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T Takeya

Publications and source records attributed to T Takeya.

At least 55 records · Page 3Linked to original sources
PubMed1987

Structural features of the carboxy terminus of p60c-src that are required for the regulation of its intrinsic kinase activity.

The major structural difference between p60c-src and p60v-src resides in the carboxy-terminal region. We introduced substitution, replacement and deletion into the carboxy-terminal region of chicken p60c-src and examined the effects on its biochemical and biological properties in chicken embryo fibroblasts. The replacement of the carboxy-terminal region after amino acid position 515 of p60c-src with the corresponding region of p60v-src converted p60c-src to a transforming protein with elevated kinase activity. The substitution of Tyr527, which is a major tyrosine phosphorylation site of p60c-src, with phenylalanine resulted in an increase of kinase activity and in transforming ability. On the other hand, a deletion after position 515 abolished the kinase activity. These results revealed the structural features of the carboxy terminus of p60c-src that are required for the regulation of its intrinsic protein kinase activity.

Animals↗
PubMed1986

Amino acid substitutions sufficient to convert the nontransforming p60c-src protein to a transforming protein.

We have previously shown that Rous sarcoma virus variants that carry the cellular homolog (c-src) of the viral src gene (v-src) do not transform chicken embryo fibroblasts. We also have shown that replacement of sequences upstream or downstream from the BglI site of the cellular src gene with the corresponding regions of v-src restored transforming activity to the hybrid genes. Since there are only six amino acid changes between p60c-src and p60v-src within the sequences upstream from BglI, we constructed chimeric molecules involving v-src and c-src to determine the effect of each amino acid substitution on the biological activities of the gene product. We found that the change from Thr to Ile at position 338 or the replacement of a fragment of c-src containing Gly-63, Arg-95, and Thr-96 with a corresponding fragment of v-src containing Asp-63, Trp-95, and Ile-96 converted p60c-src into a transforming protein by the criteria of focus formation, anchorage-independent growth, and tumor formation in newborn chickens. These mutations also resulted in elevation of the protein kinase activity of p60c-src.

Amino Acid Sequence↗
PubMed1985

Two independent mutations are required for temperature-sensitive cell transformation by a Rous sarcoma virus temperature-sensitive mutant.

We molecularly cloned the src coding region of tsNY68, a mutant of Rous sarcoma virus temperature sensitive (ts) for transformation, and constructed a series of ts wild-type recombinant src genes. DNA containing the hybrid genes was transfected into chicken cells together with viral vector DNA and helper viral DNA, and infectious transforming viruses were recovered. Characterization of these recombinant viruses indicated that at least two mutations are present in the 3' half of the mutant src gene, both of which are required for ts. Nucleotide sequence analysis revealed three differences in the deduced amino acid sequence compared with the parental virus. Two of these changes, a deletion of amino acids 352 to 354 and an amino acid substitution at position 461, are responsible for the ts phenotype.

Animals↗
PubMed1984

Rous sarcoma virus variants that carry the cellular src gene instead of the viral src gene cannot transform chicken embryo fibroblasts.

The transforming activity of the cellular src (c-src) gene as well as of hybrid genes between viral and cellular src was tested by constructing derivatives of Rous sarcoma virus DNA in which all or part of the viral src gene (v-src) was replaced by the corresponding portion of the c-src gene. After these derivatives were introduced into chicken embryo fibroblasts by transfection, replication-competent virus was recovered, which induced the expression of p60src at a level equivalent to p60v-src expression in cells infected with Rous sarcoma virus wild type. Replacement of the portion of the v-src gene, either upstream or downstream of the Bgl I site, with the homologous portion of the c-src gene resulted in fully transforming viruses. On the other hand, the virus stock obtained from cells transfected with Rous sarcoma virus DNA containing the entire c-src gene had a very low titer of focus-forming virus, while it contained a high titer of infectious virus. We present evidence that the rare small foci are formed by mutant viruses generated from the original c-src-containing virus. These results indicate that overproduction of the c-src gene product does not cause cell transformation, and that this proto-oncogene is subject to a relatively high rate of mutation when incorporated in a retrovirus genome, resulting in the acquisition of transforming capacity.

