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T Tamaoki

Publications and source records attributed to T Tamaoki.

At least 127 records · Page 7Linked to original sources
PubMed1988

Guanine nucleotide binding properties of purified v-Ki-ras p21 protein produced in Escherichia coli.

We have purified the v-Ki-ras p21 protein by expression in Escherichia coli of a plasmid encoding 189 amino acids of the Kirsten murine sarcoma virus oncogene. The purified p21 (over 95%) exhibited a tenfold greater affinity for GTP (1.6 x 10(-7) M) than for GDP (1.1 x 10(-6) M). The preferential binding of v-Ki-ras p21 towards GTP was attributed to the faster dissociation rate of GDP from the protein. The affinity of GDP to v-Ki-ras p21 decreased with the depletion of Mg2+ while that of GTP did not change significantly. The dissociation constant rate of the p21-GDP complex was estimated as 2 x 10(-3) s-1 in the presence of Mg2+ and increased to 2 x 10(-2) s-1 after the removal of Mg2+. These results are discussed with respect to the role of GDP-GTP exchange in the regulation of p21 function.

Escherichia coli↗
PubMed1987

Cell-specific enhancer activity in a far upstream region of the human alpha-fetoprotein gene.

We describe experiments showing that the 5'-flanking region of the human alpha-fetoprotein (AFP) gene contains transcription control elements with characteristics of enhancers. The enhancer activity was detected and characterized by the ability to direct the expression of a linked chloramphenicol acetyltransferase (CAT) gene in transfected AFP-producing hepatoma cells in culture. The enhancer activity is cell-specific in that it occurs in hepatoma cells producing AFP, but not in non-AFP-producing hepatoma or nonhepatic cells. The active elements can direct CAT expression in conjunction with the SV40 promoter in an orientation- and position-independent manner. The sequences important for enhancer activity reside in the 400-base pair region between 3.3 and 3.7 kilobase pairs upstream of the AFP gene. This and proximal upstream regions contain multiple enhancer "core"-like sequences and other stretches of potential biological significance.

Acetyltransferases↗
PubMed1986

Staurosporine, a potent inhibitor of phospholipid/Ca++dependent protein kinase.

Staurosporine, microbial alkaloid which has been known to have antifungal activity was found to inhibit markedly phospholipid/Ca++dependent protein kinase (protein kinase C) from rat brain, with an IC50 value of 2.7 nM. However, it had little effect on the binding of 3H-phorbol-12, 13-dibutyrate (PDBu) to protein kinase C. The inhibition of protein kinase C was not competitive with phospholipid. This compound also showed the strong cytotoxic effect on the growth of HeLa S3 cells, with an IC50 value of 4 X 10(-12)M under the condition of 72 hr-exposure.

Alkaloids↗
PubMed1986

The human albumin gene. Characterization of the 5' and 3' flanking regions and the polymorphic gene transcripts.

We have characterized the 5' and 3' regions of the human albumin gene with respect to nucleotide sequences, mRNA transcription initiation and polyadenylation. There are at least two transcription initiation sites and two polyadenylation sites in the human albumin gene. The multiple transcription initiation sites are utilized almost equally. The two polyadenylation sites are used differentially, with the site proximal to the protein termination codon dominating. The "TATA" and the "CCAAT" boxes are present at about 30 and 90 nucleotides upstream of the putative cap sites. About 250 nucleotides immediately 5' to the transcription initiation sites are highly conserved among the human, rat, and chicken. Within the 1-kilobase pair 5' flanking region of the human albumin gene there are 8- and 35-base pair alternating purine/pyrimidine sequences, primarily consisting of dG and dT, and three nucleotide segments exhibiting homology to the proposed progesterone receptor-binding site. In these and other characteristics, the 5' flanking region of the human albumin gene is distinct from the corresponding region of the human alpha-fetoprotein gene. Each of the two polyadenylation sites of the human albumin gene is preceded by a AATAAA hexamer. In addition, the first polyadenylation site has several conserved sequences believed to play a role in the 3'-end formation. The second polyadenylation site lacks these signals which may explain why it is used less frequently. In spite of these differences the two polyadenylation sites show a high overall sequence similarity, suggesting that they arose by duplication. There is no major difference between the normal liver and hepatoma cells in the relative utilization of multiple sites for transcriptional initiation and polyadenylation of the human albumin gene, suggesting that the albumin mRNA polymorphism has no direct relevance to hepatocarcinogenesis.

Albumins↗
PubMed1986

Mythylation of human HLA-D/DR genes: derangement in chronic lymphocytic leukemia.

