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Biomedical subjects

K Itakura

Publications and source records attributed to K Itakura.

At least 145 records · Page 8Linked to original sources
PubMed1980

Simplications in the synthesis of short oligonucleotide blocks.

A rapid and convenient procedure has been developed for the synthesis of fully protected mono, di and trideoxyribonucleotides utilizing an aryl phosphoroditriazolide. It affords advantages over coupling strategies employing condensing reagents, such as 2,4,6-triisopropylbenzenesulfonyl tetrazolide in preparing small oligonucleotides and is relatively free of the drawbacks inherent in other approaches using bifunctional phosphorylating reagents. In particular, the synthesis of trinucleotide blocks without purification at the dimer stage is described.

Indicators and Reagents↗
PubMed1980

Solid-phase synthesis of polynucleotides. II. Synthesis of polythymidylic acids by the block coupling phosphotriester method.

Synthesis of two oligothymidylic acids, tridecamer and nonadecamer, is described by a rapid and simple solid-phase method on two kinds of polyacrylamide supports derivatized from commercially available Enzacryl Gel K-2. The syntheses were performed by the phosphotriester method using di- and tri-thymidylic acid blocks as the incoming 3'-phosphodiester component. High coupling yields were consistently obtained and the final product was isolated very easily by high performance liquid chromatography on Permaphase AAX.

Indicators and Reagents↗
PubMed1980

Solid-phase synthesis of polynucleotides. III. Synthesis of polynucleotides with defined sequences by the block coupling phosphotriester method.

Preparation of the three hexadecanucleotides, dGpTpApTpCpApCpGpApGpGpCpCpCpTpT, dCpGpApCpGpApGpCpGpTpGpApCpApCpC and cTpGpCpCpGpGpCpCpApCpGpApTpGpCpG, is described by a rapid and simple solid-phase method on polyacrylamide supports. The synthesis were performed by the extension of the method described in the previous paper using di and trinucleotides of defined sequences as an incoming 3'-phosphodiester unit. Although the coupling yields to form phosphotriester bonds are slightly lower than those for the homothymidylic acid series, pure polydeoxyribonucleotides of defined sequences can be synthesized without any major difficulty.

Base Sequence↗
PubMed1980

Solid-phase synthesis of polynucleotides. IV. Usage of polystyrene resins for the synthesis of polydeoxyribonucleotides by the phosphostriester method.

Contrary to the expectation, the Merrifield polystyrene resin, 2% cross-linked by divinylbenzene, is as efficient as the polyacrylmorpholide resin for the synthesis of polydeoxyribonucleotides using a phosphotriester method. On the Merrifield resin, the tetradecamer, dTpCpGpTpCpApApCpTpGpGpCpTpT, and the hexadecamer, dCpCpApGpTpCpApCpGpApCpGpTpTpGpT, were synthesized by the phosphotriester method using di and trinucleotide blocks as coupling units.

Base Sequence↗
PubMed1980

Equilibrium binding of inducer to lac repressor.operator DNA complex.

The characteristics of inducer binding to lactose repressor protein in the presence of a small (29 base pair) operator DNA fragment have been examined. The presence of operator DNA fragments decreases the affinity of the protein for inducer molecules. The maximum change observed is a 20-fold increase in the concentration of inducer necessary for half-saturation of the protein. The non-linearity of the Scatchard type plots and slopes of the Hill plots of the binding data indicate that inducer binding exhibits cooperative behavior in the presence of operator DNA fragments, while free repressor binds inducer in a noncooperative fashion. The experimental data for the binding of inducer to repressor both in the presence and absence of saturating amounts of operator DNA fragments were compared to curves predicted by several different models. Using the limits placed by the measured values for repressor-operator DNA-inducer interaction, it was possible to discriminate between the various models. The models which accurately predicted the inducer binding curves and conformed to the measured values for the other parameters were Monod-Wyman-Changeux and Koshland type models with the following features: 1) positive cooperativity in the binding of inducer to the repressor . operator DNA complex; 2) two sites for the binding of the operator DNA fragment to repressor protein; 3) effect of binding of each operator DNA fragment on all four subunits of the repressor. These models are consistent with all of the available data, and they indicate that the binding of the first two inducer molecules accounts for greater than or equal to 60% of the difference in affinities between free and induced repressor for operator DNA; in vivo this difference should be sufficient for induction to occur.