Animals↗
PubMed1983

Structure and sequence of the cellular gene homologous to the RSV src gene and the mechanism for generating the transforming virus.

We determined the nucleotide sequences of all coding regions and a significant part of the flanking regions of the chicken c-src gene, which is a cellular homolog of the v-src gene of Rous sarcoma virus. The c-src gene consists of 12 exons; the boundaries of the exons were determined by assuming that the amino acid sequence of its product, pp60c-src, is basically the same as that of pp60v-src. The deduced amino acid sequence of pp60c-src was very similar to that of pp60v-src, but the last 19 carboxy-terminal amino acids of pp60c-src were replaced by a new set of 12 amino acids of pp60v-src. The sequence encoding the carboxy-terminal sequence of pp60v-src was found 900 bp downstream from the termination codon of the c-src gene. We suggest that the c-src sequence was captured by a virus through recombination at both sides of the c-src gene, and that the recombinations occurred at the level of proviral DNA.

Animals↗
PubMed1982

DNA sequence of the viral and cellular src gene of chickens. 1. Complete nucleotide sequence of an EcoRI fragment of recovered avian sarcoma virus which codes for gp37 and pp60src.

Recovered avian sarcoma virus is a class of virus obtained from chicken tumors induced by mutants of Rous sarcoma virus which have a deletion in the src gene. We have determined the entire nucleotide sequence of a 3.1-kilobase EcoRI DNA fragment of molecularly cloned recovered avian sarcoma virus DNA. This DNA fragment contains part of the env gene and the entire src gene. Amino acid sequences of both gene products were deduced from the DNA sequences; the predicted amino acid sequences were verified by protein studies. An env protein (gp37) was found to be composed of 205 amino acids with three glycosylation sites. gp37 had a long stretch of hydrophobic residues near the carboxyl terminus. The src gene product, pp60src, was composed of 526 amino acids and contained the possible sites for tyrosine and serine phosphorylation. The amino acid sequences predicted in this study differ significantly from the amino acid sequence predicted previously for the Schmidt-Ruppin strain of Rous sarcoma virus.

Amino Acid Sequence↗
PubMed1982

DNA sequence of the viral and cellular src gene of chickens. II. Comparison of the src genes of two strains of avian sarcoma virus and of the cellular homolog.

The nucleotide sequence of the src gene and flanking regions of the Schmidt-Ruppin strain of Rous sarcoma virus (SR-A) was determined. The src region of SR-A was very homologous to that of recovered avian sarcoma virus (rASV1441), with only 17 differences among 1,578 nucleotides. The size of the predicted protein was 526 amino acids in both viruses, of which 6 amino acids were different. The differences in nucleotides and amino acids between the two viruses localized within the 5' two-thirds of the src coding region. There were also viruses localized within the 5' two-thirds of the src coding region. There were also some differences in the region flanking the 5' end of src. Since rASVs are considered to be recombinatns between deletion mutants of SR-A and cellular-src (c-src) sequences, several segments of c-src DNA were also sequenced to understand the molecular basis for the recombination. At 14 of 17 bases where SR-A and rASV1441 differed, rASV1441 had the same sequence as c-src. Three of these sequences corresponded to sequences of oligonucleotides which were previously identified in RNAs of nearly all isolates of rASV but which were absent in SR-A RNA. In the 5'-flanking sequences of the src gene, c-src was more similar to rASV1441 than to SR-A. These results confirm the cellular origin of the src sequences of rASVs and provide information about the possible sites of the recombination.

Animals↗
PubMed1981

Comparison between the viral transforming gene (src) of recovered avian sarcoma virus and its cellular homolog.

