Mapping↗
PubMed1995

Loss of the catalytic subunit of the DNA-dependent protein kinase in DNA double-strand-break-repair mutant mammalian cells.

The DNA-dependent protein kinase (DNA-PK) consists of three polypeptide components: Ku-70, Ku-80, and an approximately 350-kDa catalytic subunit (p350). The gene encoding the Ku-80 subunit is identical to the x-ray-sensitive group 5 complementing gene XRCC5. Expression of the Ku-80 cDNA rescues both DNA double-strand break (DSB) repair and V(D)J recombination in group 5 mutant cells. The involvement of Ku-80 in these processes suggests that the underlying defect in these mutant cells may be disruption of the DNA-PK holoenzyme. In this report we show that the p350 kinase subunit is deleted in cells derived from the severe combined immunodeficiency mouse and in the Chinese hamster ovary cell line V-3, both of which are defective in DSB repair and V(D)J recombination. A centromeric fragment of human chromosome 8 that complements the scid defect also restores p350 protein expression and rescues in vitro DNA-PK activity. These data suggest the scid gene may encode the p350 protein or regulate its expression and are consistent with a model whereby DNA-PK is a critical component of the DSB-repair pathway.

Animals↗
PubMed1995

Molecular cloning of a novel human cDNA on chromosome 1q21 and its mouse homolog encoding a nuclear protein with DNA-binding ability.

We previously reported that human chromosome 1q21 or 1q23-q24 carries a transformation suppressor gene(s) for Kirsten sarcoma virus-transformed NIH3T3 cells. In this study, we have isolated a novel human cDNA on 1q21 (designated as YL-1) as a candidate, then cloned its mouse homolog. Human and mouse YL-1 cDNA shared 96% in amino acid homology and presumably encoded a transcription factor-like polypeptide. An analysis using a specific antibody revealed the nuclear localization of YL-1 protein. The bacterially expressed YL-1 could bind to DNA. Thus, YL-1 protein possibly functions as a transcriptional regulator.

3T3 Cells↗
PubMed1995

Suppression of endometrial carcinoma cell tumorigenicity by human chromosome 18.

Presumptive tumor suppressor genes may be localized to specific chromosomes by the procedure of microcell fusion, whereby individual chromosomes derived from normal human cells are introduced into tumor cells. Allelic loss on chromosome 18 is commonly seen in endometrial carcinoma, and the DCC gene on chromosome 18q is a potential human tumor suppressor gene. In this study, we investigated the hypothesis that a gene on chromosome 18, possibly DCC, is capable of suppressing the tumorigenicity of endometrial carcinoma cells. Microcells from the mouse A9 cell clone containing one human chromosome 18 tagged with the pSV2-neo plasmid were fused with the highly tumorigenic endometrial carcinoma cell lines HHUA and Ishikawa, and G418-resistant microcell hybrids containing and extra copy of chromosome 18 were isolated. Clones isolated from the HHUA cell line were completely suppressed for tumorigenicity in nude mice, and clones from the Ishikawa line were suppressed or inhibited for tumorigenicity. In contrast, growth rates in vitro were not significantly affected in clones from either parental cell line. DCC expression was elevated in most of the suppressed hybrids. These results indicate that a gene on human chromosome 18 is capable of suppressing the tumorigenicity of endometrial carcinoma cells, and that DCC is a candidate for this endometrial carcinoma tumor suppressor gene.

Animals↗
PubMed1995

Human chromosome 11 inhibits tumorigenicity of a murine squamous cell carcinoma cell line.

Loss of heterozygosity (LOH) of mouse chromosome 7 has been consistently demonstrated in chemically induced murine squamous cell carcinomas (SCCs). The region of this chromosome presenting LOH in the mouse tumors is syntenic to human chromosome segments 11p15 and 11q. To determine whether the introduction of human chromosome (Hchr) 11 can suppress the growth of murine SCC, we injected four clones of a chemically induced murine SCC cell line bearing an Hchr 11 into athymic BALB/c nude mice. All microcell hybrid clones with Hchr 11 (CH72/Hchr 11) had latency periods twice as long as those of the parental CH72 cells and control hybrids containing a Hchr 12. Tumor-derived cells from CH72/Hchr 11 hybrids had lost centromeric and telomeric sequences from Hchr 11. All repressed cell lines grew significantly more slowly in vitro than did the controls. These results suggest that Hchr 11 contains a tumor-suppressor gene capable of inhibiting tumorigenicity in chemically induced SCC, confirming common pathways in the development of human neoplasias and the murine model.

Alleles↗
PubMed1995

Localization of metastasis suppressor gene(s) for rat prostatic cancer to the long arm of human chromosome 10.

