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T Seno

Publications and source records attributed to T Seno.

At least 55 records · Page 3Linked to original sources
PubMed1992

Regulatory sequences clustered at the 5' end of the first intron of the human thymidylate synthase gene function in cooperation with the promoter region.

A human thymidylate synthase (TS) minigene containing 5'- and 3'-flanking sequences, all the exons, and only intron 1 showed a normal frequency of stable transformation when transfected into TS-negative mutant cells, whereas minigenes in which intron 1 was replaced by intron 2 or deleted in the above construct showed only a few percent of the above frequency. Introduction of intron 1 into the above intronless or intron 2 minigene restored the transforming activities regardless of its position and orientation. Deletion analysis revealed two positive and one negative regulatory sequences in the 5' end of intron 1, each of which seemed to bind specific proteins as shown by gel shift analysis. Intron 1 also stimulated expression of a TS promoter-CAT gene construct but not that of an SV40 promoter-CAT gene construct. These results indicate that the multiple regulatory sequences clustered in intron 1 stimulate TS gene expression in concert with the 5'-flanking sequences.

Gene Expression Regulation, Enzymologic↗
PubMed1992

Complementation by a cloned human ubiquitin-activating enzyme E1 of the S-phase-arrested mouse FM3A cell mutant with thermolabile E1.

A temperature-sensitive growth mutant tsFS20 isolated from mouse FM3A cells was identified as a mutant with thermolabile ubiquitin-activating enzyme E1 by transfection with a full-length cDNA encoding the human E1 enzyme and cell-cell hybridization with an authentic E1 mutant ts85 previously isolated from FM3A cells. The resulting transformants produced thermoresistant E1 activity. Upon shift-up of temperature, asynchronously growing tsFS20 cells showed multiple points of cell-cycle arrest. At the nonpermissive temperature, tsFS20 cells that had been synchronized at the G1-S-phase progressed and accumulated in the mid-S-phase, as evidenced by the absence of G2-specific cdc2 kinase activity, while ts85 mutant cells, the widely used E1 mutant, reached the G2-phase and were arrested. Thus, the E1 mutation seemed to be involved in progression in the S-phase as well as in the G2-phase in the cell cycle. Degradation of short-lived abnormal proteins in tsFS20 cells was decreased to about 50% at the nonpermissive temperature, while the block was fully restored to the wild-type level in the transformant cells. Relevance of the unusually high incidence of the temperature-sensitive E1 mutation was discussed in terms of the E1 as a determinant of heat tolerance of cells.

Animals↗
PubMed1991

Thymidylate stress induces homologous recombination activity in mammalian cells.

We studied whether homologous recombination activity in mammalian cells could be induced by thymidylate stress (thymidylate deprivation). In vitro recombination activity in cell extracts was measured with pSV2neo-derived plasmids. When prior to the preparation of extracts, mouse FM3A cells were grown in 5-fluorodeoxyuridine (FdUrd), an inducer of thymidylate stress, the homologous recombination activity was significantly induced, as judged from an increase in the number of neomycin-resistant bacterial colonies. Maximum induction was observed in cells treated with 1 microM FUdR for 16 h. However, 3-8 h of treatment of FM3A cells with the drug followed by an additional 8-16-h incubation in its absence was sufficient to induce the recombination activity while slightly reducing their growth rates. These results indicate that thymidylate stress induces homologous recombination activity in mammalian cells as observed in Escherichia coli and in yeast.

Animals↗
PubMed1991

SAT, a 'new' low frequency blood group antigen, which may be associated with two different MNS variants.

A new private blood group antigen, SAT, was identified in an NFLD-Japanese woman as a result of testing 10,480 blood donors with a serum containing anti-NFLD and anti-SAT. Three other sera were subsequently also shown to contain anti-SAT. The donor's family showed that SAT is inherited as a dominant character and may be associated with a weak M antigen. Serological and immunochemical analysis revealed no other aberrations in the MNS system. Study of a second SAT+Japanese blood donor and his family suggested that SAT is associated with an unusual MNS variant resulting from a hybrid glycophorin comprising the N-terminus of glycophorin A and the C-terminus of glycophorin B. The propositus appears to be homozygous for the gene that produces the putative hybrid, which differs from previously described glycophorin (A-B) hybrids by expressing no S, s or U antigen. SAT antigen, therefore, may be associated with two different MNS variants in the only two families in which it has been identified.

