Search PubMed⌕ Search

Biomedical subjects

K Itakura

Publications and source records attributed to K Itakura.

At least 109 records · Page 6Linked to original sources
PubMed1983

Detection of sickle cell beta S-globin allele by hybridization with synthetic oligonucleotides.

Two 19-base-long oligonucleotides were synthesized, one complementary to the normal human beta-globin gene (beta A) and one complementary to the sickle cell beta-globin gene (beta S). The nonadecanucleotides were radioactively labeled and used as probes in DNA hybridization. Under appropriate hybridization conditions, these probes can be used to distinguish the beta A gene from the beta S allele. The DNA from individuals homozygous for the normal beta-globin gene (beta A beta A) only hybridized with the beta A specific probe; the DNA from those homozygous for the sickle cell beta-globin gene (beta S beta S) only hybridized with the beta S specific probe. The DNA from heterozygous individuals (beta A beta S) hybridized with both probes. This allele-specific hybridization behavior of oligonucleotides provides a general method for diagnosis of any genetic disease which involves a point mutation in the DNA sequence of a single-copy gene.

Alleles↗
PubMed1983

Biological expression of an Escherichia coli consensus sequence promoter and some mutant derivatives.

A prokaryotic consensus sequence promoter has been chemically synthesized and cloned in bacterial plasmid vectors. This designed sequence is biologically active and promotes efficient expression of the genes to which it is fused. It is an unusually strong promoter in vitro, capable of specifying multiple rounds of transcription even when there is a large molar excess of heparin present prior to the addition of RNA polymerase. These properties make this a useful sequence for the in vitro production of RNAs. A 2-base-pair spacer mutant and a -35 region transversion mutant have been created in vitro in the synthetic promoter by synthetic-DNA-mediated, site-specific mutagenesis. The spacer mutant has a marginal in vivo effect on promoter strength but virtually abolishes the in vitro heparin resistance. The -35 region transversion changes a highly conserved nucleotide into the statistically least preferred base. This mutation has no marked effect on in vivo or in vitro promoter strength.

Base Sequence↗
PubMed1983

Isolation of a cDNA clone for human X-linked 3-phosphoglycerate kinase by use of a mixture of synthetic oligodeoxyribonucleotides as a detection probe.

We have obtained a cDNA clone encoding most of human X-linked 3-phosphoglycerate kinase (PGK; ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3). Total mRNA was prepared from human adenocarcinoma-derived cell line LS174T and used for cDNA preparation. Double-stranded cDNA was inserted, after tailing with oligo(dC), into the plasmid vector pBR327 and cloned in Escherichia coli K-12. Transformants were screened by colony hybridization with a mixture of 32P-labeled oligodeoxyribonucleotides. A pool of hexadecamers complementary to all 32 possible sequences encoding amino acids 291-296 of X-linked PGK was used for the initial screen. One clone among 2,500 gave a strong positive signal. Plasmid DNA from this clone was purified and characterized by hybridization first to the hexadecamer probe mixture and then to an undecamer probe consisting of a mixture of four sequences. The cloned fragment hybridizes preferentially to DNA from human cells with five X chromosomes. DNA sequence analysis has established that the 1.2-kilobase-pair fragment encodes PGK from amino acid 121 through the COOH terminus.

Amino Acid Sequence↗
PubMed1983

Sequence dependence of hydrogen exchange kinetics in DNA duplexes at the individual base pair level in solution.

The kinetics for hydrogen exchange at individual base pairs in self-complementary deoxydodecanucleotide duplexes have been estimated from NMR saturation recovery measurements on the resolved imino protons as a function of temperature. The imino protons of dA . dT base pairs in the center of the fully alternating d(C-G-C-G-T-A-T-A-C-G-C-G) duplex exchange a factor of 2- to 3-fold faster than the corresponding protons at the same positions in the partially alternating d(C-G-C-G-A-A-T-T-C-G-C-G) duplex. These exchange parameters are a direct measure of the rate constants for transient opening of individual dA . dT base pairs in the dodecanucleotide duplexes and demonstrate faster opening kinetics for the "TATA" box region compared to the related "AATT" segment.

