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J Inazawa

Publications and source records attributed to J Inazawa.

At least 181 records · Page 10Linked to original sources
PubMed1994

Molecular cloning, characterization, and chromosomal localization of a novel protein-tyrosine phosphatase, HPTP eta.

Protein-tyrosine phosphatases (PTPases) are considered to play an important role in signal transduction. We previously identified partial sequences of three novel PTPases in a human leukemic cell line. F-36P. We describe here cloning, characterization, and chromosomal localization of one of the newly identified PTPases, termed as HPTP eta (human protein-tyrosine phosphatase eta). The deduced amino acid sequence was composed of an extracellular region homologous to fibronectin type III repeats, a transmembrane region, and a cytoplasmic region containing a single PTPase-like domain. Based on its primary structure, this clone belongs to type-III receptor-type PTPases. The PTPase-like domain showed PTPase activity when expressed in Escherichia coli. Antibody against the extracellular region detected a protein of 220 to 250 kD in human hematopoietic cell lines expressing HPTP eta mRNA. The antibody also recognized a protein of approximately the same molecular weight in COS cells transfected with HPTP eta cDNA, indicating that the antibody specifically recognized HPTP eta gene product and that the cloned cDNA contained full-length coding region. The chromosomal localization determined by fluorescence in situ hybridization showed that the HPTP eta gene was located at chromosome 11p11.2 on the short arm of chromosome 11, which is frequently lost or deleted in human carcinomas.

Amino Acid Sequence↗
PubMed1994

Isolation, characterization and structural organization of the gene and pseudogene for the dihydrolipoamide succinyltransferase component of the human 2-oxoglutarate dehydrogenase complex.

In the present study, the dihydrolipoamide succinyltransferase gene of the 2-oxoglutarate dehydrogenase complex was isolated from a human genomic DNA library and its entire nucleotide sequence was determined. This gene was approximately 23 kbp in size with 15 exons and 14 introns. All of the donor and acceptor splice sites of this gene conformed to the GT/AG rule. A guanine residue 43 bases upstreams of the ATG initiating translation codon was the transcription initiation site of the human dihydrolipoamide succinyltransferase mRNA. Sequence analysis of the promoter-regulatory region showed the presence of a CAAT-box-like sequence but the presence of a TATA-box-like sequence was not evidenced. Also located in this region were sequences resembling glucocorticoid-responsive and cAMP-responsive elements, and an Sp1 binding site. No nucleotide sequence corresponding to the E3-binding and/or E1-binding domain was found in any region of the gene. Therefore, the exon coding for the E3-binding and/or E1-binding domain may have been lost from the gene during evolution. Moreover, a processed pseudogene of dihydrolipoamide succinyltransferase was isolated and sequenced. The nucleotide sequence of the pseudogene is 93% similar to the sequence of the human dihydrolipoamide succinyltransferase cDNA, but the pseudogene is not functional for base changes, deletions and insertions of the pseudogene. Southern-blot analysis showed the presence of a single copy of this gene and a single copy of a pseudogene in the human genome. In addition, a possible relationship between dihydrolipoamide succinyltransferase and familial Alzheimer's disease is discussed.

Acyltransferases↗
PubMed1994

BST-1, a surface molecule of bone marrow stromal cell lines that facilitates pre-B-cell growth.

Bone marrow stromal cells are essential for B-lymphocyte development. However, how stromal cells regulate B lymphopoiesis is not clear. In this paper, we report the molecular cloning of a stromal cell line-derived glycosyl-phosphatidylinositol-anchored molecule, BST-1, that facilitates pre-B-cell growth. The deduced amino acid sequence of BST-1 exhibited 33% identity with CD38. BST-1 was expressed in a wide range of tissues and in umbilical vein endothelial cells, whereas it was scarcely expressed in a variety of hematopoietic cell lines. The gene for BST-1 was assigned to chromosome 14q32.3, where immunoglobulin heavy-chain genes are clustered. BST-1 expression was enhanced in rheumatoid arthritis patient-derived bone marrow stromal cell lines that were previously shown to have an enhanced ability to support the growth of a pre-B-cell line as compared with stromal cell lines derived from healthy donors.

