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J Miyoshi

Publications and source records attributed to J Miyoshi.

34 records · Page 2Linked to original sources
PubMed1993

The murine cot proto-oncogene: genome structure and tissue-specific expression.

We cloned and analyzed the murine cot proto-oncogene and examined its tissue-specific expression in fetal, newborn and adult mice. Genomic cot DNA consists of eight exons, spanning more than 25 kb, and all intron-exon borders are well conserved as compared to the human homolog. Analysis of the full-length cot cDNA revealed that it contained an open reading frame of 1,401 nucleotides, like human cot proto-oncogene. The sequence identity between murine and human cot gene is 84.4% at the nucleotide level and 93.9% at the deduced amino acid level. On northern blot analysis of poly (A)+ RNA, the cot message was detected at 2.9 kb in size. Expression of the cot gene was observed in many tissues from fetal to adult mice, though the level of expression was low in all tissues examined.

Amino Acid Sequence↗
PubMed1992

Oncogenic activation of murine mos protein kinase by DNA rearrangement of its N-terminal coding region.

An activated c-mos oncogene was detected by DNA transfection assay of hamster SHOK cells with DNAs from X-ray-induced mouse osteosarcoma. It was molecularly cloned by the cosmid rescue method and found to form transformed foci of SHOK cells. Genomic DNA sequencing revealed that in this oncogene the N-terminal coding region of the mouse proto-mos gene was deleted and replaced by a hamster-derived sequence in the primary transformant, suggesting that activation was due to the rearrangement during transfection. The gene product was about 37 kDa and was immunoprecipitated with anti-mos antibody from a lysate of a SHOK cell transfectant. This truncated mos (t-mos) gene transformed SHOK cells more effectively than v-mos. A chimeric gene construct of this hamster-derived upstream sequence and normal mouse c-mos also transformed SHOK cells at a lower level, whereas neither t-mos nor the chimeric c-mos gene transformed NIH3T3 cells appreciably. The high transforming efficiency of t-mos in SHOK cells was due not only to truncation of the coding region but also to its integration under a putative promoter sequence derived from the hamster genome. This is the first report of detection of an activated c-mos gene by DNA transfection assay.

Amino Acid Sequence↗
PubMed1992

Differences in effects of oncogenes on resistance of gamma rays, ultraviolet light, and heat shock.

The effects of viral or activated cellular oncogenes on sensitivity to gamma rays, ultraviolet light, and heat shock were examined in SHOK (Syrian hamster Osaka-Kanazawa) cells and their transfectants. Resistance to gamma rays was conferred by the introduction of v-mos or c-cot genes, which coded serine/threonine kinase. Cells transfected with v-mos and c-cot genes increased their resistance to ultraviolet light and heat shock compared to their parent cells (SHOK cells). Of the activated ras genes, the N-ras gene developed a SHOK cell phenotype resistant to gamma rays and ultraviolet light. The Ha-ras gene produced SHOK cells resistant to ultraviolet light and heat shock, while introduction of the Ki-ras gene did not affect sensitivity. The v-erbB gene was found to be involved in the development of resistance to heat shock. Transfection with neo, c-myc, and v-fgr genes had little or no effect on cell survival. The karyotypes of SHOK cells and oncogene-containing cells were compared. No alterations were seen after the introduction of a foreign gene. Using cell cycle analysis, we found no apparent difference between SHOK cells and their transfectants. These results suggest that activation of serine/threonine kinase may be involved in common processes occurring after gamma-ray, ultraviolet-light, and heat-shock treatment, and that each oncogene may have a different effect on the development of a resistant phenotype.

Animals↗
PubMed1991

A novel point mutation at codon 146 of the K-ras gene in a human colorectal cancer identified by the polymerase chain reaction.

In this report, point mutations of the K-ras gene at codon 146 were analyzed in 25 cases of colon cancer, 4 cases of lung cancer, and 41 cases of lymphoid malignancy. A codon 146 mutation substituting threonine (ACA) for alanine (GCA) was detected in the tumor tissue of a patient with colon cancer and was not detected in the normal tissue of the same patient. Any additional mutations of the ras gene family were not detected in this patient. These results suggest that the codon 146 mutation of the K-ras gene could be involved in the development of naturally occurring human malignancies.

Antisense Elements (Genetics)↗
PubMed1991

Structure and transforming potential of the human cot oncogene encoding a putative protein kinase.

