Deoxyribonucleic acids (DNA) and related compounds. XIII. Synthesis of DNA duplexes containing a ribosome binding site.
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Base Sequence↗
Biomedical subjects
E Ohtsuka
Publications and source records attributed to E Ohtsuka.
Specificity of the interaction between lambda cro repressor protein and operator DNA fragments.
Cro repressor protein is known to interact with specific sites in the operator DNA. The cro protein of lambda phage was isolated and the mode of its interaction with three different DNA fragment, lambda-OR3 17mer, phi 80-OR2 19mer and CAP binding site 22-mer, were examined by the use of proton NMR. Some of the imino proton resonances of lambda-OR3 shifted and were broadened remarkably on addition of lambda-cro protein, which indicated the induction of conformational change with complexation. In the spectrum of phi 80-OR2 which has a six base pair sequence common to lambda-OR3 the signals of the common base pairs revealed slight shifts on addition of lambda-cro protein. The imino proton signals of the CAP site DNA, however, did not show any change at all on mixing with lambda-cro. Combining the data of photo CIDNP of lambda-cro, we could postulate the mode of interaction between lambda-cro repressor and operator DNA.
Bacteriophage lambda↗
[Synthesis of genes].
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Amino Acid Sequence↗
Synthesis and expression of the native RNase T1 gene and several mutant genes.
RNase T1 gene and several mutant genes were constructed by joining of chemically synthesized deoxyoligonucleotides. These genes were inserted into an expression vector and expressed as fused protein in E. coli. RNase T1 and its mutant enzymes were liberated by cyanogen bromide treatment and their activities were measured.
Amino Acid Sequence↗
Synthesis and properties of novel nucleic acid probes.
2'-O-Methyl derivatives of common ribonucleosides were prepared from appropriately protected nucleosides via the methylation of the 2'-hydroxyl groups with methyl iodide or diazomethane. These derivatives were converted into 2'-O-methylribonucleotide units and the guanosine derivative was linked to a polymer support. Thus, oligo(2'-O-methylribonucleotides) having the sequence of AmCmUmUmAmCmCmUmGm was synthesized in a stepwise manner by using the phosphotriester method. Thermal stabilities (Tm's) of the hybrid between the 2'-O-methyl ribooligomer and the complementary ribooligomer and of the related hybrids are compared. The synthesis of ribooligomer by a new approach is also described.
Base Sequence↗
Sequence specificity of ozone-degradation of bases in supercoiled plasmid DNA.
It was found that ozone reacted preferentially with thymine and guanine residues located in the specific region in pBR322 DNA. The sequence analysis of the region including the cleavage site produced by ozonization of ccDNA showed that ozone-modification proceeded in the single stranded region formed by cruciform-formation in supercoiled DNA.
Base Sequence↗
Synthesis and conformational studies of ribooligonucleotides which contain an alternating C-G sequence and show unusual circular dichroism spectra.
The poly [r(C-G)] duplex shows an unusually large negative CD band in the long wavelength region. In order to elucidate this phenomenon, r(C-G-C-G) and r(C-G-C-G-C-G) were synthesized by a phosphotriester method and their properties were examined by UV, CD, 1H and 31P NMR spectroscopy. These ribooligomers form self-duplexes at low temperature, the CD spectra of which show negative bands at around 290 nm and positive bands at around 265 nm. The results of 1H nuclear Overhauser effect experiments, 1H chemical shift-temperature profiles of base protons, and the sharp singlet observed for all H1' protons are consistent with a normal A-RNA structure but not with a Z-DNA like structure. The CD-temperature profiles and 31P NMR spectra support this conclusion. These results indicate that RNA duplexes with an alternating C-G sequence can give an unusually large negative CD band in the long wavelength region despite their right-handed helical structure.
Base Sequence↗
Molecular cloning and the complete nucleotide sequence of cDNA to mRNA for S-100 protein of rat brain.
The complete nucleotide sequence of mRNA for beta-subunit of rat brain S-100 protein was determined from recombinant cDNA clones. The sequence was composed of 1488 bp which included the 276 bp of the complete coding region, the 120 bp of the 5'-noncoding region and the 1092 bp of the 3'-noncoding region containing two polyadenylation signals. In addition, the poly(A) tail was also found. The amino acid sequence deduced from the nucleotide sequence was homologous to the amino acid sequence of bovine S-100 beta subunit except 4 residues showing species differences. From the viewpoint of evolutionary implications, the homology between the nucleotide sequence of S-100 and those of rat intestinal Ca-binding protein (ICaBP) and calmodulin (CaM) was examined. A dot-blot hybridization of poly(A) RNA from the developing rat brains using a labeled cDNA showed a rapid increase in S-100 mRNA at 10-20 postnatal days. The presence of S-100 mRNA in C-6 glioma cells is also described.
