Deoxyribonucleic acids and related compounds. XXII. Synthesis of genes for human nerve growth factor and its fused protein.
Explore the source record for details and available documents.
Amino Acid Sequence↗
Biomedical subjects
E Ohtsuka
Publications and source records attributed to E Ohtsuka.
An NMR study on the structure of OR3 in the lambda cro-OR3 complex.
1H-NMR spectra of lambda-OR3 17mer in the complex with lambda-cro has been analysed and the change in its conformation on the complexation has been deduced. The changed conformation is retained until the duplex melts above 55 degrees C. Increase in heat stability of the cro dimer was observed. The result was compared with those of other DNA fragments phi 80-OR2 19 mer and the CRP binding site 22mer mixed with lambda-cro.
Amino Acid Sequence↗
Synthesis and expression of a synthetic gene for the activated human c-Ha-ras protein.
A 576 base-pair DNA encoding human c-Ha-ras protein (p21) with a valine codon at position 12 has been synthesized by ligation of chemically synthesized oligodeoxyribonucleotides. The duplex was inserted into a plasmid with the E. coli tryptophan promoter and was expressed in E. coli efficiently.
Amino Acid Sequence↗
Chemical synthesis of the T4 endonuclease V gene and its expression in Escherichia coli.
A gene coding for T4 endonuclease V was constructed by joining chemically-synthesized oligodeoxyribonucleotides. This gene was inserted into an expression vector and expressed in E. coli.
Amino Acid Sequence↗
Misreading of 8-hydroxydeoxyguanosine-containing DNA in in vitro DNA replication.
Explore the source record for details and available documents.
8-Hydroxy-2'-Deoxyguanosine↗
Synthesis and expression of a human growth hormone (somatotropin) gene mutated to change cysteine-165 to alanine.
We have mutated a synthetic human growth hormone (hGH) gene specifically at the codon for Cys-165 to a codon for Ala by replacement of synthetic deoxyoligonucleotides corresponding to this site. This modification prevented the formation of a disulfide bond between Cys-53 and Cys-165 in the hGH molecule. This mutated protein, [Ala165]hGH was expressed at the same level as the intact hGH, 4 X 10(5) molecules per cell under the control of the tryptophan promoter in Escherichia coli, and retained similar immunological activity to intact hGH. The limited digestion pattern of the mutated protein with human plasmin suggests that the tertiary structure of [Ala165]hGH resembles to that of the intact hGH molecule. [Ala165]hGH revealed full biological activity as examined by the body weight increase of hypophysectomized rats.
Alanine↗
Interaction of methionine-specific tRNAs from Escherichia coli with immobilized elongation factor Tu.
The interaction of three different Met-tRNAsMet from E. coli with bacterial elongation factor (EF) Tu X GTP was investigated by affinity chromatography. Met-tRNAfMet which lacks the base pair at the end of the acceptor stem binds only weakly to EF-Tu X GTP, while Met-tRNAmMet has a high affinity for the elongation factor. A modified Met-tRNAfMet which has a C1-G72 base pair binds much more strongly to immobilized EF-Tu X GTP than the native aminoacyl(aa)-tRNA with non-base-paired C1A72 at this position, demonstrating that the base pair including the first nucleotide in the tRNA is one of the essential structural requirements for the aa-tRNA X EF-Tu X GTP ternary complex formation.
Chromatography, Affinity↗
Ozonolysis of supercoiled pBR322 DNA resulting in strand scission to open circular DNA.
Treatment of supercoiled pBR322 DNA with ozone resulted in the conversion of closed circular DNA to open circular DNA. Restriction analysis of the resulting open circular DNA showed that ozonolysis in the absence of salt caused single strand cleavage at specific sites.
Chemical Phenomena↗
Synthesis and hybridization of dodecadeoxyribonucleotides containing a fluorescent pyridopyrimidine deoxynucleoside.