Base Sequence↗
PubMed1983

Ventilatory response to carbon dioxide in bronchial asthma and chronic obstructive lung disease.

A comparative study was made of ventilatory and airway occlusion pressure (P0.1; a parameter reflecting respiratory center output) responses to carbon dioxide between 11 patients with bronchial asthma and 10 chronic obstructive lung disease (COLD). Increments in ventilatory volume (VE) produced by a rise in end-tidal CO2 pressure (PETCO2), i.e. delta VE/BSA/delta PETCO2, were smaller in 4 patients with hypercapnic COLD than in 6 normal subjects. On the other hand, increments in P0.1 produced by an elevation of PETCO2 (i.e. delta P0.1/delta PETCO2) tended to be diminished in patients with hypercapnic COLD. Higher values for both VE/BSA and P0.1 were observed in 6 patients with normocapnic COLD, but the differences from corresponding control values failed to achieve statistical significance due to a large variance. In 11 patients with bronchial asthma without attack, VE/BSA elevated significantly at PETCO2 levels of 50 and 60 torr, but values of delta VE/BSA/delta PETCO2 were virtually same as those in normal subjects.

Asthma↗
PubMed1982

Point mutagenesis of the ovalbumin gene promoter sequence and its effect on in vitro transcription.

The role of the eucaryotic T-A-T-A box (Hogness box) homology sequence located approximately 30 base pairs upstream from the RNA initiation site has been further examined by oligodeoxynucleotide-directed site-specific mutagenesis. A method was employed which allows insertion of nucleotide-specific mutations into virtually any double-stranded, recombinant plasmid DNA. A synthetic mixed oligonucleotide, bearing defined multiple nucleotide substitutions at a single site, was used both as a specific mutagen during primed DNA repair synthesis in vitro, as well as a highly sensitive hybridization probe for the identification of the mutated cloned DNA. Using this methodology, an A leads to G transition mutation was introduced into the second position of the ovalbumin gene T-[A]-T-A box, and the effect of modifying this highly conserved nucleotide on the expression of the mutant DNA was analyzed in a cell-free transcription system. Comparison of two allelic ovalbumin genes, slightly divergent upstream from the TATA box, resulted in identical in vitro transcription efficiencies. While correct initiation of ovalbumin RNA transcripts was not affected, the efficiency of gene expression using the mutant template, compared to either the corresponding wild-type sequence or the allelic gene, was markedly reduced. These results suggest that it is the T-A-T-A box sequence which plays a role in the efficient initiation of RNA transcription in vitro and further supports the implication that this region may serve a promoter-related function in eucaryotic transcription.

Animals↗
PubMed1982

An alternate method for synthesis of double-stranded DNA segments.

Recent progress in the chemical synthesis of DNA has now made it possible to rapidly synthesize single-stranded DNAs over 40 bases in length. We have taken advantage of these longer DNAs in assembling and cloning a 132-base pair gene segment coding for amino acids 126 through the stop codon of human leukocyte interferon alpha 2. The method used involves DNA polymerase I-mediated repair synthesis of synthetic oligonucleotide substrates having short stretches of complementary sequence at their 3' termini. In the presence of DNA polymerase I and the four deoxyribonucleoside triphosphates, those primer-templates are converted to full length double-stranded DNAs. The economy in chemical synthesis using this approach is substantial with a greater than 40% reduction in the amount of chemical synthesis required as compared with the conventional approach. We describe in detail this methodology for the biochemical assembly of long gene segments from synthetic oligodeoxyribonucleotides.

Cloning, Molecular↗
PubMed1982

DNA conformation, dynamics, and interactions in solution.

The conformation and dynamics of the d(CGCGAATTCGCG) duplex, its analogs containing mismatched base pairs and helix interruptions, and its complexes with actinomycin and Netropsin, bound separately and simultaneously, have been investigated by nuclear magnetic resonance spectroscopy in aqueous solution. Structural information has been deduced from chemical shift and nuclear Overhauser effect parameters, while the kinetics have been probed from line width and saturation recovery experiments on proton and phosphorus markers at the individual base pair level. These studies lead to an improved understanding of the role of nucleic acid sequence on the structure, flexibility, and conformational interconversions in the duplex state. The nuclear magnetic resonance measurements readily identify helix modification and antibiotic binding sites on the nucleic acid and estimate the extent to which the observed conformational and dynamic perturbations are transmitted to adjacent base pair regions.