DNA, Bacterial↗
PubMed1980

Crystal structure analysis of a complete turn of B-DNA.

DNA is probably the most discussed and least observed of all biological macromolecules. Although its role in biology is a central one, with many examples such as operators and restriction sites where specific base sequences have control functions or interact with specific enzymes, the structures that DNA can adopt have been based until now only on sequence-averaged fibre diffraction patterns. Recent improvements in triester synthesis methods have made possible the preparation of sufficient homogeneous DNA of predetermined sequence for crystallization and X-ray structure analysis. We report here the first single-crystal structure analysis of more than a complete turn of right-handed B-DNA, with the self-complementary dodecamer sequence d(CpGpCpGPApApTpTpCpGpCpG) or CGCGAATTCGCG.

Base Sequence↗
PubMed1980

Chemical DNA synthesis and recombinant DNA studies.

Chemically synthesized DNA has been used in many recombinant DNA studies. These uses have included the total synthesis and cloning of functional genes, the cloning and expression of natural genes, and editing of changing genes by directed mutation.

Base Sequence↗
PubMed1980

Directed deletion of a yeast transfer RNA intervening sequence.

Many eukaryotic genes contain intevening sequences, segments of DNA that interrupt the continuity of the gene. They are removed from RNA transcripts of the gene by a process known as splicing. The intervening sequence in a yeast tyrosine transfer RNA (tRNA Tyr) suppressor gene was deleted in order to test its role in the expression of the gene. The altered gene and its parent were introduced into yeast by transformation. Both genes exhibited suppressor function, showing that the intervening sequence is not absolutely essential for the expression of this gene.

Base Sequence↗
PubMed1980

High-salt d(CpGpCpG), a left-handed Z' DNA double helix.

The DNA tetramer d(CpGpCpG) or CGCG crystallizes from high-salt solution as a left-handed double helix, the Z' helix. Its structure differs from that of the other known left-handed helix, Z-DNA, by a Cl'-exo sugar pucker at deoxyguanosines rather than C3'-endo, and these represent two alternative solutions to the same steric constraint arising from the syn glycosyl bond orientation. The apparent molecular basis for the Z to Z' transition in going from intermediate to high salt is substitution of a bound anion for water at guanine amino groups, and consequent charge repulsion of anions and backbone phosphates.

Alcohols↗
PubMed1980

Association of B cell alloantigen with juvenile onset diabetes mellitus in the Japanese.

Sixty-four Japanese insulin dependent juvenile onset diabetes mellitus (JOD) were studied in relation to HLA-A, B, and DR. Significant deviations were observed. HLA-Bw54 was increased (PF = 49.2%, RR = 6.4) and HLA-B5 was decreased (PF = 7.9%, RR = 0.19). Using radioimmunoassay, two HLA-DR antigens were investigated. Hon 7 antigen, so-called MT3 (WIA4x7), which has linkage disequilibrium between HLA-BW54, is highly associated (PF = 96.9%, RR = 27.8) with JOD found in the Japanese.

Adolescent↗
PubMed1980

Transcription and processing of a yeast tRNA gene containing a modified intervening sequence.

