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Biomedical subjects

E Ohtsuka

Publications and source records attributed to E Ohtsuka.

At least 217 records · Page 12Linked to original sources
PubMed1987

A new solid-phase synthesis of oligoribonucleotides by the phosphoro-p-anisidate method using tetrahydrofuranyl protection of 2'-hydroxyl groups.

Six nonaribonucleotides containing the 5'-splice site, one complementary nonamer and an octadecamer containing the 3'-splice site have been synthesized on a polymer support using the phosphoro-p-anisidate method. A 5'-linked 2'-O-tetrahydrofuranyl-N-protected nucleoside 3'-(o-chlorophenyl)phosphoro-p-anisidate was used as the starting nucleotide, and the chain elongated in the 3'-direction by removing the p-anisidate protecting group with isoamyl nitrite under neutral conditions. The octadecamer has been synthesized using dinucleotide blocks and a 3'-terminal trinucleotide.

Base Sequence↗
PubMed1987

Base sequence-specific interactions of operator DNA fragments with the lambda-cro repressor coupled with changes in their conformations.

The mechanism of interaction of the operator DNA with the lambda-cro repressor protein was investigated using proton n.m.r. and photo CIDNP. Three kinds of DNA duplexes, the lambda-OR3 17-mer, phi80-OR2 19-mer and CRP binding site 22-mer, were prepared, and all of their imino proton resonances of the complexes with lambda-cro were assigned to individual base pairs. By monitoring the assigned signals of the DNA fragments and lambda-cro, it was found that in the complex of lambda-cro with lambda-OR3, two subunits of the cro dimer bind to the right and left halves of the OR3, respectively, and the bidentate binding induces a structural distortion in the middle of the 17-mer. lambda-cro itself also undergoes a conformational change including loosening of the dimeric form. In the complex of lambda-cro with phi 80-OR2, which has a 6-bp sequence common to that of lambda-OR3, one subunit of the cro dimer seems to bind specifically to the common part. However, there is only a slight conformational change in the cro dimer. In the mixture of the CRP binding site 22-mer and lambda-cro, soft contact without any conformational change was observed between them.

Base Sequence↗
PubMed1987

Construction of an excretion vector and extracellular production of human growth hormone from Escherichia coli.

A new excretion vector, pEAP8, was constructed to develop an excretion system for Escherichia coli. This plasmid, derived from pEAP37, carried the weakly activated kil gene of plasmid pMB9 [Kobayashi et al., J. Bacteriol. 166 (1986) 728-732] and the penicillinase promoter and signal region of an alkalophilic Bacillus sp. to excrete foreign gene products. A gene for human growth hormone (hGH) was joined to this signal sequence through the HindIII site. The recombinant plasmid p8hGH1 thus constructed, was introduced into E. coli. The hybrid protein which was produced in E. coli carrying p8hGH1 was processed during transport through the inner membrane, with the mature hGH being excreted into the medium through the outer membrane which was made permeable by the action of the kil gene. The N-terminal amino acid sequence and the biological activity of the extracellular hGH were consistent with those of the authentic hGH.

Animals↗
PubMed1987

Efficient cleavage by alpha-thrombin of a recombinant fused protein which contains insulin-like growth factor I.

The gene for insulin-like growth factor I (IGF-I) was constructed from chemically synthesized deoxyoligonucleotides and expressed in Escherichia coli, under the control of a trp promoter, as a set of fusion proteins which were connected with a portion of human growth hormone through the recognition sequence for a sequence-specific protease, either blood coagulation factor Xa or alpha-thrombin. Upon induction with 3-indoleacrylic acid, fusion proteins accumulated with a yield of 10-30% of the total protein. A fusion protein connected through a tetradecapeptide (Asp-Asp-Pro-Pro-Thr-Val-Glu-Leu-Gln-Gly-Leu-Val-Pro-Arg) was efficiently and correctly cleaved by alpha-thrombin, and the purified IGF-I possessed somatomedin-like activity, as determined by the enhancement of sulfation of glycosaminoglycans in cultured costal chondrocytes from rabbits.

Biological Assay↗
PubMed1987

Sequence-dependent hydrolysis of RNA using modified oligonucleotide splints and RNase H.

To cleave RNA molecules using RNase H in a site-specific manner, a short deoxyoligonucleotide (3-5mer) joining with 2'-O-methyl oligonucleotide(s) was designed as a DNA splint to be used. Model experiments were carried out using ribooligonucleotide substrates (9 and 18 mer). It was found that the use of this type of splints (9 mer) causes a unique cleavage by RNase H. For example, when 3'm (GA)d(AGAA)m(GGU)5' was used as a hybridization strand, 32pUCUUUCUUCUUCCAGGAU was cleaved specifically between U11 and C12 to yield 32pUCUUUCUUCUU. This method will have a variety of applications for the study of RNA.