HLA-DR antigens are expressed as differentiation markers in certain human leukemias. To investigate whether DNA methylation plays a role in expression of DR genes in leukemia, we analyzed methylation patterns of the DR-alpha and D/DR-beta genes in the DR antigen-positive and -negative B-cell lines, in normal adults and in chronic lymphocytic leukemia (CLL) patients using Southern blot hybridization of DNA digested with Msp I and Hpa II. The DR-alpha and D/DR-beta genes of a DR antigen positive B-cell line, T5-1, were heavily methylated, while those of DR antigen-negative variant, 6.1.6, were hypomethylated. Blood cells collected from four normal adults contained different levels of DR-alpha and D/DR-beta mRNAs, but their relative amounts were about the same among the individuals. By contrast, the relative amounts of these mRNAs in CLL cells varied widely, indicating aberrant expression of one or both of these genes in CLL. The DR-alpha gene in four normal adults and six CLL patients produced only a 3 kb hybridizable band after Msp I digestion. Normal adult DR-alpha genes were resistant to Hpa II digestion, suggesting that all Hpa II sites are methylated. In contrast, digestion of CLL DNA with Hpa II yielded various bands of larger sizes which differed among the CLL patients, suggesting that Hpa II sites are differentially methylated in the CLL DNA. In the case of D/DR-beta genes, normal adult DNA gave Msp I bands which were slightly polymorphic among four individuals tested. In contrast, CLL DNA showed a high degree of restriction fragment length polymorphism (RFLP) on Msp I digestion. We speculate that the high RFLPs in the CLL DNA may result from differential methylation in CpG clusters in the D/DR-beta genes, and that this characteristic may be of use for diagnosis of CLL.

Adult↗
PubMed1985

Expression of intact Ki-ras p21 protein in Escherichia coli.

We have constructed recombinant plasmids capable of expressing in Escherichia coli the intact ras p21 protein encoded by Kirsten murine sarcoma virus. The Ki-ras gene was inserted into an expression vector carrying the E. coli tryptophan promoter and E. coli lipoprotein transcriptional terminator. The resulting plasmids direct the synthesis of large quantities of p21 protein, which represented 20% of the total cellular protein. The Ki-ras p21 protein is immunoprecipitated with monoclonal antibody to p21, and exhibits guanine nucleotide binding activity and autophosphorylation activity. The purified Ki-ras p21 expressed in E. coli has shown to have intact N-terminal and C-terminal amino acid sequences predicted by the nucleotide sequences and migrate as -23K in SDS/polyacrylamide gels.

Amino Acid Sequence↗
PubMed1985

The human alpha-fetoprotein gene. Sequence organization and the 5' flanking region.

The human alpha-fetoprotein (AFP) gene was isolated into three overlapping clones in bacteriophage lambda vectors and its sequence organization analyzed by restriction endonuclease mapping and nucleotide sequencing. The human AFP gene is about 20 kilobase pairs long and contains 15 exons and 14 introns. The overall organization of the human AFP gene is similar to that of the mouse AFP gene, with all but two exons showing identical sizes. Nucleotide sequences at all exon/intron junctions display similarity to the consensus boundary sequence (Breathnach, R., and Chambon, P. (1981) Annu. Rev. Biochem. 50, 349-383), with the GT-AG rule applied to the splicing point. The cap site maps 44 nucleotides upstream from the translation initiation site. The "TATA box" is located 27 nucleotides upstream from the putative cap site and is flanked by sequences with dyad symmetry. The TATA box can thus be placed in the loop portion of a possible stem-loop structure formed by intrastrand base-pairing. Other characteristic nucleotide sequences in the 5' flanking region include a CCAAC pentamer, a 14-base pair (bp) enhancer-like sequence, and a 9-bp sequence homologous to the glucocorticoid responsive element. A long (90 bp) direct repeat and several alternating purine/pyrimidine sequences are also present in the 5' flanking region. A 736-bp sequence of the 5' flanking region adjacent to the cap site of the human AFP gene shows a 61% similarity with the corresponding region of the mouse AFP gene. There are two Alu family sequences and two poly(dT-dG) repeats in the human AFP gene that show different distribution patterns from those in the mouse AFP gene.

Amino Acid Sequence↗
PubMed1985

Isolation of cDNA clones encoding a T-cell receptor beta-chain from a beef insulin-specific hybridoma.

cDNA clones coding for a T-cell receptor beta-chain were isolated from a beef insulin/IAd-reactive T-cell hybridoma, A20.2.15, and its complete amino acid sequence was deduced. This beta-chain gene utilizes the same V beta segment as a thymocyte beta-chain gene (86T1) and rearranges to the 5' proximal J-C locus (J beta 1-C beta 1), thus providing definitive evidence of a beta-chain gene from a functional hybridoma that utilizes C beta 1. The amino acid sequence of the V beta gene in A20.2.15 is identical to 86T1, thus suggesting the absence of somatic mutation in the beta-chain of A20.2.15. Southern blot analysis revealed a somatic DNA rearrangement unique to the A20.2.15 hybridoma. The expression of this gene in the hybridoma was confirmed by RNA dot hybridization. All 24 beta-chain clones so far isolated from the A20.2.15 hybridoma contained C beta 1, suggesting that the beta-chain gene of the fusion partner BW5147 is not expressed in the hybridoma.