A new transforming gene has been molecularly cloned from hamster SHOK cells transformed with DNA extracted from a human thyroid carcinoma cell line and named the cot (cancer Osaka thyroid) oncogene. cDNA sequencing disclosed that this oncogene codes for a protein with 415 amino acid residues, and computer matching showed 42 to 48% similarity matches with serine protein kinases. Its gene product was identified as a 52-kDa protein by transcription and translation in vitro. Expression of cot cDNA under transcriptional control by a retroviral long terminal repeat induced morphological transformation of NIH 3T3 cells as well as SHOK cells. Protein kinase activity associated with constructed p60gag-cot was detected by immune complex kinase assay with anti-gag antiserum. The cot oncogene was overexpressed in transformed SHOK cells and found to have a rearranged 3' end in the last coding exon, which probably resulted in a deletion and an altered C' terminus in the transforming protein. This DNA rearrangement appeared to have occurred during transfection of the tumor DNA into hamster SHOK cells and not in the original thyroid tumor.

Amino Acid Sequence↗
PubMed1991

Identification and characterization of protein products of the cot oncogene with serine kinase activity.

The products of the cot gene, a novel oncogene isolated by DNA transfection assay using the hamster cell line SHOK, were identified as 46 kDa and 52 kDa proteins by using anti-peptide antibodies. The 46 kDa and 52 kDa proteins both showed autophosphorylation activity at serine residues. The two forms of the Cot protein were suggested to differ in their amino-terminal structures as a result of alternative initiation of translation. Subcellular fractionation revealed that the 46 kDa and 52 kDa proteins are both predominantly localized in the cytosol. These Cot proteins are the fourth oncogene products with serine kinase activity identified.

Amino Acids↗
PubMed1990

Alteration of N-ras gene mutation after relapse in acute lymphoblastic leukemia.

We investigated N-ras activation in childhood acute lymphoblastic leukemia (dALL) by the polymerase chain reaction (PCR) and the oligonucleotide hybridization method. The frequency of point-mutation of the N-ras gene was not high (2 of 15), and one positive case who relapsed was analyzed in detail. Although N-ras gene activation was detected at both onset and relapse, the mutation sites were different. At onset, Gly (GGT) was changed to Ser (AGT) at codon 12, and at relapse, Gly (GGT) to Asp (GAT) was observed at the same codon. In addition, the DNA at relapse showed a remarkably higher transforming activity than the DNA at onset on two independent recipient cell lines. The identical cell surface phenotype and the same rearrangement patterns of both the immunoglobulin (Ig) heavy chain and T-cell receptor (TCR) gamma chain genes indicated that the leukemic cells at onset and those at relapse were derived from the same precursor cell. Therefore, this case supports the concept that ras activation is not the event initiating leukemogenesis, but may be involved in leukemic progression.

Base Sequence↗
PubMed1990

Hamster cell line suitable for transfection assay of transforming genes.

We established a subclone, SHOK, from the GHE-L cell line, an immortal line derived from a primary culture of Syrian hamster embryo cells, as a recipient cell line useful for the detection of oncogenes by transfection. SHOK cells were almost as susceptible as NIH 3T3 cells to focus formation by many oncogenes, including v-raf, v-Ha-ras, v-Ki-ras, or activated c-Ha-ras. The susceptibility of SHOK to focus formation was higher than that of NIH 3T3 for v-mos but was lower for v-fps, v-fgr, v-src, v-sis, and v-abl. When DNAs extracted from 27 human and murine tumors were tested for focus formation, 5 DNAs were positive in NIH 3T3 cells, whereas 9 were positive in SHOK cells at the primary transfection. Using SHOK cells as recipients of tumor cellular DNA, we isolated another oncogene and a c-Ki-ras2 gene mutated at codon 146 that were difficult to detect in NIH 3T3 cells. SHOK cells have a low rate of spontaneous transformation, produce easily distinguishable foci, and maintain a stable karyotype in transformed cells. In addition to being useful for the screening of human tumor DNAs, SHOK cells will be useful for the isolation of oncogenes from murine tumors because of their hamster origin.

Animals↗
PubMed1989

Differential requirements of gag and gamma-actin domains for transforming potential of Gardner-Rasheed feline sarcoma virus.

The oncogene of Gardner-Rasheed feline sarcoma virus (GR-FeSV) encodes the 70-kilodalton protein containing gag(p15), gamma-actin, and fgr domains. To determine the role of these domains in the biological activity of P70gag-actin-fgr, we have constructed in-frame deletion and insertion mutants of GR-FeSV. We found, first, that the gamma-actin region could be deleted without affecting the transforming ability of these constructs, although an insertion mutant in the middle of the gamma-actin domain (map position 671) was partially defective in transformation and specifically had a reduced level of in vitro autophosphorylation activity. Second, mutations affecting the C-terminal third of the gag region appeared to abolish the ability to transform NIH 3T3 cells and autophosphorylation activity. These results suggest that the gamma-actin domain is not essentially required for the transforming activity of GR-FeSV but that it may take part in maintaining the conformational integrity of P70gag-actin-fgr and that the gag(p15) domain might have a critical role in modulating the function of P70gag-actin-fgr.

Actins↗
PubMed1988

A simple and useful method for simultaneous screening of elevated levels of expression of a variety of oncogenes in malignant cells.

