Studies on Degradation of d-Biotin by Microorganisms

During the course of our investigations on the metabolism of d-biotin by microorganism, it has been found that some strains of fungi belonging to the genera Rhodotorula, Penicillium and Endomycopsis, are able to degrade d-biotin oxidatively into various biotin vitamers. The present work was undertaken to characterize these vitamers. The vitamers formed were separated by the ion exchange column chromatography, into Fraction A (d-biotin sulfoxide), Fraction B (unknown vitamer II), Fraction C (d-biotin) and Fraction D (unknown vitamer I). Rf values of vitamer I and vitamer II were found to be different from those of the known biotin vitamers. The vitamers I and II did not support the growth of Lactobacillus arabinosus and Saccharomyces cerevisiae, but did support that of Bacillus subtilis. This degradation reaction occurred rather favorably in high aerobic condition.     

Purification of Cytosine Deaminase from Serratia marcescens

Cytosine deaminase was purified about 900-fold from the cell-free extract of Serratia marcescens. The purification procedure included heat treatment, ammonium sulfate fractionation, ethyl alcohol fractionation, DEAE-cellulose and hydroxylapatite column chromatography, and Sephadex G–200 gel filtration.

The enzyme was homogeneous by the criteria of ultracentrifugation and acrylamide gel electrophoresis. The molecular weight was determined to be approximately 580,000 and the molecule was composed of equimolecular weight of 8 subunits.

The enzyme catalyzed the stoichiometric conversion of cytosine into uracil and ammonia.     

Studies on Biosynthesis of Biotin by Microorganisms

During the course of the study on the production of biotin from desthiobiotin by microorganisms, the present authors have found that some strains of molds produced an unknown biotin-vitamer (BS-factor) from desthiobiotin. The present investigation was undertaken to clarify the characteristics of the unknown vitamer. The unknown vitamer produced from desthiobiotin was isolated in crystalline form from culture filtrate of Aspergillus oryzae. The compound isolated was identified as 4-methyl-5-(ω-carboxybutyl)-imidazolidone-2 by the physico-chemical procedures.

The biosynthesis of biotin-vitamers by resting cell system of Bacillus sphaericus was studied.

It was found that pimelic acid was essential substrate in biosynthesis of biotin-vitamers and that some amino acids and organic acids stimulated the biosynthesis of biotin-vitamers from pimelic acid. Alanine was found to be most effective. It was assumed that, in the presence of pimelic acid, some amino acids, especially alanine, and some organic acids play an important role in the biosynthesis of biotin-vitamers.

The main component of the biotin-vitamers synthesized by the resting cell system was identified as desthiobiotin. The existence of a small amount of unknown biotin-vitamer, an avidin-uncombinable substance, which was assumed to be 7-keto-8-amino-pelargonic acid, was also observed. True biotin was hardly observed in any conditions tested.     

Mapping of a major locus controlling seed dormancy using backcrossed progenies in wheat (Triticum aestivum L.).

Seed dormancy is an important factor regulating preharvest sprouting (PHS) but is a complex trait for genetic analysis. We previously identified a major quantitative trait locus (QTL) controlling seed dormancy on the long arm of chromosome 4A (4AL) in common wheat. To transfer the QTL from the dormant lines 'OS21-5' and 'Leader' into the Japanese elite variety 'Haruyokoi', which has an insufficient level of seed dormancy, backcrossing was carried out through marker-assisted selection (MAS) using PCR-based codominant markers. Nineteen BC5F2 plants with homozygous alleles of 'OS21-5' or 'Haruyokoi' were developed and evaluated for seed dormancy under greenhouse conditions. The seeds harvested from plants with 'OS21-5' alleles showed a clearly high level of dormancy compared with seeds from plants with 'Haruyokoi' alleles. Additionally, the dormancy phenotype of BC3F3 seeds harvested from 128 BC3F2 plants with homozygous alleles of 'Leader' or 'Haruyokoi' showed a clear difference between these alleles. The QTL on 4AL confers a major gene, Phs1, which was mapped within a 2.6 cM region. The backcrossed lines developed in this study can be important sources for improving PHS resistance in Japanese wheat and for analyzing the mechanism of seed dormancy. MAS was useful for the development of near-isogenic lines in this complex trait, to facilitate the molecular dissection of genetic factors.     

Preparation of DNA-modified nanoparticles and preliminary study for colorimetric SNP analysis using their selective aggregations

DNA-modified nanospheres were prepared by anchoring amino-terminated oligodeoxynucleotides (ODNs) with carboxylates onto a colored polystyrene sphere surface through amido bonds. About 220 ODN molecules were immobilized onto a nanosphere 40 nm in diameter. Preliminary studies using the microspheres with 1 μm diameter reveal that the specificity of hybridization was retained after modification. Three kinds of differently colored (RGB, red/green/blue) nanospheres bearing unique ODNs on their surface were prepared for detecting the p53 gene. Each ODN is complementary to a different part in the 45mer sample that is a part of a conservative region of the p53 gene containing one of the hot spots. In a binary system using spheres R and G, the wild-type 45mer made the aggregates with yellow emission as the result of mixing both colors. The mutant 45mer containing one nucleotide displacement did not give such aggregates with distinct colors. The study of fluorescence resonance energy transfer (FRET) showed that spheres R and G directly contact each other in the aggregates with the wild type. The RGB ternary system gave aggregates with specific colors corresponding to the added ODN samples, wild type or mutant. In addition, in the presence of both samples, all of the spheres formed aggregates with white emission as a consequence of mixing three primary colors of light. This means that the present technique should allow us to conduct an allele analysis.