ADP-ribosyl Cyclase↗
PubMed1994

Chromosomal localization of the protein tyrosine phosphatase G1 gene and characterization of the aberrant transcripts in human colon cancer cells.

We have recently described the isolation of the human PTPG1 gene which encodes a member of intracellular protein tyrosine phosphatases that may be candidates for tumor suppressor genes. In order to investigate the abnormality of the PTPG1 transcript in various human cancer cell lines, we have analyzed the consensus catalytic region of PTPG1 cDNA, using the reverse transcription polymerase chain reaction. In a colorectal carcinoma cell line, DLD-1, we found three aberrant transcripts. Sequencing analysis revealed that one had a missense point mutation and the remainders contained 77 bp and 173 bp deletions, respectively. These alterations might directly affect their phosphatase activities. Our findings provide the first evidence for the aberrant transcripts of the protein tyrosine phosphatase in human cancer cells, and suggest that the aberration of PTPG1 gene might be involved in the tumorigenesis. Moreover, the human PTPG1 gene is localized on chromosome 7q11.23, a region with frequent abnormalities implicated in some human cancers.

Amino Acid Sequence↗
PubMed1994

Molecular cloning of a human transmembrane-type protein tyrosine phosphatase and its expression in gastrointestinal cancers.

To determine the expression of various protein-tyrosine phosphatases (PTPs) in human gastric cancers, cDNAs encoding conserved PTP domains were amplified by reverse transcriptase polymerase chain reaction from KATO-III cell mRNA and sequenced. Among 72 polymerase chain reaction clones, one of the cDNA sequences encoded a novel potential PTP (stomach cancer-associated PTP, SAP-1). The full length (3.9 kilobases) of the SAP-1 cDNA was further isolated from the KATO-III cell cDNA library and the WiDr cell cDNA library. The predicted amino acid sequence of the SAP-1 cDNA showed that mature SAP-1 consisted of 1093 amino acids and a transmembrane-type PTP, which possessed a single PTP-conserved domain in the cytoplasmic region. The extracellular region of SAP-1 consisted of eight fibronectin type III-like structure repeats and contained multiple N-glycosylation sites. These data suggest that SAP-1 is structurally similar to HPTP beta and that SAP-1 and HPTP beta represent a subfamily of transmembrane-type PTPs. SAP-1 was mainly expressed in brain and liver and at a lower level in heart and stomach as a 4.2-kilobase mRNA, but it was not detected in pancreas or colon. In contrast, among cancer cell lines tested, SAP-1 was highly expressed in pancreatic and colorectal cancer cells. The bacterially expressed SAP-1 fusion protein had tyrosine-specific phosphatase activity. Immunoblotting with anti-SAP-1 antibody showed that SAP-1 is a 200-kDa protein. In addition, transient transfection of SAP-1 cDNA to COS cells resulted in the predominant expression of a 200-kDa protein recognized by anti-SAP-1 antibody. SAP-1 is mapped to chromosome 19 region q13.4 and might be related to carcinoembryonic antigen mapped to 19q13.2.

Amino Acid Sequence↗
PubMed1994

Myelomonocytic crisis with t(5;17) and a p53 mutation in a patient with chronic myelogenous leukemia.

We report a 64-year-old Japanese man with chronic myelogenous leukemia (CML) who expired with myelomonocytic crisis. Cytogenetic analyses of chronic phase (CP) and accelerated phase (AP) cells revealed a Philadelphia chromosome and an isochromosome for the long arm of chromosome 17, i(17q). This karyotype was replaced by another karyotype in blast crisis (BC), resulting in near triploidy with t(5;17) (p15;p11) and loss of chromosome 17 pter-->p11. Interphase fluorescent in situ hybridization studies with a chromosome 17 specific alpha satellite DNA probe confirmed the presence of a clonal change in BC. In addition, single-strand conformation polymorphism analysis and PCR-direct sequencing of BC cells revealed a point mutation at codon 203 of the p53 gene, GTG to GAG (Val to Glu), and loss of the normal allele. In contrast, no alterations of the p53 gene were found in CP and AP cells. Therefore, progression of CML in this patient appeared to be related to loss of 17p, as well as a mutation in the p53 gene.

