Yasunobu Takeshima

Establishment of a Pure Culture of the Hitherto Uncultured Unicellular Cyanobacterium Aphanothece sacrum, and Phylogenetic Position of the Organism

ABSTRACT Aphanothece sacrum, an edible freshwater unicellular cyanobacterium, was isolated by using novel synthetic media (designated AST and AST-5xNP). The media were designed on the basis of the ratio of inorganic elements contained in A. sacrum cells cultured in a natural pond. The isolated strain exhibits unicellular rod-shaped cells ∼6 μm in length that are scattered in an exopolysaccharide matrix, a feature similar to that of natural A. sacrum. DNA analysis of the isolated strain revealed that it carried two ferredoxin genes whose deduced amino acid sequences were almost identical to previously published sequences of ferredoxins from natural A. sacrum. Analysis of the 16S rRNA gene and ferredoxin genes revealed that A. sacrum occupies a phylogenetically unique position among the cyanobacteria.

Chemically-Synthesized Gene Encoding Modified Human Superoxpde Dismutase: Its Construction, Expression and Properties of the Product

The gene encoding modified human superoxide dismutase (h-SOD) with 153 amino acid residues was constructed by chemical synthesis using the phosphoramidite method. The gene was designed so as to use bacterial codons for expression in prokaryotes and to introduce several unique restriction sites for further mutagenesis by the cassette exchange method. The distance between Shine-Dalgarno sequence and initiation codon was adjusted to maximum expression by using synthesized oligonucleotide. In addition, Cys 6 of h-SOD was changed to Ala to improve instability of native h-SOD. Synthesized structural gene of h-SOD was expressed in E. coli after induction of isopropyl beta-D-thiogalactoside by inserting the gene into the expression vector pKK223-3 having tac promoter. The gene that has 10 base pairs between Shine-Dalgarno sequence and initiation codon showed the most efficient expression. The gene produced three active SOD isomers as revealed by chromatofocusing. The main isomer was purified to homogeneity and characterized. The h-SOD-Ala6 showed similar properties to those of native h-SOD with respect to molecular weight, subunit structure, absorption spectrum, but the modified SOD was more resistant to heat denaturation than was native h-SOD; half-denaturing temperature was shifted by 10 degrees C. Thus, the exchange of Cys 6 to Ala of h-SOD increased a stability of the enzyme.