Partially self-complementary dodecadeoxyribonucleotides containing a fluorescent nucleoside, 3-beta-D-2'-deoxyribofuranosyl-2, 7-dioxopyrido[2, 3-d]pyrimidine (pyridopyrimidine deoxynucleoside, dF) were synthesized by the phosphotriester solidphase method. A dodecanucleotide d(GGGAAFGTTCCC) pairing the analog and guanine at the centre of the chain showed a higher melting temperature than the corresponding G-C paired duplex. A similar comparison between A-T and A-F suggested that weaker hydrogen bonds exist when adenine and pyridopyrimidine residues are paired.
Circular Dichroism↗
Modification of the amino acid acceptor stem of E. coli tRNAMetf by ligation of chemically synthesized ribooligonucleotides.
The single-stranded region of the amino acid acceptor stem corresponding to the 3'-end of E. coli tRNAMetf was replaced by ligation of chemically synthesized ribooligonucleotides, in order to change the length of the single-stranded CCA terminus. The chemically synthesized ribooligomers, CCA, ACCA, AACCA and CAACCA, were ligated to nuclease-treated E. coli tRNAMetf, which lacked the ACCA sequence at the 3'-end. The methionine acceptor activities of these modified tRNAs were examined using E. coli methionyl-tRNA synthetase. Ligation of the chemically synthesized pentamer (AACCA) to the acceptor terminus restored the methionine acceptor activity, whereas ligation of the hexamer (CAACCA) or trimer (CCA) to the acceptor terminus did not Modification of the acceptor terminus had no effect on the formylation of accepted methionine.
Amino Acids↗
Replacement and insertion of nucleotides at the anticodon loop of E. coli tRNAMetf by ligation of chemically synthesized ribooligonucleotides.
Insertion of the four major nucleotides at the 5'-side of the anticodon triplet of E. coli tRNAMetf was performed by joining of the half molecules obtained by limited digestion with RNase A and the chemically synthesized tetranucleotide pN-C-A-U using RNA ligase. Insertion of U-U at the 5'-side or A and A-A at the 3'-side of the anticodon were also performed using U-U-C-A-U, C-A-U-A and C-A-U-A-A. The constant U next to the 5'-side of the anticodon was replaced with A and C by ligation of A-C-A-U and C-C-A-U to the 5'-half molecule which had been treated with periodate plus lysine, followed by joining to the 3'-half. These modified tRNAs were tested for their ability to accept methionine with the methionyl-tRNA synthetase of E. coli. The affinity of these analogs for the synthetase decreased more extensively when the insertion was at the 3'-side of the anticodon triplet. Insertion of mononucleotides at the 5'-side or replacement of the constant U next to the 5'-side of the anticodon did not affect aminoacylation drastically. This may mean that the 3'-side of the anticodon loop of tRNA is one of the major recognition sites for the methionyl-tRNA synthetase.
Anticodon↗
An alternative approach to deoxyoligonucleotides as hybridization probes by insertion of deoxyinosine at ambiguous codon positions.
Two deoxyoligonucleotide probes (23-mer and 26-mer) carrying deoxyinosine residues (I) at positions corresponding to ambiguous nucleotides derived from amino acid sequence have been synthesized by the phosphotriester method using a polymer support. The 23-mer and 26-mer corresponded to the mRNA for 8 amino acids from gastrin and 9 amino acids from cholecystokinin, respectively. The dIs have been used where the base in the third position of the amino acid codon is ambiguous. These deoxyoligonucleotides were used as probes for hybridization with colonies containing the corresponding cDNAs or genes. The hybrid formed between a gastrin clone and the 23-mer that harbors 5 dIs was dissociated at 50-55 degrees C, suggesting that deoxyinosine did not significantly effect the stabilization or destabilization of the DNA duplex. A similar result was obtained using the 26-mer that contains 5 dIs and a phage clone DNA of the cholecystokinin gene. Thus oligonucleotide probes with deoxyinosine residues at ambiguous points seem to be useful as hybridization probes for cloning genes for proteins containing amino acids with degenerate codons.