Age Factors↗
Recognition by restriction endonuclease EcoRI of deoxyoctanucleotides containing modified sugar moieties.
The deoxyribooctanucleotide d(G-G-A-A-T-T-C-C), containing the recognition sequence for EcoRI, d(G-A-A-T-T-C), and analogs containing modified sugar moieties were tested for their activity in cleavage with EcoRI. These analogs, with replacement in the third position from the 5' end, were synthesized using 9-beta-D-arabinosyladenine (aA), 2'-deoxy-2'-fluoroadenosine (Afl) and adenosine (rA). Duplex formation by the three analogs was confirmed by measurements of ultraviolet/temperature profiles. It was found that EcoRI cleaved these duplexes less efficiently than d(G-G-A-A-T-T-C-C). The adenosine-containing analog d(G-G)-rA-d(A-T-T-C-C) was cleaved much more slowly than d(G-G)-aA-d(A-T-T-C-C) and d(G-G-Afl-A-T-T-C-C). The corresponding ribooctamer G-G-A-A-U-U-C-C showed a higher melting temperature than the deoxyoctamers but its duplex was not cleaved by this enzyme. An analog with 2'-deoxy-2'-fluoroguanosine at the second position was cleaved by the endonuclease faster than the natural deoxyoctamer.
Binding Sites↗
Conformation of ribooligonucleotide duplexes containing an alternating C-G sequence which show an unusual circular dichroism spectrum.
The poly[r(G-C)] duplex shows an unusually large negative band in the long wavelength region of the CD spectrum. In order to elucidate this phenomenon, r(C-G-C-G) and r(C-G-C-G-C-G) were synthesized chemically and their properties were examined by UV and CD, and 1H and 31P NMR spectroscopy. These ribooligomers form a self-complementary duplex at low temperature, the CD spectrum of which shows a negative band at around 290 nm and a positive band at around 265 nm with almost equal magnitudes. The proton resonances in the 1H NMR spectra of the oligo[r(C-G)] duplexes were assigned by nuclear Overhauser effect experiments. The chemical shift-temperature profiles of the base proton signals and the sharp singlets observed for all H1' protons are consistent with a normal A-RNA structure but not with a Z-DNA like structure. Moreover, a 500-MHz two-dimensional nuclear Overhauser effect experiment recorded for r(C-G-C-G-C-G) shows that all guanine bases adopt the normal anti-conformation. CD-temperature profiles and 31P NMR spectra of oligo[r(C-G)]s support this conclusion. These results indicate that duplexes of oligo- and polyribonucleotides containing alternating C-G sequences can give an unusually large negative CD band in the long wavelength region despite their right-handed helical structure.
Base Sequence↗
Synthesis of a gene for human growth hormone and its expression in Escherichia coli.
A gene coding for human growth hormone, which consists of 192 amino acids, was chemically synthesized. The synthesis entailed ligating 78 deoxyribooligonucleotides, which had been synthesized on polymer supports by the phosphotriester method with frequently occurring amino acid codons of Escherichia coli. The chemically synthesized gene was inserted into an E. coli plasmid downstream from the E. coli trp promoter, with a modified ribosome-binding region carried on pBR322. E. coli cells transformed with this recombinant plasmid synthesized 2.9 X 10(6) molecules per cell of human growth hormone upon induction. The induced polypeptide was identical with natural human growth hormone in size and in immunological properties, as well as in biological activity as examined by the tibial test with hypophysectomized rats.
Amino Acid Sequence↗
Molecular cloning and nucleotide sequence of cDNA coding for rat brain cholecystokinin precursor.
A mixture of 14-mer oligodeoxynucleotides was used for the screening of a cDNA clone coding for a cholecystokinin (CCK) precursor from a cDNA library for rat brain microsomal poly(A)RNA. The longest insert is 718 bp long which was verified to contain a nearly full-length cDNA sequence coding for rat CCK precursor, because the size of CCK mRNA was estimated to be about 850 bases long by Northern blotting analysis. Sequence analysis revealed 110 bp in the 5'-untranslated region, 345 bp in the amino acid coding region corresponding to the CCK precursor and 263 bp in the 3'-noncoding region which contains polyadenylation signal AUUAAA and the poly(A) sequence. The precursor may contain a 28 amino acid signal peptide and 12 additional amino acids at the carboxyl terminus.
Amino Acid Sequence↗
High resolution NMR study of CAP binding site 22mer in H2O solution.