Base Sequence↗
PubMed1982

Solid phase synthesis of polynucleotides. VI. Further studies on polystyrene copolymers for the solid support.

A simple solid phase method for the synthesis of oligodeoxyribonucleotides has been developed using the phosphotriester approach. Mononucleotide coupling units are sequentially added to the polystyrene copolymer with 1% divinylbenzene and two kinds of oligonucleotides, d(CACGACCCCTCCACGT) and d(AACTGGTATTACTGGGCG), are synthesized in a relatively high yield. One cycle of the mononucleotide addition is about 70 minutes, and this method is particularly suitable for the automation of the synthesis upon availability of an automatic synthesizer.

Base Sequence↗
PubMed1982

The molecular structure of d(ICpCpGpG), a fragment of right-handed double helical A-DNA.

The DNA tetramer d(ICpCpGpG) or ICCGG crystallizes as a double-stranded 4-base pair (bp) segment of an A helix. Two such tetramer helices are packed together in the crystal with local helix axes nearly coincident, simulating an 8-bp helix, and four such octamers make up the tetragonal unit cell. Restrained energy and reciprocal space refinement has led to an R factor of 20.5% at 2.1 A resolution. The ICCGG helix has a twist corresponding to 10.7 bp per turn, a 19 degree base tilt and a 2.3 A rise per base pair along the helix axis. The mean propeller twist of 18 degree is comparable with, and has the same rotational sense as that observed in the B-DNA dodecamer CGCGAATTCGCG at similarly high alcohol concentration. Backbone phosphate groups in A-DNA are extensively hydrated, including a network across the opening of the major groove, whereas base edge N and O groups in major and minor grooves are less hydrated than in B-DNA. The minor groove spine of hydration observed in B-DNA is totally absent. These observations of relative hydration confirm and extend the model for the B- to A- helix transition proposed earlier on the basis of the B helix structure.

Base Composition↗
PubMed1982

HLA-linked susceptibility gene of Takayasu Disease.

The HLA-A, B, DR and MB antigens were investigated in patients suffering from Takayasu disease (Aortitis syndrome). Out of twenty-one HLA-A and B antigens tested, only HLA-Bw52 was significantly deviated (30147, PF = 63.8%, RR = 7.8) from the controls (14/76, PF = 18.4%). Since in the Japanese, HLA-Bw52 is in positive linkage disequilibria with HLA-DR2 and MB1, the association of the DR2 and MB1 antigens with Takayasu disease was studied. The HLA-DR2 antigen was significantly increased (23/30, PF = 76.7%,, RR = 6.0) in patients compared with the control (18/51, PF = 35.3%). Moreover, an almost perfect association of MBI (29/30, PF = 96.7%, RR = 12.6) with Takayasu disease was demonstrated. This finding supports the hypothesis that the genes in the HLA-D region play a major role in determining the susceptibility to Takayasu disease.

Aortic Arch Syndromes↗
PubMed1982

The use of synthetic oligodeoxyribonucleotides to produce specific deletions in the araBAD promoter of Escherichia coli B/r.

Two oligodeoxyribonucleotides were chemically synthesized and used to specifically mutate the regulatory region of the araBAD operon in Escherichia coli B/r. One oligodeoxyribonucleotide introduced a 3-bp deletion in the araC activator binding site, the other a 3-bp deletion in the CRP-cAMP binding site. The mutations were introduced onto an ara insert cloned in an M13 vector using the synthetic oligodeoxyribonucleotides as primers and the (+) strand of an M13 mp2::ara hybrid phage as a template in an in vitro polymerization reaction. Hybridizations using the original synthetic oligodeoxyribonucleotide as a radioactive probe identified phage containing the desired deletion. The mutant ara inserts were subcloned into a stable plasmid for functional analysis. Transcription studies performed on strains containing the mutant ara plasmids demonstrated that both mutations reduced the amount of araBA mRNA synthesized in the presence of L-arabinose.

Arabinose↗