Base Sequence↗
Photo CIDNP study on the complex formation of lambda cro protein with OR3.
A photo CIDNP spectrum of lambda cro repressor protein showed that one of the three tyrosines and His 35 are quite accessible to the photosensitive dye. For the remaining two tyrosine residues one is slightly accessible, but the other is inaccessible. In comparison with the result of differential nitration at tyrosine side chains followed by the peptide analysis, it can be concluded that Tyr 26 is mostly exposed and Tyr 51 is slightly exposed on the surface of the cro dimer. On the addition of OR3 17mer, His 35 and Tyr 26 are no longer accessible to the dye, which indicates that they are involved in interaction. However, a similar phenomenon was observed by adding CAP binding site 22mer. The interaction mechanism will be discussed.
Bacteriophage lambda↗
Expression of a synthetic human growth hormone gene in yeast.
A synthetic human growth hormone (hGH) gene was efficiently expressed under the control of the repressible acid phosphatase promoter in yeast (Saccharomyces cerevisiae). More than 10(6) molecules of hormone were formed per cell despite the fact that the gene was constructed with codon preference for Escherichia coli.
Alkaline Phosphatase↗
Use of the deoxyinosine-containing probe to isolate and sequence cDNA encoding the fusion (F) glycoprotein of Sendai virus (HVJ).
A synthetic 20-mer based on the known amino acid (aa) sequence of the N-terminus of Sendai virus F1 polypeptide was synthesized. Using this dI-probe, which contained deoxyinosines at all six ambiguous codon positions, we isolated clones carrying cDNAs for the F mRNA of Sendai virus. Nucleotide (nt) sequence analysis revealed a long open reading frame (ORF) that encodes a protein of 565 aa. Thus, this type of dI-probes should prove useful for selecting cDNA clones, when the aa sequence is known and is characterized by high codon redundancy.
Amino Acid Sequence↗
Molecular cloning of the human cholecystokinin gene by use of a synthetic probe containing deoxyinosine.
A synthetic DNA based on the known amino acid sequence of the brain/gut peptide cholecystokinin (CCK) was synthesized. This DNA contained deoxyinosines at ambiguous codon positions and was used as a probe to isolate the CCK gene directly from a human genomic library. Nucleotide sequence analysis of the isolated gene revealed that human preprocholecystokinin consists of 115 amino acid residues, with 11 amino acids in common with the human gastrin precursor, another member of the gastrin-CCK family, and that the coding region is separated by a single, long intron. CCK appears to be encoded by a single-copy gene in the haploid human genome, as revealed by genomic Southern hybridization analysis, suggesting that the same gene is expressed both in gut and brain.
Amino Acid Sequence↗
Dynamic behavior of the imino protons of the gamma OR3 17mer in H2O solution studied by high-resolution NMR.
The imino proton resonances of gamma OR3 17mer in water were observed at 500 MHz with the time-shared Redfield pulse train. All of the 17 imino proton resonances could be assigned specifically to individual base pairs by utilizing the trace of NOE connectivities between the imino and adenine C2H protons and between imino protons themselves. AT1 and 17 showed abnormally high chemical shifts in comparison with the other AT pairs. On raising the temperature, broadening of the signal occurred in a sequential manner from the terminals except for AT10 and AT11, which were broadened at a lower temperature than GC12. The relaxation rates of the imino protons were measured by the inversion recovery method. The rates at higher temperatures represent the exchange rates of the imino protons. From the temperature dependences, activation energies of about 15 kcal/mol for the AT imino protons and 23-26 kcal/mol for the GC imino protons were obtained.
Bacteriophage lambda↗
Deoxyribonucleic acids and related compounds. XII. Polymer support synthesis of a 46-mer duplex containing the promoter of galactose operon of Escherichia coli.
Explore the source record for details and available documents.
DNA, Bacterial↗