Alleles↗
PubMed1994

Mapping of the 8q23 translocation breakpoint of t(8;13) observed in a patient with multiple exostoses.

A detailed cytogenetic map was constructed around the chromosomal breakpoint of t(8;13) observed in a patient with multiple exostoses. The order of seven loci defined by cosmid clones mapped to 8q23 was determined by means of two-color fluorescence in situ hybridization (FISH) on elongated prophase chromosomes, and localizations of these markers relative to the breakpoint were examined. The results indicated that loci defined by cC18-553 and cC18-1512 flank the breakpoint. By pulsed-field gel electrophoresis of DNA digested with BssHII and Southern hybridization with cC18-1512, DNA from the patient showed a band which was not observed in DNA isolated from either parent. As the normal size of this BssHII fragment is 600 kb, the chromosomal breakpoint probably lies less than 600 kb away from cC18-1512.

Cell Line↗
PubMed1994

Detailed analysis of loss of heterozygosity on chromosome band 17p13 in breast carcinoma on the basis of a high-resolution physical map with 29 markers.

We have constructed a physical map of chromosome band 17p13, using 29 markers that had been localized to 17p13 by means of fluorescence in situ hybridization (FISH) and analysis by pulsed-field gel electrophoresis (PFGE). The map spans nearly 8 Mb of genomic DNA, and the estimated average distance between each marker is roughly 290 kb. The p13 band of chromosome 17 is thought to contain a putative tumor suppressor gene in addition and distal to TP53. Deletion mapping in a large number of breast carcinomas indicated that the tumor suppressor gene lies between the loci defined by cC117-708 (D17S878) and p144D6 (D17S34), which are an estimated 7 Mb apart. Our results should contribute to construction of a contig map of chromosome band 17p13 with cosmid and/or YAC (yeast artificial chromosome) clones, and to isolation of the putative tumor suppressor gene.

Breast Neoplasms↗
PubMed1994

Fluorescence in situ hybridization analysis of chromosomal localization of three human cytochrome P450 2C genes (CYP2C8, 2C9, and 2C10) at 10q24.1.

Chromosomal localization of three human cytochrome P450 genes belonging to the CYP2C subfamily (CYP2C8, 2C9, and 2C10) was identified by fluorescence in situ hybridization (FISH). An original MP-8 clone was used as a DNA probe for the assignment of the CYP2C10 gene, while two cDNA probes, a 1.37 kb fragment of CYP2C8 and a 1.19 kb fragment of CYP2C9, were obtained after amplifying the predicted fragments (MP-20 and MP-4 clones, respectively) by polymerase chain reaction using a single human liver cDNA library. The results showed that three human CYP2C8, 2C9, and 2C10 cDNAs were located at the same subchromosomal region, 10q24.1.

Aryl Hydrocarbon Hydroxylases↗
PubMed1994

Characterization of an 800 kb region at 3p22-p21.3 that was homozygously deleted in a lung cancer cell line.

We have characterized a homozygous deletion at 3p22-p21.3 found in a lung cancer cell line ACC-LC-5. Three yeast artificial chromosomes (YACs) were isolated from a YAC library by hybridization with two cosmid probes (cCI3-1994 and cCI3-1999) representing loci that were homozygously deleted in five lung cancer cell lines. Cloning both ends of the region deleted in the cell line ACC-LC-5 revealed the deletion was an interstitial deletion within the chromosomal arm. A cosmid contig map covering the entire region corresponding to the homozygous deletion was constructed by means of Southern hybridizations. From these results and analyses by pulsed-field gel electrophoresis, this interstitial deletion on chromosome 3p22-21.3 in this cell line was estimated to be nearly 800 kb long and is smallest among five cell lines containing the homozygous deletion. The cosmid clones representing this region will contribute important new resources for isolating the putative tumor suppressor gene(s) on chromosome 3p22-21.3.