The tRNA(3) (Leu) gene from yeast contains an intervening sequence of 32 nucleotides not present in mature tRNA. This sequence is transcribed and subsequently removed during the maturation of the RNA. To probe the involvement of this region of the gene in transcription and processing of the pre-tRNA(3) (Leu), the yeast DNA was cloned in plasmid pBR322 and a 21-base-pair DNA fragment corresponding to the lac operator was inserted into the intervening sequence. Insertion was done at a cleavage site for the restriction endonuclease Hpa I that occurs 19/20 base pairs from the 5' end of the intervening sequence. The parent and modified plasmids were then transcribed in a Xenopus germinal vesicle extract. RNA-fingerprint analysis of the transcription products revealed that both the tRNA(3) (Leu) gene and its modified counterpart were accurately transcribed. Transcription products corresponding to mature tRNA(3) (Leu) and pre-RNA(3) (Leu) with the normal and lac-containing intervening sequence were identified. Precursors extended at their 5' and 3' ends were also present. Both parent and modified genes were transcribed efficiently, and the various products accumulated in similar amounts, indicating that no deleterious effects on transcriptional competence, stability of the transcripts, or processing result from insertion of the 21-base-pair lac operator DNA. Incubation of pre-tRNA molecules that contained intervening sequences but were 5' and 3' mature with a yeast ribosomal wash fraction resulted in excision of the intervening sequence and, in the presence of ATP, ligation of the resulting half-tRNA molecules. The presence of RNA complementary to lac operator DNA neither inhibited the excision and splicing activities nor altered the site of the junction.

Base Sequence↗
PubMed1980

Solid-phase synthesis of polynucleotides: V. Synthesis of oligodeoxyribonucleotides by the phosphomonotriazolide method.

Synthesis of two oligodeoxyribonucleotides of defined sequences, an undecamer [d(TGCACCATTCT)] and a dodecamer [d(TGGAGCCACTAT)], and tetradecathymidylic acid was described by a simple solid-phase method on a polystyrene resin. The synthesis was performed by the stepwise addition of deoxynucleoside 3'-phosphomonotriazolide to the resin, in the presence of a nucleophilic catalyst, 4-dimethylaminopyridine or N-methylimidazole. Quantitative coupling yield was consistently obtained for each cycle and the desired product was a major peak in the analysis of the final reaction by high performance liquid chromatography on Permaphase AAX.

Base Sequence↗
PubMed1980

HLA heterozygosity in children and old people.

When the distribution of four different antigens in young and old people were compared, statistical differences were observed between the two groups. The data are thus compatible with the hypothesis that HLA heterozygosity enhances surveillance.

Adolescent↗
PubMed1979

Direct expression in Escherichia coli of a DNA sequence coding for human growth hormone.

DNA coding for human growth hormone was constructed by using chemically synthesised DNA in conjunction with enzymatically prepared cDNA. This 'hybrid' gene was expressed in Escherichia coli under the control of the lac promoter. A polypeptide was produced having the size and immunological properties characteristic of mature human growth hormone.

Base Sequence↗
PubMed1979

Hybridization of synthetic oligodeoxyribonucleotides to phi chi 174 DNA: the effect of single base pair mismatch.

Oligodeoxyribonucleotides complementary to the DNA of the wild type (wt) bacteriophage phi chi 174 have been synthesized by the phosphotriester method. The oligomers, 11, 14, and 17 bases long, are complementary to the region of the DNA which accounts for the am-3 point mutation. When hybridized to am-3 DNA, the oligonucleotides form duplexes with a single base pair mismatch. The thermal stability of the duplexes formed between wt and am-3 DNAs has been measured. The am-3 DNA:oligomer duplexes dissociate at a temperature about 10 degrees C lower than the corresponding wt DNA:oligomer duplexes. This dramatic decrease in thermal stability due to a single mismatch makes it possible to eliminate the formation of the mismatched duplexes by the appropriate choice of hybridization temperature. These results are discussed with respect to the use of oligonucleotides as probes for the isolation of specific cloned DNA sequences.

Bacteriophage phi X 174↗
PubMed1979

Expression in Escherichia coli of chemically synthesized genes for human insulin.

Synthetic genes for human insulin A and B chains were cloned separately in plasmid pBR322. The cloned synthetic genes were then fused to an Escherichia coli beta-galactosidase gene to provide efficient transcription and translation and a stable precursor protein. The insulin peptides were cleaved from beta-galactosidase, detected by radioimmunoassay, and purified. Complete purification of the A chain and partial purification of the B chain were achieved. These products were mixed, reduced, and reoxidized. The presence of insulin was detected by radioimmunoassay.

Amino Acids↗