High resolution proton NMR were measured for the deoxyoligonucleotide 22mer duplex corresponding to the CAP (catabolite gene activator protein) binding site of lac promotor. The spectra in the lower field region than the water resonance were taken with the time-shared Redfield pulse method by using a JEOL 500 MHz NMR spectrometer. In the imino proton region 18 peaks were separately observed, but the area intensity at 10 degrees C corresponds to 20 protons. By selective irradiation at each peak position NOEs (nuclear Overhauser effects) were observed between the imino and adenine C2H protons and between imino proton themselves. By tracing sequential NOE train carefully, 17 imino proton signals could be unambiguously assigned to each base pair except five AT base pairs at terminals. With the elevation of temperature the peaks showed gradual broadening and disappeared, which indicates the stepwise base pair opening of the duplex. Referring to the above peak assignments it can be concluded that GC20 and AT4 pairs close to terminals relax first and the base pair opening proceeds toward central GC13 and 14.
Base Sequence↗
Synthesis and expression of RNase T1 gene.
In order to obtain knowledges about structure-function relationship of ribonuclease T1, we synthesized a structural gene for RNase T1 and several its modified genes. Using amino acid codons frequently used in Escherichia coli we designed genes consist of 328 X 2 bases. Synthesis of oligodeoxynucleotides with 9-20 base lengths was performed by 1% polystyrene supported triester approach and resulting 42 oligomers were joined together using T4 DNA ligase. The product was analyzed and utilyzed to construct expression vectors, which produced effectively fused proteins.
Amino Acid Sequence↗
Isolation and nucleotide sequence of a partial cDNA clone for bovine opsin.
Bovine cDNAs were cloned by using a mixture of 18-base-long synthetic deoxyribonucleotides as a hybridization probe. The longest cDNA clone (pBO-1) contained an 811-bp insert that included the 434 bp of the coding region corresponding to the C-terminal 144 amino acid residues of opsin peptide and the 377 bp of the 3'-untranslated region. The size of opsin mRNA was determined as 23 S by Northern blot hybridization. Bovine liver DNA gave rise to a single band of 2.8 kb, 1.1 kb and 7.9 kb each with Eco RI, Hind III and Bam HI, respectively, by Southern blot hybridization with pBO-1 as probe. Therefore, bovine opsin gene may occur once per haploid genome.
Amino Acid Sequence↗
Essential structure of E. coli promoter: effect of spacer length between the two consensus sequences on promoter function.
A promoter with the consensus sequence(TTGACA and TATAAT) at -35 and -10 regions was constructed, and the distance between the two consensus sequences was independently altered at two restriction sites. In an in vitro transcription system, a maximum activity was observed at the spacer length of 17 base-pairs regardless of the sites of space adjustment. Evidence was also provided that the start point of transcription was fixed by the consensus sequence at the -10 region.
Base Composition↗
Modification of the anticodon triplet of E.coli tRNAMetf by replacement with trimers complementary to non-sense codons UAG and UAA.
E. coli tRNAMetf was hydrolyzed with RNase A using a limited amount of the enzyme to give two half molecules lacking the anticodon trimer and 3'-terminal dimer. Chemically synthesized trimers CUAp and UUAp were joined to the 5'-half molecules by phosphorylation with polynucleotide kinase plus ATP followed by treatment with RNA ligase. These modified tRNAMetf species had anticodons complementary to the termination codons UAG and UAA. Two half fragments were joined by a similar procedure to yield a molecule lacking the anticodon trimer and the 3'-dimer. Methionine acceptor activity of these tRNA was tested under conditions in which the CAU inserted control tRNAMetf accepted methionine. It was found that all three modified molecules were not recognized by the methionyl-tRNA synthetase from E.coli. The other sixteen amino acids were not incorporated with partially purified aminoacyl-tRNA synthetases.
Codon↗
Studies on transfer ribonucleic acids and related compounds. XLV. Block condensation of ribooligonucleotides containing 2'-O-tetrahydrofuranyl-5'-O-dimethoxytritylnucleosides.
2'-O-Tetrahydrofuranyl-5'-O-dimethoxytrityl-N-protected nucleosides were phosphorylated to give the 3'-(o-chlorophenyl) phosphates which were then condensed with 3',5'-unprotected nucleosides to elongate the chain in the 3'-direction. The 5'-dimethoxytrityl group of these oligonucleotides was selectively deblocked by treatment with zinc bromide. The rate of removal of the dimethoxytrityl group differed in each nucleotide. A dodecamer containing a termination codon UAG, U(AGU)3AG, was synthesized by elongating the chain in the 5'-direction using the selective dedimethoxytritylation followed by condensation of protected oligonucleotide blocks.
Base Sequence↗