Genetic and phenotypic polymorphisms of the A subunit of Coagulation factor XIII in Japanese population

To clarify whether genetic polymorphisms in exon 14 of Coagulation factor XIII A-subunit gene (FXIIIA) affect phenotype expressions, we studied genetic polymorphisms in exon 14 of FXIIIA in a Japanese population and the relationship between the genetic polymorphisms and phenotype expression. Genetic polymorphisms in exon 14 of FXIIIA of 144 unrelated Japanese were analyzed by single-strand conformation polymorphism. Plasma FXIIIA antigen concentrations, FXIII activities, and phenotype were also determined by two-dimensional electrophoresis. The frequencies of the three genotypes, the homozygote (AD), the homozygote (BC) and the heterozygote (AD/BC), were 77.1, 0.7, and 22.2%, respectively. The gene frequencies of AD and BC were 0.88 and 0.12. We detected AD (GTT x GAG) and BC (ATT x CAG) at codon 650 and 651 of exon 14. There were no significant differences of FXIIIA antigen concentrations and FXIII activities between these genotypes. We detected three pl differences among them as being pls of 5.3, 5.6, 5.8 in the homozygote (AD) and the heterozygote (AD/BC), and a pl of 5.8 in the homozygote (BC). These polymorphisms affected isoelectric mobility, but did not affect protein levels, enzyme activities, or the molecular weight of FXIII.     

Probing biosynthesis of plant polyketides with synthetic N-acetylcysteamine thioesters

Recombinant chalcone synthase (CHS) from Scutellaria baicalensis accepted cinnamoyl diketide-NAC and cinnamoyl-NAC as a substrate, and carried out sequential condensations with malonyl-CoA to produce 2',4',6'-trihydroxychalcone. Steady-state kinetic analysis revealed that the CHS accepted the diketide-NAC with less efficiency, while cinnamoyl-NAC primed the enzyme reaction almost as efficiently as cinnamoyl-CoA. On the other hand, it was for the first time demonstrated that the diketide-NAC was also a substrate for recombinant polyketide reductase (PKR) from Glycyrrhiza echinata, and converted to the corresponding beta-ketohemithioester. Furthermore, by co-action of the CHS and the PKR, the NAC-thioesters were converted to 6'-deoxychalcone in the presence of NADPH and malonyl-CoA.     

Saccharide production from methanol by transposon 5 mutants derived from the extracellular polysaccharide-producing bacterium Methylobacillus sp. strain 12S

A CH₃OH-utilizing bacterium that has the ability to produce extracellular polysaccharide (EPS) was isolated from a soil sample, and was identified as the obligate methylotroph Methylobacillus sp. strain 12S on the basis of its 16S rDNA sequence and growth-substrate specificity. The EPS produced by strain 12S was purified and the sugar composition was analysed by GC-MS and HPLC to reveal that the EPS was a heteropolymer composed of glucosyl, galactosyl, and mannosyl residues in the molar ratio 3:1:1. In order to produce mono- and/or oligosaccharides by single-step fermentation from CH₃OH, stain 12S was mutagenized by transposon 5. Among eleven EPS-deficient mutants, three strains were found to accumulate significant amounts of reducing sugars in the media. The amounts of the reducing sugars produced by the mutants (>ca. 700 mg glucose equivalent/l) were >11–22 times higher than those produced by the wild-type strain (<ca. 60 mg glucose equivalent/l). The GC-MS analysis showed that all the mutants accumulated glucose, erythrose, threose and a disaccharide-like compound in the media. Received: 25 August 1999 / Received revision: 15 March 2000 / Accepted: 24 March 2000     

Genetic variants of hemoglobin in canine erythrocytes*

Genetic polymorphism of hemoglobin was found in the erythrocytes of dogs of seven Japanese native breeds by using starch gel electrophoresis. Analysis of parentage records of the dogs revealed that the phenotypic variation of hemoglobin is controlled by one autosomal locus with two codominant alleles, Hb^A and Hb^B. The allele Hb^A occurred only in Japanese native breeds except Shikoku. The frequency of Hb^A in the Japanese breeds was low and 0.08. All the dogs belonging to 25 European breeds and 5 oriental origin (except Japan) breeds examined in this experiments had the homozygous genotype constitution Hb^B/Hb^B.     

Establishment of a novel tissue culture method, stem-disc culture, and its practical application to micropropagation of garlic (Allium sativum L.)