Recovered avian sarcoma viruses are recombinants between transformation-defective mutants of Rous sarcoma virus and the chicken cellular gene homologous to the src gene of Rous sarcoma virus. We have constructed and analyzed molecular clones of viral deoxyribonucleic acid from recovered avian sarcoma virus and its transformation-competent progenitor, the Schmidt-Ruppin A strain of Rous sarcoma virus. A 2.0-megadalton EcoRI fragment containing the entire src gene from each of these clones was subcloned and characterized. These fragments were also used as probes to isolate recombinant phage clones containing the cellular counterpart of the viral src gene, termed cellular src, from a lambda library of chicken deoxyribonucleic acid. The structure of cellular src was analyzed by restriction endonuclease mapping and electron microscopy. Restriction endonuclease mapping revealed extensive similarity between the src regions of Rous sarcoma virus and recovered avian sarcoma virus, but striking differences between the viral src's and cellular src. Electron microscopic analysis of heteroduplexes between recovered virus src and cellular src revealed a 1.8-kilobase region of homology. In the cellular gene, the homologous region was interrupted by seven nonhomologous regions which we interpret to be intervening sequences. We estimate the minimum length of cellular src to be about 7.2 kilobases. These findings have implications concerning the mechanism of formation of recovered virus src and possibly other cell-derived retrovirus transforming genes.

Alpharetrovirus↗
PubMed1979

Total synthesis of a tyrosine suppressor transfer RNA gene. XVII. Transcription, in vitro, of the synthetic gene and processing of the primary transcript to transfer RNA.

Primer- and promoter-dependent transcription of the synthesis gene had been studied. Primer-dependent transcription gave, as a major product, an end-to-end transcript which was strand-specific. The transcript was characterized rigorously by two-dimensional separation and analysis of the oligonucleotides formed on digestion with T1-RNase and pancreatic RNase and by nearest neighbor analyses of the oligonucleotides obtained when different alpha-32P-labeled ribonucleoside triphosphates were used as substrates. Minor products accompanying the major transcript were characterized similarly. The major transcript, when treated with an Escherichia coli S-100 extract, was processed to the tRNATyr with correct 5'- and 3'-ends. The nucleolytic cleavages occurring at the 3'-end were characterized. In promoter-dependent transcription, transcription of a restriction fragment containing phi80psu+III gene and the synthetic gene with and without the promoter were compared. Transcription of the synthetic gene was promoter-dependent and strand-specific, the initiation of transcription occurring at the same point as previously found in vivo. Although the synthetic gene contains only 16 base pairs corresponding to the natural sequence following the C-C-A end, processing of the transcript at the 3'-end occurred normally, the endonucleolytic cleavage being followed by exonucleolytic cleavages. The products of promoter-dependent transcription were completely characterized. An examination of the base modifications of the primary transcript during treatment of the latter with E. coli S-100 extract showed couplete modification of uridine to pseudouridine and partial methylation of uridine to ribosylthymine in TpsiCG sequence and partial formation of pseudouridine in the anticodon loop. However, hardly any formation of 2'-O-methylguanosine or of 2-methylthio-6-isopentenyl adenosine could be detected.

Base Sequence↗
PubMed1979

Total synthesis of a tyrosine suppressor transfer RNA gene. XVI. Enzymatic joinings to form the total 207-base pair-long DNA.

The total synthesis of a 207-base pair-long DNA, which is biologically functional as a tyrosine suppressor transfer RNA gene, has been completed. The synthesis involved the enzymatic joining of the previously synthesized duplexes. Thus, the duplex corresponding to the promoter region [P] (Sekiya, T., Brown, E.L., Ramamoorthy, B., Fritz, H.-J., Gait, M.J., Lees, R.G., Ryan, M.J., Khorana, H.G., and Norris, K.E. (1979) J. Biol. Chem. 254, 5781-5786) was jointed to Duplex [I] (Caruthers, M.H., Kleppe, R., Kleppe, K., and Khorana, H.G. (1976) J. Biol Chem. 251, 658-666) to form [P + I]. Separatively, Duplex [III + IV + Vb] was prepared from the previously described Duplexes [III], [IV], and [Vb]. (Loewen, P.C., Miller, R.C., Panet, A., Sekiya, T., and Khorana, H.G. (1976) J. Biol. Chem. 251, 642-650; Sekiya, T., Besmer, P., Takeya, T., and Khorana, H.G. 1976) J. Biol. Chem. 251, 634-641; Ramamoorthy, B., Lees, R.G., Kleid, D., and Khorana, H.G., (1976) J. Biol Chem. 251, 676-694). The product [P + I], was joined to Duplex [II] (Panet, A., Kleppe, R., Kleppe, K., and Khorana, H.G. (1976) J. Biol. Chem. 251, 651-657) and then to [III + IV + Vb] without isolation of the intermediates. In all the above joinings, the duplexes carried 32P-labeled phosphate groups at the appropriate 5'-ends. The total DNA and the intermediate duplexes were all characterized by their relative mobilities in electrophoresis on polyacrylamide gel slabs, by nearest neighbor analysis, and by degradation to 5'-nucleotides of radioactively labeled joined products. Two succeeding papers describe the transcription in vitro and the suppressor activity in vivo, of the synthetic gene now described.