Animals↗
PubMed1991

[Aging and exocrine pancreatic function evaluated by the recently standardized secretin test].

The authors studied the relationship between aging and exocrine pancreatic function by the secretin test which was recently standardized by the Japanese Society of Gastroenterology. Pancreatic juice was collected at 10 min intervals for 60 minutes after a bolus intravenous injection of secretin (Secrepan, Eisai Co., Ltd., 100 U/body) through a quadruple-lumen doudenal tube equipped with double balloons. Exocrine pancreatic function was evaluated by three parameters: secretory volume, maximal bicarbonate concentration or bicarbonate output, and enzyme (amylase and lipase) output. Control subjects consisted of 65 outpatients presenting with mild vague abdominal symptoms who fulfilled the following three criteria: 1) good general condition with no known diseases; 2) no abnormality in the liver, bile duct, pancreas, kidney and metabolism judged from blood chemistry, urine and stool analysis, upper GI series, abdominal ultrasonography (US), and endoscopic retrograde cholangiopancreatography (ERCP); 3) alcohol consumption less than 25 g/day. Control subjects were divided into three groups: 15 subjects below 40 years of age (group A), 32 subjects from 40 to 65 years (group B), and 18 subjects of 65 years and above (group C). Nineteen patient with chronic pancreatitis were also studied. The group C showed significantly lower values in secretory volume, bicarbonate output, and enzyme output than group A and B. Enzyme output showed a gradual decrease with aging. However, secretory volume and bicarbonate output showed a gentle convex curve with a peak around age 40 and a rather steep down-slope after late 50s. The degree of the decrease was significantly more marked in volume and bicarbonate output than in enzyme output in group C.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1990

Structural and functional analysis of the human thymidylate synthase gene.

The complete nucleotide sequence of the human thymidylate synthase (TS) gene was determined. The biologically active unit spans about 16 kilobase pairs (kbp) and is composed of seven exons and six introns. The promoter region and the major transcriptional start sites were located within about 400 base pairs (bp) and 160-180 bp, respectively, upstream from the ATG initiation codon. A minigene consisting of a 5'-flanking sequence of about 4 kbp, a coding sequence of about 1.7 kpb was sufficient for cell cycle-dependent expression of TS when introduced into a TS-deficient mutant of rodent cells. The 5'-flanking region does not contain a "TATA," "CAAT," or the consensus sequence for Sp1 binding (GC box), whereas intron 1 contains three GC boxes. The gene is G + C-rich and the CpG-rich region extends from 5'-flanking region to intron 1. These sequence characteristics were compared with those of the mouse TS gene and other cell cycle-regulated genes.

Animals↗
PubMed1990

Gangliosides and sialoglycoproteins carrying a rare blood group antigen determinant, Cad, associated with human cancers as detected by specific monoclonal antibodies.

Two murine monoclonal antibodies, 2A3D2 and 2D11E2 (both IgM), which are directed to the gangliosides and sialoglycoproteins related to a rare blood group antigen, Cad, were obtained by using a ganglioside mixture prepared from human hepatocellular carcinoma cells (PLC/PRF/5) as the immunogen. These two monoclonal antibodies detected multiple ganglioside antigens present in the PLC/PRF/5 cells, and the major antigenic ganglioside was characterized as IV4GalNAc beta-GD1a, which has the carbohydrate structure GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----3GalNAc beta 1---- 4(NeuAc alpha 2----3)Gal beta 1----Cer. The two antibodies also reacted with GM2 (GalNAc beta 1----4[NeuAc alpha 2----3]Gal beta 1----4Glc beta 1----Cer) and a Cad-active lactoseries ganglioside (IV4GalNAc beta-sialosylparagloboside, GalNAc beta 1----4[NeuAc alpha 2----3]Gal beta 1----4GlcNAc beta 1---- 3Gal beta 1----4Glc beta 1----Cer), which have carbohydrate structures related to IV4GalNAc beta-GD1a. Beside gangliosides, both antibodies recognized the carbohydrate determinant carried by glycophorin A on very rare Cad-positive human RBC; the structure of which is GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----3(NeuAc alpha 2---- 6)GalNAc alpha 1----Ser/Thr. From these findings, it is clear that monoclonal antibodies 2A3D2 and 2D11E2 both recognize the nonreduced carbohydrate terminus composed of three sugar residues, GalNac beta 1----4(NeuAc alpha 2----3)Gal beta 1----R, and are useful for detecting the Cad-related antigen in cells and tissues. By using these monoclonal antibodies, it was revealed that many cultured human hepatocellular carcinoma cell lines and cancer tissues taken from patients with hepatocellular carcinoma contain both Cad-active glycoprotein antigens and related gangliosides, while normal liver tissues contain no appreciable amount of either species of antigen. The Cad-active glycoprotein antigens in cultured human hepatocellular carcinoma cells appeared as triplet bands having molecular weights of 92,000, 75,000, and 61,000, under either reducing or nonreducing conditions in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Essentially the same triplet proteins were observed in as many as 4 of 9 cases (44%) of cancer tissue from patients with hepatocellular carcinoma, but not in neighboring cirrhotic tissues or normal livers tissues. These results suggest that the rare blood group antigen Cad is associated with human cancers, especially hepatocellular carcinoma.