To examine the role of human chromosome 10 in development of prostatic cancer, we introduced human chromosome 10 into highly metastatic rat prostatic cancer cells by microcell-mediated chromosome transfer. Microcell hybrid cells introduced with human chromosome 10 showed suppression of the metastatic ability to the lung to some extent without any suppression of tumorigenicity, although the tumor growth rate decreased slightly. To minimize the region that contains metastasis suppressive activity, the hybrid cells in metastasis foci of lung were established in culture and reanalyzed for portions of human chromosome 10 retained in the metastasis tissues. Cytogenetic and molecular analyses demonstrated that loss of the region between 10cen and D10S215 on human chromosome arm 10q was related to expression of the metastatic phenotype. These results demonstrate that the region between 10cen and D10S215 on human chromosome arm 10q contains at least one of the metastasis suppressor genes for rat prostatic cancer.

Animals↗
PubMed1995

Normal human chromosome 2 induces cellular senescence in the human cervical carcinoma cell line SiHa.

For identification of the chromosome carrying cellular senescence-inducing activity, normal human chromosome 2, 3, 6, 7, 9, 11, or 12 tagged with a selectable marker gene (neo) was introduced into the human cervical carcinoma cell line SiHa via microcell-mediated chromosome transfer. Seventy-six percent (158/207) of the G418-resistant clones obtained by the transfer of chromosome 2 showed a remarkable change in morphology (cells were flat), and 93% (147/158) of them ceased to divide (senesced) prior to 6-9 population doublings, whereas most of the clones generated by the transfer of other chromosomes exhibited a morphology similar to that of the parental cells and continued to grow. Chromosome analyses suggested that cells which escaped from senescence contained only a small fragment derived from the transferred chromosome 2, whereas the transferred chromosomes were apparently intact in most of the continuously growing microcell hybrids with introduction of other chromosomes. These results indicate that the normal human chromosome 2 carries a gene or genes that induce cellular senescence in SiHa cells.

Animals↗
PubMed1995

Forced expression of YL-1 protein suppresses the anchorage-independent growth of Kirsten sarcoma virus-transformed NIH3T3 cells.

The YL-1 gene, encoding a novel nuclear protein with transcription factor-like features, has been isolated from the human chromosome 1q21, one of the regions supposedly carrying a transformation suppressor gene(s) for Kirsten sarcoma virus-transformed NIH3T3 (DT) cells. To test the suppressive activity of the YL-1 gene product, we forced the expression of human YL-1 cDNA in DT cells. The anchorage-independent growth (colony-forming ability in soft agar medium) was markedly suppressed in cells highly expressing the exogenous human YL-1 protein. Moreover, the soft agar clones, which were rarely originated from these cells, expressed reduced levels of exogenous YL-1 or none, with or without the loss/rearrangement of the introduced cDNA. In control experiments, cells carrying an introduced vector alone or an antisense-strand expression plasmid grew in soft agar as efficiently as parental DT cells. In contrast to the suppression of anchorage-independent growth, the forced expression of YL-1 did not effect the transformed phenotypes in adherent culture and tumorigenicity in nude mice. These findings not only indicated that the YL-1 protein functions as a transformation suppressor, but also suggest that it may be important for elucidating anchorage independence under separate genetic control from other transformed phenotypes.

3T3 Cells↗
PubMed1995

Cosmids and transcribed sequences from chromosome 11q23.

To obtain cosmid markers and transcribed sequences from a specific chromosome region, a series of radiation-reduced hybrids (RHs) containing various regions of human chromosome 11 was prepared from microcell hybrid A9 (neo11) cells containing a normal human chromosome 11 tagged with pSV2neo at 11p11.2. Among 15 radiation hybrid clones isolated, RH(11)-9 which contains a q23 fragment in addition to the neo integration site, was used for the construction of a cosmid library. Cosmid clones having human DNA sequences were screened, and localized by Southern hybridization with the radiation hybrid panel. Fifty-nine cosmids were assigned to 11q23 and 6 cosmids to 11p11.2. Exon amplification proceeded with 23 of the 59 cosmids and 16 putative exons were cloned. Three of them were identical to those constituting a known gene which locates on q23 (ATDC), and the others were unknown. Thus, the RHs containing various subchromosomal fragments of chromosome 11 were useful for constructing region-specific DNA markers. The RH(11)-9 cells and putative exons also facilitate the positional cloning of genes in the 11q23 region.

Animals↗
PubMed1995

Genetic heterogeneity of chromosome 11 associated with tumorigenicity in HeLa D98-OR cells.

D98-OR is a tumorigenic subline of HeLa cells. We isolated nine subclones from D98-OR and examined their tumorigenicity in nude mice. Three, two, and four subclones were highly, weakly, and nontumorigenic, respectively. While they all contained two copies of intact chromosome 11, restriction fragment length polymorphism (RFLP) analysis revealed that the allelic composition of this chromosome differed among them. The highly tumorigenic subclones were heterozygous for the 11p and 11q loci, whereas those that were weakly or nontumorigenic were homozygous. Thus, the loss of one chromosome 11 with the duplication of another associated with the reduced tumorigenicity. Taken together with previous reports, our results indicate that a putative tumor suppressor gene on chromosome 11 controls tumorigenic expression in a gene dosage-dependent manner, and most importantly, suggested that the functional inactivation of the gene requires only a "one-hit" mutation.

Animals↗
PubMed1995

Genetic complementation of the immortal phenotype in group D cell lines by introduction of chromosome 7.