Base Sequence↗
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↗
PubMed1979

Physiological changes in the treatment of acrophobia (fear of height).

In order to investigate the physiological changes produced by the treatment of acrophobic patients body movement and Microvibration were measured before and after treatment. Eighteen acrophobic patients were assigned at random to 1 of the 2 groups: a treatment group (n = 8) and a non-treatment group (n = 10). The control group consisted of 16 healthy volunteers. Body movement area while viewing a slide of a high place or imagining a high place in the treatment group decreased significantly after treatment. Body movement of the control group showed almost no changes, and that of the non-treatment group was situated between the above-mentioned 2 groups. Simple body movements of the acrophobic patients without any stimulation of height were bigger than those of the control subjects. MV pattern of the treatment group had a tendency to improve under psycho-therapy. Acrophobic patients had more abnormal MV patterns than the normal subjects.

Adult↗
PubMed1979

Location of a ColE1 deoxyribonucleic acid region that affects the plaque-forming ability of lambda-ColE1 hybrid bacteriophage.

The plaque-forming ability of a hybrid phage between plasmidColE1 and phage lambda carrying amber mutations in genes O and P was inhibited by the presence of ColE1 in suppressor-deficient Escherichia coli cells. ColE1 deoxyribonucleic acid regions concerned with this inhibition were examined by using various deletion and transposon insertion derivatives of ColE1, and it was found that the presence of the deoxyribonucleic acid region extending between 420 and 613 base pairs upstream from the initiation site of ColE1 deoxyribonucleic acid replication (J. Tomizawa, H. Ohmori, and R. E. Bird, Proc. Natl. Acad. Sci. U. S. A. 74: 1865--1869, 1977) was essential for this function.

Bacteriophage lambda↗
PubMed1979

Relationships among differentiated T-cell subpopulations. IV. Preferential induction of delayed footpad reaction to xenografts and its radioresistant nature.

Immune response in mice to xenogeneic cells was characterized by positive delayed footpad reaction and negative macrophage migration inhibition. Mice immunized with allogeneic cells exhibited negative delayed footpad reaction and positive macrophage migration inhibition. Cytotoxic T lymphocytes were detected only in mice immunized with allogeneic tumour cells. Delayed footpad reaction against xenogeneic cells was radioresistant and the immune lymphocytes responsible for such a reaction were presumed to have some relation to xenograft rejection.

Animals↗
PubMed1978

Biofeedback modification of frontal EMG in normal subjects.

We carried out a controlled study on the voluntary control of the frontalis muscle by biofeedback procedures employing 20 normal subjects. Subjects were randomly divided into two groups of 10: (1) the biofeedback group and (2) the control group. Each of the two groups received five training sessions of about 40 minutes' duration each on different days. The results obtained are as follows: (1) In the biofeedback group, mean EMG levels decreased progressively and markedly from 2. 16 muVp-p min the first session to 1.54 muVp-p min in the last session. On the contrary, the control group did not show constant decreases in EMG levels over sessions. (2) The changes in the heart rate did not correlate with the changes in EMG activity. (3) The changes in the respiratory rate correlated with the changes in EMG activity.

Adult↗