Manabu Sugimoto

A simple and efficient method for the oligonucleotide-directed mutagenesis using plasmid DNA template and phosphorothioate-modified nucleotide.

We have developed a simple and efficient method for oligonucleotide-directed mutagenesis with double-stranded (plasmid) DNA as a template. The template was simply and rapidly prepared by cell lysis and the following DNA denaturation with alkali. The chain elongation was performed with phosphorothioate-modified nucleotide at 37 degrees C. After the selective digestion of original DNA with NciI and exonuclease III, the desired mutated gene was obtained at a high frequency (about 70%).

Spin-orbit effect on the magnetic shielding constant using the ab initio UHF method. Electronic mechanism in the aluminum compounds, A1X4− (X = H, F, Cl, Br and I)

Abstract The 27 Al NMR chemical shifts of the compounds A1X 4 − (X = H, F, Cl, Br and I) are studied theoretically by the ab initio UHF/finite perturbation (FP) method including a previously propsed spin-orbit (SO) interaction. When the SO interaction is included, the calculated chemical sshifts agree well with experiment. The SO effects become large in the heavier halogen compounds, AlBr 4 − .and AlI 4 − . The paramagnetic term and the SO term are important in the chemical shifts of these compounds. The paramagnetic term is governed by the Al valence p electron mechanism and the SO term arises from the Fermi contact interaction in the Al valence s-orbital. The twofold halogen dependences, namely the normal halogen dependence and the inverse halogen dependence, observed for those compounds arise from the SO effect and the p-electron mechanism, respectively.

Basis set dependence of magnetic shielding constant calculated by the Hartree-Fock/finite perturbation method

Abstract We investigate the basis set dependence and the gauge origin dependence in the calculations of Se and Cd nuclear magnetic shielding constants of several selenium and cadmium compounds. We improve the basis set systematically by adding the first-order higher angular momentum basis functions (FOBFs) to the conventional basis sets, as proposed previously, in the ab initio Hartree-Fock/finite perturbation method. The calculated results become almost independent on the employed basis sets when this improvement is done. Furthermore, it is shown that the mechanisms of the chemical shifts are basis-set independent. The dominances of the p-hole mechanism in the Se chemical shift and the p-electron mechanism in the Cd chemical shift are valid for all the basis set examined in this paper.

SYNTHESIS OF BIOLOGICALLY ACTIVE SELENIUM-CONTAINING AMINO ACIDS AND PEPTIDES

Abstract We here describe the synthesis of selenium amino acids with O-acetylhomoserine sulfhydrylase, partially purified from bakers yeast. The enzyme was found to catalyze the syntheses of L-selenocystine and L-selenohomocystine from sodium diselenide with the corresponding acetyl-derivatives of serine and homoserine, respectively. L-Serine O-sulfate also serves as a substrate of the β-replacement reaction. Sodium diselenide is less efficient as a substituent donor than the physiological substrate, sodium sulfide and inhibits the enzyme at high concentrations. Therefore, limited amounts of sodium diselenide were added to the reaction mixture to increase the yield (about 60%). This provides a facile method to produce optically active seleno-cystine and selenohomocystine. In addition, we developed a convenient method for the synthesis of a new selenium-containing amino acid, L-selenodjenkolic acid (3,3′-methyl-enediselenobis(2-amino-propionic acid)) from L-selenocystine thus prepared. This amino acid und...

Construction of Mouse Glutathione Peroxidase Gene and Its Expression

The essentiality of selenium can be ascribed mostly to the presence of enzymes containing selenocysteine (SeCys) as an integral moiety. Recently, the genes of mammalian glutathione peroxidase (GSHPx) and Escherichia coli formate dehydrogenase were cloned, and it was shown that a nonsense opal codon, TGA, encodes SeCys. However, the mechanism of the gene expression is still obscure. We here describe the construction of chimeric fused genes of mouse GSHPx and E. coli β-galactosidase (β-gal), and its expression in E. coli. DNA fragments of GSHPx gene which include the TGA codon encoding the SeCys residue, were inserted into the portion of lacZ’ gene which encodes the amino terminal moiety of β-gal. When E. coli JM109 cells were transformed with the fused genes, β-gal activity appeared in the transformants under anaerobic conditions. These indicate that the UGA was readthrough to give the protein containing SeCys residue in-frame under anaerobic conditions. Moreover, selenium was incorporated into the fused proteins produced anaerobically.