Human immortal cell lines have been classified into at least four (A-D) genetic complementation groups by cell-cell hybrid analysis, i.e., a hybrid derived from different groups becomes mortal. Recently we have demonstrated that introduction of human chromosome 7 suppresses indefinite division potential in the non-tumorigenic human immortalized fibroblast lines KMST-6 and SUSM-1, both assigned to complementation group D. By extending our microcell-mediated chromosome transfer, we found that chromosome 7 also suppresses division potential in the human hepatoma line HepG2 (again, assigned to group D). Chromosome 7 was thus shown to suppress indefinite growth in the above group D cell lines irrespective of their cell types, or whether they are tumorigenic or not. Since chromosome 7 had no such effect on representative cell lines derived from complementation group A, B or C, these results indicate that the senescence gene(s) commonly mutated in the group D cell lines is located on chromosome 7.

Base Sequence↗
PubMed1995

Restoration of the cellular senescence program and repression of telomerase by human chromosome 3.

Telomeres, at the end of chromosomes, shorten with each cell division, resulting in cellular senescence. Tumor cells, unlike normal somatic cells, express a telomerase that maintains the telomere length. Deletion of a gene(s) on chromosome 3 is common in human renal cell carcinoma (RCC) and reintroduction of a normal chromosome 3 into an RCC immortal cell line restored the program of cellular senescence. The loss of indefinite growth potential was associated with the loss of telomerase activity and shortening of telomeres in the RCC cells with a normal chromosome 3. However, microcell hybrids that escaped from senescence and microcell hybrids with an introduced chromosome 7 or 11 maintained telomere lengths and telomerase activity similar to those of the parental RCC23. Thus, restoration of the cellular senescence program by chromosome 3 is associated with repression of telomerase function in RCC cells.

Base Sequence↗
PubMed1995

Subchromosomal mapping of a putative transformation suppressor gene on human chromosome 1.

We previously reported that the introduction of a normal human chromosome 1 via microcell-mediated chromosome transfer suppressed the transformed phenotypes, including anchorage-independent growth, of Kirsten murine sarcoma virus-transformed NIH3T3 (DT) cells. Soft-agar clones derived from DT-#1 cells (DT cells with an intact transferred human chromosome 1) exclusively failed to retain an intact form of this chromosome. Thus, a gene(s) with a suppressive activity on this chromosome had probably been lost. We therefore attempted to identify a commonly deleted region on human chromosome 1 in these soft-agar clones. Although eight of the 9 soft-agar clones examined still contained regions on this chromosome, to a greater or lesser degree, four loci on 1q21 and 1q23-q24 were commonly lost in all of them. Furthermore, the soft-agar clones had growth properties similar to those of DT cells. Thus, chromosome and DNA analyses suggested that human 1q21 and/or 1q23-q24 carries a transformation suppressor gene(s) which controls the transformed phenotypes of DT cells.

3T3 Cells↗
PubMed1995

Cloning and nucleotide sequence of the gene for acidocin 8912, a bacteriocin from Lactobacillus acidophilus TK8912.

Acidocin 8912 is a bacteriocin produced by Lactobacillus acidophilus TK8912. The acidocin 8912 structural gene, acdT, was cloned and determined. It was located on the 14-kb plasmid pL103 and encoded a 46 amino acid precursor including a 20 amino acid N-terminal extension. The precursor sequence of the acdT gene shows a conservation of the general structural characteristics of the bacteriocin precursors from some lactic acid bacteria.

Amino Acid Sequence↗
PubMed1995

Isolation and characterization of acidocin A and cloning of the bacteriocin gene from Lactobacillus acidophilus.

Acidocin A, a bacteriocin produced by Lactobacillus acidophilus TK9201, is active against closely related lactic acid bacteria and food-borne pathogens including Listeria monocytogenes. The bacteriocin was purified to homogeneity by ammonium sulfate precipitation and sequential ion-exchange and reversed-phase chromatographies. The molecular mass was determined by high-performance liquid chromatography gel filtration to be 6,500 Da. The sequence of the first 16 amino acids of the N terminus was determined, and oligonucleotide probes based on this sequence were constructed to detect the acidocin A structural gene acdA. The probes hybridized to the 4.5-kb EcoRI fragment of a 45-kb plasmid, pLA9201, present in L. acidophilus TK9201, and the hybridizing region was further localized to the 0.9-kb KpnI-XbaI fragment. Analysis of the nucleotide sequence of this fragment revealed that acidocin A was synthesized as an 81-amino-acid precursor including a 23-amino-acid N-terminal extension. An additional open reading frame (ORF2) encoding a 55-amino-acid polypeptide was found downstream of and in the same operon as acdA. Transformants containing this ORF2 became resistant to acidocin A, suggesting that ORF2 encodes an immunity function for acidocin A. The 7.2-kb SacI-XbaI fragment containing the upstream region of acdA of pLA9201 was necessary for acidocin A expression in the acidocin A-deficient mutant, L. acidophilus TK9201-1, and other Lactobacillus strains.

Amino Acid Sequence↗