Base Sequence↗
PubMed1994

Human Fas ligand: gene structure, chromosomal location and species specificity.

Fas ligand (FasL) is a 40 kDa type II membrane protein belonging to the tumor necrosis factor family, which induces apoptosis by binding to its receptor, Fas. In this report, we isolated the chromosomal gene for human FasL. The human FasL gene consists of approximately 8.0 kb and is split into four exons. The human FasL gene was mapped on chromosome 1q23 by in situ hybridization against human metaphase chromosomes. Human FasL cDNA was isolated by the reverse polymerase chain reaction of mRNA prepared from human activated peripheral blood lymphocytes. Human FasL is a type II membrane protein consisting of 281 amino acids with a calculated M(r) of 31,759. It has an identity of 76.9% at the amino acid sequence level with mouse FasL. Both human and mouse recombinant FasL expressed in COS induced apoptosis in the cells expressing either human Fas or mouse Fas, indicating that FasL fully cross-reacts between human and mouse. A comparison of human and mouse FasL chromosomal genes indicated that a approximately 300 bp sequence upstream of the ATG initiation codon is highly conserved between them. Several transcription cis-regulatory elements such as SP-1, NF-kappa B and IRF-1 were recognized in this region.

Adult↗
PubMed1994

Human arylhydrocarbon receptor: functional expression and chromosomal assignment to 7p21.

We isolated the human arylhydrocarbon receptor (AhR) cDNA from a human lung cDNA library, by using mouse AhR cDNA as a labeled probe. The nucleotide sequence of cloned human AhR cDNA is identical to the previously reported human AhR sequence [Dolwick et al. (1993), Mol. Pharmacol. 44, 911-917] from cell line HepG2. The overall amino acid identity with mouse AhR from cell line Hepa-1 is 72.5%. The human AhR expressed either in COS-7 cells or in a reticulocyte lysate in vitro translation system showed specific dioxin-binding activity and Arnt-dependent DNA-binding activity. Chromosomal localization of the AhR gene was determined to be chromosome 7p21 by fluorescent in situ hybridization and DNA blot hybridization using 23 human x mouse or Chinese hamster hybrid cell DNAs.

Amino Acid Sequence↗
PubMed1994

High resolution ordering of DNA markers by multi-color fluorescent in situ hybridization of prophase chromosomes.

To improve resolution for physical ordering of adjacent DNA loci, prophase chromosomes were used for multi-color fluorescent in situ hybridization (FISH). The prophase chromosomes were prepared from cultured lymphocytes by a thymidine synchronization, bromodeoxyuridine release technique and then treating the synchronized cultures with topoisomerase II inhibitors ICRF154 or ICRF193. Almost all mitotic figures exhibited highly elongated prophase chromosomes without significant reduction of the mitotic index. Using multi-color FISH with these prophase chromosomes, we were able to distinguish signals for loci separated by as little as 50 kb, and determine their orientation. Furthermore, using this prophase ordering system, we confirmed the linear order and defined the orientation of seven cosmid markers within a 360-kb region surrounding D10S102, a locus that is closely linked to the disease locus in families segregating an allele causing multiple endocrine neoplasia IIA (MEN2A). This prophase FISH system, by rapidly and precisely providing the linear order of loci that are very close, can expedite construction of fine cytogenetic maps and contribute to positional-cloning studies in which the precise ordering of DNA loci in a target region is critical.

Cells, Cultured↗
PubMed1994

Molecular cloning and analysis of the human Tec protein-tyrosine kinase.

Mouse Tec is a non-receptor type protein-tyrosine kinase and is highly expressed in many hematopoietic cell lines. To investigate the roles of the Tec kinase in the human hematopoietic system, we isolated cDNAs encoding the human Tec kinase. The human tec cDNAs can encode a peptide of 631 amino acid residues with a calculated molecular mass of 73,624. The predicted human Tec protein is highly homologous to those of the members of the Tec family including mouse Tec type IV (94% homology), mouse Tsk/Itk (60%), and human Btk (57%). The homology between human Tec and other members of the Tec family can be observed not only in the Src homology 3 (SH3), SH2, and kinase domains, but also in the N-terminal unique domain. Northern blot analysis demonstrated that the major transcripts of tec could be detected at 2.6 kb and 3.6 kb in a wide range of human hematopoietic cell lines including myeloid, B-, and T-cell lineages. Interestingly, high expression of the tec gene could be detected in all of the three patients examined with myelodysplastic syndrome. The human tec gene was mapped by fluorescence in situ hybridization (FISH) to chromosome 4p12.