Base Sequence↗
PubMed1986

Studies on nucleic acid interactions. I. Stabilities of mini-duplexes (dG2A4XA4G2-dC2T4YT4C2) and self-complementary d(GGGAAXYTTCCC) containing deoxyinosine and other mismatched bases.

The thermal stability of DNA duplexes containing deoxyinosine in a pairing position in turn with each of the four major deoxynucleotides has been investigated by measuring ultraviolet-absorbance at different temperatures. d(G2A4 X A4G2) and d(C2T4YT4C2) were prepared by the solid-phase phosphotriester method. When X is deoxyinosine, the Tm values of the duplexes are in the order Y = dC greater than dA greater than dG greater than dT greater than dU. The Tm of other duplexes containing dG, dA and dT at X were also measured. Self-complementary duplexes d(GGGAAINTTCCC) showed the same order of stability with N being dC, dA, dG and dT. Thermal stabilities of duplexes containing dG instead of dI were compared with other matched and mismatched duplexes. The Tm values of sequence isomers containing purine-pyrimidine combinations were compared. Self-complementary duplexes containing G-C and A-T in the central positions showed Tm values ca. 10 degrees higher than those containing C-G and T-A in the same positions. Thermodynamic parameters and circular dichroism spectra of these oligonucleotides were compared.

Base Composition↗
PubMed1986

Modification of Glu 58, an amino acid of the active center of ribonuclease T1, to Gln and Asp.

Glu 58 is one of the amino acids which participates in its catalytic action of ribonuclease T1. We mutated this residue to Gln 58 or Asp 58 by genetic engineering using chemically synthesized genes. The mutant enzymes were expressed in E. coli as fused proteins and purified to homogeniety on SDS-PAGE after cleavage with cyanogen bromide. Their activities in hydrolyzing pGpC were reduced to 10% in the Asp 58 mutant and about 1% in the Gln 58 mutant compared to that of the wild-type enzyme. These results suggest that Glu 58 is important but not essential for catalysis of ribonuclease T1.

Aspartic Acid↗
PubMed1986

Structure- and sequence-specificity of ozone degradation of supercoiled plasmid DNA.

Ozone-reactive sites on the nucleobase moieties in supercoiled pBR322 DNA were investigated by using sequencing procedures. Ozonolysis in the absence of salt resulted in degradation of thymine residues in the A + T rich region located at 3100-3400bp. In the presence of salt, such as NaCl or MgCl2, a conformational change of plasmid DNA was induced. Subsequently the thymine and guanine residues in the loop of the cruciform located at 3120bp and 3220bp were degraded. In addition, central thymine residues present in sequences GTA, GTT and ATA were also degraded.

Base Sequence↗
PubMed1986

Comparison of the antigenicities of native human growth hormone (hGH) and three forms of recombinant hGHs using monoclonal antibodies.

A panel of hybridomas producing antibodies specific for human growth hormone (hGH) were prepared by using a recombinant hGH [methionylsomatotropin (r-hGH)] as an immunogen. Thirteen representative monoclonal antibodies which showed different reactivity patterns were used to analyze the antigenicities of four different forms of hGHs by RIA inhibition studies. Native hGH and r-hGH showed almost the same antigenicities with these monoclonal antibodies. A Cys-substituted recombinant hGH (r-hGH-165) retained the epitopes recognized by 11 monoclonals but not those recognized by two monoclonals. All except one of the monoclonals showed little or no reactivity with a recombinant hGH fragment (r-hGH-AB). On the basis of these results, the differences in the structures and antigenicities of the recombinant hGH proteins were discussed.

Animals↗
PubMed1986

Cloning a defined region of DNA using a limited action of DNA polymerase: application to dissection of hepatitis B virus surface antigen gene.

Using dodecadeoxynucleotides as primers for DNA synthesis and 3'-o-chlorophenyl-phosphorylated dodecadeoxynucleotides as "stoppers" for chain elongation, pre-defined regions of a gene previously cloned in M13 single-stranded (ss) DNA phage were converted into double-stranded (ds) DNA utilizing the action of the Klenow fragment of Escherichia coli DNA polymerase I (PolIk). The resulting ds DNA was freed from the ss region by S1 nuclease treatment. This method can be used to obtain DNA fragments of any size with pre-defined 5' and 3' ends. About 15% of the input ss DNA template molecules are converted into ds DNA fragments. This technique was used to synthesize several DNA fragments from different portions of the hepatitis B virus surface antigen (HBsAg) gene. The products were then ligated into a yeast plasmid vector that carries the E. coli lacZ gene which is located downstream from the yeast acid-phosphatase promotor. Using this system, several fragments of HBsAg were produced in the form of beta-galactosidase fused protein.