Animals↗
PubMed1990

Regional assignment of the human thymidylate synthase (TS) gene to chromosome band 18p11.32 by nonisotopic in situ hybridization.

The human thymidylate synthase (TS) gene was regionally assigned to chromosome band 18p11.32 by nonisotopic in situ hybridization using biotinylated cDNA (1.1 kb insert) and genomic DNA (6.8 kb insert) probes of the human gene. There have been two provisional assignments for the TS gene to 18pter-q12 and 18q21-qter. The present result confirmed the first of these and further localized the TS gene to the telomeric region of the short arm of chromosome 18. The TS gene appears to be a novel telomeric anchor point for the construction of both physical and genetic linkage maps of human chromosome 18.

Chromosomes, Human, Pair 8↗
PubMed1990

High level of aphidicolin resistance with multiple mutations in mouse FM3A cell mutants.

Spontaneous mutants of mouse FM3A cells (AC1, AC2, and AC3), highly resistant to aphidicolin (3000-, 2500-, and 300-fold increase in resistance, respectively), were isolated by multistep selection. The DNA synthesizing activity in permeabilized cells of all three mutants was substantially resistant to aphidicolin, like that in intact cells. The DNA polymerase activity in nuclear extracts in AC1 and AC3, but not AC2, was resistant to aphidicolin. Partially purified DNA polymerase alpha from AC3, but not from AC1 or AC2, showed resistance to aphidicolin. The apparent Ki value for aphidicolin of AC3 polymerase alpha was three to four times that of the enzyme from the parent cells, but the apparent Km values of the enzyme for dCTP and dTTP were normal. All the mutants showed cross-resistance to both arabinofuranosyladenine and arabinofuranosylcytosine. The AC3 mutant had expanded deoxyribonucleoside triphosphate pools. On two-dimensional polyacrylamide gel electrophoresis, AC1 gave a new protein (mol wt 40 kDa). The aphidicolin-resistance trait was reversible in AC2, unlike in AC1 and AC3. These results show that in mammalian cells there are at least two mechanisms of aphidicolin-resistance that involve an altered DNA polymerase alpha that is resistant to aphidicolin and simultaneous expansion of the four DNA-precursor pools.

Animals↗
PubMed1990

Purification and characterization of alpha-L-fucosidase from Bacillus circulans grown on porcine gastric mucin.

Bacillus circulans isolated from soil was found to produce two types of alpha-L-fucosidase differing in substrate specificity. One was able to liberate L-fucose from porcine gastric mucin (PGM), but not from artificial substrates, including p-nitrophenyl and methyl alpha-L-fucosides, while the other acted not on PGM but on p-nitrophenyl alpha-L-fucoside. The production of the former enzyme was enhanced about 150 times as much by PGM added to the medium as by glucose. The alpha-L-fucosidase acting on PGM was purified from the culture fluid obtained with PGM medium by ammonium sulfate fractionation and subsequent column chromatography. The purified enzyme was found to be homogeneous by PAGE and its molecular weight was estimated to be approximately 285,000. The optimum pH was found to be 5.5 to 6.5 and the stable pH range was 4.5 to 9.0. The enzyme decomposed various blood group O(H) active substances such as PGM, human milk and human saliva, and moreover acted on A-, B-, and O-erythrocytes. The enzyme was shown to cleave alpha-(1----2)-, (1----3)-, and (1----4)-L-fucosidic linkages in various glycoproteins and oligosaccharides, but failed to hydrolyze alpha-(1----6)-L-fucosic linkages in 6-O-alpha-L-fucopyranosyl-N-acetylglucosamine and intact bovine thyroglobulin.

