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Starch debranching enzyme (R-enzyme or pullulanase) was purified to homogeneity from developing endosperm of rice (Oryza sativa L. cv. Fujihikari) using a variety of high-performance liquid chromatography columns, and characterized. A cDNA clone encoding the full length of the rice endosperm debranching enzyme was isolated and its nucleotide sequence was determined. The cDNA contains an open reading frame of 2958 bp. The mature debranching enzyme of rice appears to be composed of 912 amino acids with a predicted relative molecular mass (Mr) of 102,069 Da, similar in size to its Mr of about 100,000 Da estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The amino acid sequence of rice debranching enzyme is substantially similar to that of bacterial pullulanase, while it bears little similarity to that of bacterial isoamylase or to glycogen debranching enzymes from human muscle and rabbit muscle. Southern blot analyses strongly suggest that the debranching enzyme gene is present as a single copy in the rice genome. Analysis by restriction fragment length polymorphism with a probe including the 3'-untranslated region of cDNA for rice debranching enzyme confirmed that the debranching enzyme gene is located on chromosome 4.

Background: Mycotoxins are fungal secondary metabolites commonly present in feed and food, and are widely regarded as hazardous contaminants. Citrinin, one of the very well known mycotoxins that was first isolated from Penicillium citrinum, is produced by more than 10 kinds of fungi, and is possibly spread all over the world. However, the information on the action mechanism of the toxin is limited. Thus, we investigated the citrinin-induced genomic response for evaluating its toxicity.

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Our earlier studies showed that the surface of developing and calcifying enamel changes its pH alternatively along the tooth axis when stained with pH indicating dyes. Based on the pH conditions, the enamel at this stage was distinguished as neutral zone (N1 and N2) and acid zone (A1 and A2). The aim of the present study was to correlate changes of pH with proteolytic activity and crystal size of the calcifying bovine enamel. Specimens of developing bovine enamel were separated into four maturing stages using pH staining methods. Crystal chemistry of the developing enamel was investigated using thermogravimetry (TGA), ICP emission spectrometry, X-ray diffractometry (XRD) and infrared spectroscopy (IR). Previous biochemical analysis of proteolytic enzyme activity from enamel indicated that the optimal pH of the major protease was approximately pH 6.0, coinciding with the pH of the A1 zone. IR, TGA and XRD analyses showed that most of the organic components of the enamel decomposed at 580 degrees C. Higher levels of carbonate were observed in the secretory stages than in mature enamel. The Ca/P molar ratio of the enamel apatite was lower than the stoichiometric value of 1.67. These results suggest that growth and maturation of enamel apatite crystals is related to a decrease in the carbonate level and appear to be related to the alternative calcification and decomposition of enamel proteins.

A 2275-marker genetic map of rice (Oryza sativa L.) covering 1521.6 cM in the Kosambi function has been constructed using 186 F2 plants from a single cross between the japonica variety Nipponbare and the indica variety Kasalath. The map provides the most detailed and informative genetic map of any plant. Centromere locations on 12 linkage groups were determined by dosage analysis of secondary and telotrisomics using >130 DNA markers located on respective chromosome arms. A limited influence on meiotic recombination inhibition by the centromere in the genetic map was discussed. The main sources of the markers in this map were expressed sequence tag (EST) clones from Nipponbare callus, root, and shoot libraries. We mapped 1455 loci using ESTs; 615 of these loci showed significant similarities to known genes, including single-copy genes, family genes, and isozyme genes. The high-resolution genetic map permitted us to characterize meiotic recombinations in the whole genome. Positive interference of meiotic recombination was detected both by the distribution of recombination number per each chromosome and by the distribution of double crossover interval lengths.

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