Bacterial Proteins↗
PubMed1986

Molecular cloning and sequence determination of cDNAs for alpha subunits of the guanine nucleotide-binding proteins Gs, Gi, and Go from rat brain.

We have cloned cDNAs encoding alpha subunits of the guanine nucleotide-binding proteins Gs, Gi, and Go and determined their nucleotide sequences. Purified preparations of Gi and Go alpha subunits (Gi alpha and Go alpha) from rat brain were completely digested with trypsin, and peptides were subjected to amino acid sequence analysis. By screening of a cDNA library from rat C6 glioma cells with a synthetic probe corresponding to a 17 amino acid sequence, a clone encoding the sequence of Go alpha was obtained. Then, the library was rescreened with a Go alpha cDNA probe to isolate several strongly or weakly hybridizing clones. cDNAs encoding the complete sequences of Gi alpha and Gs alpha were thus obtained. From nucleotide sequence analysis, the amino acid sequences of Gs alpha and Gi alpha were deduced; they contain 394 and 355 amino acid residues (including the initiator methionine), respectively. The calculated molecular weights for Gs alpha and Gi alpha were 45,663 and 40,499, respectively. The Go alpha clone encoded a sequence of 310 amino acid residues that lacked the NH2 terminus. The homology of the alpha subunits of Gs, Gi, Go, transducin, and ras-encoded protein is discussed.

Animals↗
PubMed1986

Inquiries into the structure-function relationship of ribonuclease T1 using chemically synthesized coding sequences.

The genes for ribonuclease T1 and its site-specific mutants were chemically synthesized and introduced to Escherichia coli. All enzymes were fusion products produced by joining the synthetic gene at specific restriction sites to the synthetic gene for human growth hormone in a plasmid containing the E. coli trp promoter. The fusion protein from this plasmid contained 66% of the amino-terminal sequences of the human growth hormone, which were recognizable immunologically. RNase T1 or its mutants were cleaved from the fusion protein with cyanogen bromide. The synthetic RNase T1 endowed with the revised wild-type triad Gly-Ser-Pro, residues 71-73, was fully functional, readily hydrolyzing pGpC bonds, whereas a mutant enzyme having the originally reported, erroneous triad Pro-Gly-Ser was totally inactive. Various amino acid substitutions were also introduced to the guanosine recognition region comprised of residues 42-45, Tyr-Asn-Asn-Tyr. Substitution of either of the tyrosine residues noted above with phenylalanine had no dramatic effect on the enzyme's function. Replacement of asparagine-43 with arginine or alanine also caused only a small change in the hydrolyzing activity--a mutant enzyme maintained greater than 50% of the wild-type activity. In sharp contrast, when aspartic acid or alanine was substituted for asparagine-44, the activity was dramatically reduced to a few percent of the wild-type activity.

Amino Acid Sequence↗
PubMed1986

Amino acid sequence of mammalian elongation factor 2 deduced from the cDNA sequence: homology with GTP-binding proteins.

Complementary DNA clones, pHEW1 and pRE2, coding for hamster and rat polypeptide chain elongation factor 2 (EF-2), respectively, were isolated and sequenced. It was shown that the cDNA insert in pHEW1 contains a 2574-base-pair open reading frame coding for an 857-amino acid polypeptide with Mr 95,192, excluding the initiation methionine. Comparative studies of sequence homology among EF-2 and several GTP-binding proteins show that five regions in the amino-terminal position of EF-2, corresponding to about 160 amino acids, show homology with GTP-binding proteins, including protein synthesis elongation and initiation factors, mammalian ras proteins, and transducin. The carboxyl-terminal half of EF-2 contains several regions that have 34-75% homology with bacterial elongation factor G. These results suggest that the amino-terminal region of EF-2 participates in the GTP-binding and GTPase activity whereas the carboxyl-terminal region interacts with ribosomes. Finally, the sequence provides direct evidence that diphthamide (2-[3-carboxy-amido-3-(trimethylammonio)propyl]histidine), the site of ADP-ribosylation by diphtheria toxin, is produced by post-translational modification of a histidine residue in the primary translational product.

Amino Acid Sequence↗