ABO Blood-Group System↗
PubMed1989

Family study and frequency of blood group with strong B transferase accompanied by decreased A and H antigens.

Subgroups of type A blood, named A1, A2, and A1-A2 intermediate (Aint), are specifically characterized by their peculiar A alleles and have their own A1-, A2- or Aint-forms of alpha-N-acetyl-D-galactosaminyltransferase (A-transferase). It is known, however, that certain type A2B persons exhibit A1-transferase. The reason may be an unusual alpha-galactosyltransferase (B-transferase). This strong B-transferase competes with A-transferase for the substrate, H antigen, so as to decrease the A and H antigens on the red cells. We studied this blood group over three generations and found that the strong B-transferase is, in fact, inherited with the B gene and is dominant over normal B-transferase. In AB blood groups in Tokyo, the frequency of people with a strong B-transferase is 5% for A1B and 22% for A2B. This enzyme does not always cause weak H or A antigens.

ABO Blood-Group System↗
PubMed1989

Two types of mouse FM3A cell mutants deficient in 5-aminoimidazole-4-carboxamide ribonucleotide transformylase and their transformants isolated by human chromosome-mediated gene transfer.

We isolated three adenine auxotrophic mutants (Ade1, Ade2 and Ade3) of mouse FM3A cells deficient in 5-aminoimidazole-4-carboxamide ribotide transformylase (EC 2.1.2.3) activity. Ade1 and Ade3 but not Ade2 also lacked inosinicase (EC 3.5.4.10) activity. While Ade2 and Ade3 complemented each other, Ade1 complemented neither Ade2 nor Ade3, suggesting that two complementation groups exist in these mutants. We introduced human genes into the Ade2 and Ade3 cells by chromosome-mediated gene transfer. All the transformants tested were found to produce the human transformylase and inosinicase, and identical DNA bands containing human Alu sequences were detected in the transformants of Ade2 and Ade3. These mutants seem to have arisen by mutation in the same gene or adjacent genes, since only human chromosome 2 was capable of rescuing the genetic defects in all these mutants.

Acyltransferases↗
PubMed1989

Identification and characterization of an L1 family sequence with a very long open reading frame in the third intron of the human thymidylate synthase gene.

A long L1 repetitive sequence (3.6 kilobase pairs) was found in the third intron of the human thymidylate synthase gene. This L1 family sequence is unique in that it possesses the longest open reading frame (1.7 kilobase pairs) of all L1 family members identified in sequences associated with specific genes that have been cloned thus far. Furthermore, the amino acid sequence deduced from the open reading frame of the L1 sequence was found to be highly homologous (90%) to that encoded by a known human teratocarcinoma L1 RNA species, and to contain several blocks of sequences homologous to ones in RNA-dependent DNA polymerases of various origins.

Amino Acid Sequence↗
PubMed1989

Human thymidylate synthase gene: isolation of phage clones which cover a functionally active gene and structural analysis of the region upstream from the translation initiation codon.

Two genomic DNA fragments partially encoding human thymidylate synthase (TS) [EC 2.1.1.45] were previously cloned in lambda phage from the mouse cell transformant, but had no transforming activity on mouse TS-negative mutant cells. In this study, an additional genomic DNA for human TS was cloned and demonstrated to have the transforming activity in combination with one of the two previously cloned DNAs and to produce human TS mRNA. The two transforming genomic DNAs overlapped and covered a region of 23 kb in total. Using fragments from one of these DNAs, the structure of the 1.2-kb region around the ATG initiator codon of the TS gene was analyzed in relation to regulatory sequences of the gene. Sequence determination demonstrated the presence of an unusual inverted repeat consisting of a triple tandem repeat of a 28-bp sequence and an inverted sequence of the same length. These sequences can form three possible, stable, stem-loop structures, which may be interconvertible. Based on S1 nuclease mapping data and a line of circumstantial evidence, we deduced two major mRNA cap sites within the inverted sequence. Comparison of the human and mouse sequences upstream from the ATG initiator codon revealed many significant blocks of sequence homology, especially in the regions around the deduced cap sites.

Animals↗