Amino Acid Sequence↗
PubMed1985

Optimal alignments of biological sequences on a microcomputer.

An algorithm and a program have been developed which enable optimal alignments of biological sequences on an 8-bit microcomputer. The compiled program can process sequences up to 1000 residues on a Commodore 64. Since this program was written originally in the BASIC language, it may readily be adapted to other microcomputers with small changes.

Algorithms↗
PubMed1985

Detection of messenger RNAs of alpha-fetoprotein and albumin in a human hepatoma cell line by in situ hybridization.

Detection of RNA transcripts within individual cells by in situ hybridization provides a powerful means for identifying specific cell types actively transcribing specific genes. We have applied this technique in order to analyze expression of the alpha-fetoprotein and albumin genes in a human hepatoma cell line, HuH-7. Using either 3H- or 35S-labeled human alpha-fetoprotein complementary DNA clone as a probe, we found that essentially all HuH-7 cells contained alpha-fetoprotein mRNA, although in various amounts. This correlated well with the presence of alpha-fetoprotein in all cells as detected by the peroxidase-antiperoxidase immunoenzyme method. The intracellular concentration of albumin, on the other hand, was below the level of detection by the peroxidase-antiperoxidase method. Consistent with this observation, we could not detect albumin mRNA with 3H-labeled albumin complementary DNA probes. However, the use of 35S-labeled probes having higher specific activities and higher efficiency of grain development resulted in the detection of albumin mRNA in a small percentage of HuH-7 cells. A variety of parameters involved in the in situ hybridization technique was examined to establish conditions suitable for demonstrating the presence of high- and low-copy numbers of mRNA in various cell and tissue preparations.

Albumins↗
PubMed1984

Tandem arrangement of the albumin and alpha-fetoprotein genes in the human genome.

A genomic clone containing human albumin mRNA sequences was isolated from a lambda phage gene library derived from a human hepatoma cell line that produces alpha-fetoprotein (AFP) and albumin. This clone was also found to hybridize with the 5'-flanking region of the human AFP gene. Restriction mapping and nucleotide sequencing revealed that the albumin and AFP genes are present in tandem, in the same transcriptional orientation, with the albumin gene 14.5 kb upstream of the AFP gene. We have also isolated a genomic clone carrying both the albumin and AFP gene sequences from human fibroblasts, which produce neither AFP nor albumin. This DNA showed a restriction map that was indistinguishable from that of the clone obtained from the hepatoma described above, demonstrating that no gross rearrangements of the intergenic DNA sequence are involved in control of expression of the AFP and albumin genes.

Bacteriophage lambda↗
PubMed1983

Secretion and glycosylation of alpha-foetoprotein by the mouse yolk sac.

Secretion and glycosylation of alpha-foetoprotein (AFP) by mouse yolk sac were studied by using yolk-sac explants cultured in vitro. Yolk-sac explants rapidly incorporated [35S]methionine into AFP, whereas radioactively labelled AFP was not found in the medium until 30 min after incubation was initiated. Electrophoretic analysis revealed that microheterogeneity of AFP synthesized in explants increased in parallel with the gestational age of the yolk sacs. The change in microheterogeneity was noted by the formation of increasingly acidic forms. Only the most acidic forms of AFP were found to be present in the medium on each gestational day studied. Tunicamycin reduced the incorporation of glucosamine into AFP with a concomitant decrease in molecular weight and microheterogeneity. However, the relative amount of AFP released into the medium was not altered by the presence of tunicamycin. The presence of under-glycosylated AFP in the medium indicates that glycosylation of AFP is not essential for its secretion from the yolk sac. In light of these and previous findings, it is suggested that the glycosylation of AFP may be important for the turnover of this glycoprotein in serum.

Animals↗
PubMed1983

alpha-Fetoprotein and albumin mRNA levels in liver regeneration and carcinogenesis.

Using a titration procedure, we measured the proportion of alpha-fetoprotein (AFP) and albumin mRNA in normal, regenerating, and preneoplastic rat livers. AFP mRNA constitutes approximately 0.006% of the polysomal polyadenylated RNA of normal livers and this proportion increases only slightly before the onset of DNA synthesis in liver regeneration induced by partial hepatectomy or CCl4 injury. In either model of liver regeneration, the proportion of AFP mRNA in polysomal RNA is highest approximately 24 h after the peak of DNA synthesis. The increase in the proportion of AFP mRNA in polysomal RNA is relatively small during liver regeneration (2-4-fold) but is larger (30-50-fold) in preneoplastic livers of rats fed a choline-deficient diet containing 0.1% ethionine. In contrast to those changes in AFP mRNA, albumin mRNA levels remain unchanged during liver regeneration and double in preneoplastic livers. Our results indicate that the concept of "retrodifferentiation" as it applies to liver regeneration and certain types of hepatic neoplasia needs reevaluation.

Albumins↗