Amino Acid Sequence↗
PubMed1994

N-ras mutation and karyotypic evolution are closely associated with leukemic transformation in myelodysplastic syndrome.

We performed a longitudinal analysis of the karyotypes and N-ras gene configuration of bone marrow cells in 35 patients with myelodysplastic syndrome (MDS). Karyotypic evolution was found in eight patients, and was associated with disease progression, including leukemic transformation, in all the patients. We identified N-ras mutations in six patients, using a polymerase chain reaction (PCR) technique, in which oligonucleotide primers were constructed with induced mismatches, followed by endonuclease digestion. Direct sequencing confirmed single base substitutions at codon 12 in two patients and at codon 13 in four. The incidence of N-ras gene mutations was significantly higher in the karyotypically evolved group (five of eight patients) than in the stable group (one of 27 patients). All of five patients harboring both karyotypic evolution and an N-ras mutation showed concomitant disease progression to overt leukemia or refractory anemia with excess of blasts in transformation (RAEB-T). Two of four patients with either karyotypic evolution or N-ras mutation and six of 26 patients without any of these alterations also progressed to overt leukemia. Our results indicate that the accumulation of these genetic alterations is closely associated with leukemic transformation of MDS, although other genetic alterations may also play a key role in the remaining patients.

Adult↗
PubMed1994

Molecular cloning of a novel non-receptor tyrosine kinase, HYL (hematopoietic consensus tyrosine-lacking kinase).

We identified a novel non-receptor tyrosine kinase from a human megakaryoblastic cell line, UT-7, by means of a PCR-based cloning method. The HYL gene contained a SH2 and SH3 domain and a tyrosine kinase catalytic domain. The deduced amino acid sequence of the protein encoded by this gene was most homologous to CSK (c-src kinase). This gene and CSK shared some unique structural properties such as the absence of a myristylation signal and phosphorylation sites of tyrosine residues corresponding to tyrosines 416 and 527 of chicken p60c-src. Unlike CSK, the SH3 domain of HYL was unique since the ALYDY motif was absent. Northern blot analysis revealed a 2.2 kb transcript in various myeloid cell lines but not in adult tissues except for the brain and the lung, whereas CSK mRNA was ubiquitously expressed. The expression of HYL was upregulated when these myeloid cells were differentiated by induction with phorbol myristate acetate. We named this gene, hematopoietic consensus tyrosine-lacking kinase, HYL. The HYL gene was assigned to chromosome 19 at band p13. It is suggested that HYL plays a significant role in the signal transduction of hematopoietic cells.

Amino Acid Sequence↗
PubMed1993

Human dihydrolipoamide succinyltransferase: cDNA cloning and localization on chromosome 14q24.2-q24.3.

We isolated cDNA for dihydrolipoamide succinyltransferase from a human fibroblast cDNA library in lambda gt11. The cDNA revealed that the human dihydrolipoamide succinyltransferase lacked a sequence motif of an E3 and/or E1 binding site. This suggests that the human dihydrolipoamide succinyltransferase possesses a unique structure consisting of two domains in contrast with the dihydrolipoamide acyltransferases of other alpha-keto acid dehydrogenase complexes. In addition, we found that the human dihydrolipoamide succinyltransferase gene is located on chromosome 14 at q24.2-q24.3 and that a sequence related to the dihydrolipoamide succinyltransferase gene is located on chromosome 1 at p31. Interestingly, the gene for the dihydrolipoamide acyltransferase of the branched chain alpha-keto acid dehydrogenase complex is also located on chromosome 1p31 (Zneimer et al. (1991) Genomics 10, 740-747).

Acyltransferases↗