Abnormal expression of various oncogenes has been implicated in the development of many malignant tumors. Although RNA blotting methods have been used to measure abnormal expression, they involve the time-consuming process of individually labeling the oncogene probes. To simplify this process we have attempted to develop a new method, termed simultaneous screening, which is based on the synthesis of radiolabeled cDNA corresponding to the mRNA population of malignant cells and on hybridization with various oncogene probes, immobilized on a membrane filter. This method circumvents the time-consuming process of the prevailing RNA blotting methods and is also sensitive enough to detect accurately a five- to ten-fold level of expression of rare mRNA (approximately 10 copies per cell). Overexpression of ten oncogenes was detected in a variety of malignant cells and mitogen-stimulated cells with this method. These results suggest that our simultaneous screening method can be used to examine the overexpression of oncogenes.

DNA↗
PubMed1985

Isolation and characterization of an activated C-H-ras-1 gene from a squamous-cell lung carcinoma cell line.

We determined a complete nucleotide sequence of an activated form of the c-H-ras-1 proto-oncogene cloned from the human cell line (QG56), using the DNA transfection technique and NIH3T3 cells as recipients. This cell line was established from a squamous-cell lung carcinoma of a Japanese patient, and the activated gene had 2 nucleotide substitutions. One substitution of a thymidine for an adenosine was found at position 1069 of the 2898 nucleotide sequence in a restriction endonuclease (SacI) fragment, which corresponds to the second base of the 61st codon of the gene encoding P21 protein. This nucleotide replacement was assumed to be responsible for the transforming activity. Another substitution of a guanosine for an adenosine which was detected at position 746 in the first intron was thought to be a genetic polymorphism unassociated with the transforming activity. Comparison of the various lengths of restricted fragments suggested that the activity was markedly influenced by certain sequences flanking the c-H-ras-1 gene.

Animals↗
PubMed1984

The human c-Ha-ras2 is a processed pseudogene inactivated by numerous base substitutions.

The human c-Ha-ras2 gene, one of two known members of the Harvey ras family, is reportedly located on the X-chromosome and has lost introns (1, 2). There has heretofore been no information on its precise gene structure and oncogenic potential. We have determined the nucleotide sequence of the c-Ha-ras2 and demonstrate that it is a processed pseudogene surrounded by several direct repeats and contains numerous base substitutions as well as a notable mutation (AGT at codon 12 of the p21 protein) responsible for oncogenic conversion of the known ras genes (3-8).

Base Composition↗
PubMed1982

Transposon Tn3-mediated replicon fusion.

To investigate inter-replicon transposition of Tn3, we used the cosmid-phage lambda packaging system coupled with density gradient fractionation and isolated recombinant molecules of different sizes. Cosmids derived from ampicillin-resistance-transducing phage were classified into four groups: (1) cosmid-Tn3 donor cointegrates considered as Tn3 transposition intermediates, (2) similar cointegrates carrying deletions of one copy of Tn3 and of adjacent cosmid DNA sequences, (3) cosmids carrying a single Tn3 insertion, and (4) cosmids carrying two independent Tn3 insertions. Genetic and biochemical studies indicated that cosmids isolated from ampicillin-resistance transductants were derived from the authentic cosmid-Tn3 donor cointegrate intermediates.

Bacteriophage lambda↗
PubMed1982

Characterization of a ColE1-like plasmid isolated from Shigella sonnei.

A multicopy plasmid, 4.7 kb in size, was isolated from Shigella sonnei and named pKY1. This plasmid produces a colicin E1-like bacteriocin (colicin E1*) in E. coli cells. The cells harboring pKY1 are immune not only to this bacteriocin but also to colicin E1, and the cells harboring Co1E1 show immunity to colicin E1* as well. Although these two plasmid DNAs have different cleavage maps and are compatible with each other, pKY1 shows partial DNA homology with ColE1 DNA. In this paper, we report the isolation and properties of several Tn3 inserted pKY1 mutants, and propose a preliminary genetic map of pKY1. It was also found that this plasmid is not capable of self-transmission and is poorly mobilized by the F factor.

Chromosome Mapping↗
PubMed1979

DNA sequences of the integration sites and inverted repeated structure of transposon Tn3.

The nucleotide sequence of the "inverted repeat" structure of the transposon Tn3 was determined by the DNA sequencing procedure developed by Maxam and Gilbert(1). The sequence, 38 base pairs long, is as follows: 5'-GGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAG..(Tn3) 3'-CCCCAGACTGCGAGTCACCTTGCTTTTGAGTGCAATTC.. The integration of Tn3 is associated with a directly repeated sequence of 5 nucleotides appearing at each end of Tn3. The two directly repeated sequences so far determined are not the same. Furthermore, there is no homologous structure around the integration point of Tn3.

Base Sequence↗