A restricted part of the undeveloped stem of the garlic clove, called the “stem disc”, which is just under the basement of the immature foliage leaves, proved to be a very potent explant for the micropropagation of garlic. Twenty to thirty tissue-cultured shoots consistently were differentiated from a single clove during 1 month of culture on phytohormone-free Linsmaier and Skoog medium. In addition, more than 90% of the shoots formed bulblets in vitro during an additional 1 month of culture. Pretreatment of the garlic bulbs at 4 °C for approximately 8 weeks before preparing the stem discs enhanced both shoot development and bulblet formation. This novel method for culturing garlic was designated the stem-disc culture method. Shoot development in this type of in vitro culture apparently is divided into four stages: expansion of tissue zones surrounded by the basal parts of the immature foliage leaves, formation of dome-shaped structures, bud differentiation directly from each dome, and development into shoots and bulblets. The dome-shaped structures appeared within 5 days of the onset of culture and had developed independently into shoots approximately 1 cm high 3 weeks later. Histological observations showed that both the internal cell organization and formation process of the dome-shaped structures were similar to those in the meristem. In addition, events leading to the formation of these dome-shaped structures appeared to be initiated by vigorous cell division in the epidermis of concentric tissue zones surrounded by the basements of immature foliage leaves. The results of several field trials showed that the stem-disc culture method is useful for the production of garlic seed plants, including virus-free plantlets. Furthermore, it is a novel field cultivation system for garlic in that the seedlings produced by in vitro-induced bulblets are used as seed instead of the usual cloves. Received: 25 November 1997 / Revision received: 4 February 1998 / Accepted: 21 February 1998     

Chalcone isomerase isozymes with different substrate specificities towards 6'-hydroxy- and 6'-deoxychalcones in cultured cells of Glycyrrhiza echinata, a leguminous plant producing 5-deoxyflavonoids.

Three chalcone isomerase isozymes in Glycyrrhiza echinata (Fabaceae) were separated by chromatofocusing and partially purified to examine their substrate specificities. Two isozymes, one of which was elicitor-inducible, acted on both 6'-hydroxychalcone and 6'-deoxychalcone and presumably are involved in the legume-specific 5-deoxyflavonoid pathway, while another acted on only 6'-hydroxychalcone.     

Genetic variants of glucose phosphate isomerase (E.C. 5.3.1.9) in canine erythrocytes*

Genetic polymorphism of glucose phosphate isomerase (GPI) was found in the erythrocytes of dogs of six Japanese breeds by using starch gel electrophoresis. Analysis of parentage records of dogs revealed that the phenotypic variation of erythrocyte glucose phosphate isomerase was controlled by one autosomal locus with two codominant alleles, GPI^Aand GPI^B. The allele GPI^Bwas observed in the following breeds: San'in-Shiba, Shinshu-Shiba, Shikoku, Kai and Kishu, but not in Hokkaidoes and Akitas. All the dogs belonging to 25 European breeds, 5 oriental or China-origin (except Japan) breeds examined in this experiments had the genotype constitution GPI^A/GPI^A, whereas one Dalmation dog was heterozygous GPI^A/GPI^B.     

Improved conditions for the transformation by electroporation of the extracellular polysaccharide-producing methylotroph Methylobacillus sp.

The transformation efficiency of Methylobacillus sp. strain 12S, using electroporation, was unaffected by the growth phase of the cells but competent cells grown at 21 °C had a 1.9 × 10³ times higher transformation efficiency than those grown at 30 °C. Heat shock treatment further increased the transformation efficiency up to 7 times.     

Affinity chromatography of nucleosides and nucleic acid base derivatives with nucleic acid bases or nitrobenzeneboronic acid substituted silicas

Nucleic acid bases such as adenine and uracil, and nitrobenzeneboronic acid substituted silicas were prepared by the reaction of chloromethylbenzene substituted silica with adenine sodium salt and trimethylsilylated uracil, and nitration of benzeneboronic acid substituted silica, respectively. From the results of HPLC of nucleosides and N-ethyl derivatives of nucleic acid bases using modified silicas, hydrophobic base stacking interaction, selective hydrogen bonding interaction between purine and pyrimidine bases, and reversible cyclic boronate ester formation between diols of nucleosides with boronic acid were effective for the separation of nucleic acid related compounds. Moreover, association constants for hydrogen bonding formation of nucleic acid bases were estimated.