Cytotaxonomical studies were conducted on Pilea brevicornuta Hayata in the Ryukyu Islands and Taiwan. Karyomorphology of dwarf and creeping individuals growing in riparian habitat of Amami-Oshima Island, which is morphologically distinct from normal individu-als growing in forest floor, is the main focus of this re -port. All of the studied individuals of P. brevicornuta including dwarf ones had same chromosome number of 2n = 24. The karyotype formula is 2n = 24 = 6m + 13sm + 5st for normal individual and 2n = 24 = 6m + 12sm + 6st for dwarf individual. The chromosomes of P. brevicornuta were all small with sizes varying between 0.9 and 3.0 μm. Considering the basic chromosome number in the genus Pilea, 2n = 24 is regarded as diploid based on x = 12. Despite the high variability in morphology, P. brevicornuta had remarkably uniform karyomorphology.

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Post-translational modifications on histone tails play essential roles in modulating chromatin higher order structure. Phosphorylation of histone H3 at serine 10 (H3S10ph) is associated with chromosome condensation. However direct evidence and the molecular mechanism underlying chromosome condensation induced by H3S10ph in human is unclear. Herein, we employed in vitro reconstituted nucleosomal arrays mimicking H3S10ph by substitution S10 to aspartic acid (H3S10D) using human recombinant histones and highly tandem repeats widom 601 sequence. Using analytical ultracentrifugation sedimentation velocity (AUC-SV) analysis and transmission electron microscopy (TEM) observation, we found that there is no significant difference in the local folding and condensation between WT H3 and H3S10D containing nucleosomal arrays. Thus, our results suggest that H3 phosphorylation at serine 10 does not directly play a role in chromatin condensation and thus higher order structure.

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Tet methylcytosine dioxygenase enzymes catalyze the conversion of DNA methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), thereby initiating DNA demethylation. In mice, Tets also regulate gene activity by binding the regulatory elements of target genes. 5hmC is associated with euchromatic regions and epigenetic reprogramming events. In this study, we examined the levels of 5mC and 5hmC in two major groups of Japanese wrinkled frogs (Rana rugosa) with morphologically distinct sex chromosome constitutions, ZZ/ZW and XX/XY. Patterns of 5mC and 5hmC, as determined by immunostaining, were indistinguishable between homologous sex chromosomes in ZZ cells and XX cells. Genome-wide conversion of 5mC to 5hmC, followed by cell cycle-mediated dilution, was induced in PHA-stimulated peripheral blood cells in adult frogs. Tet enzymes may share an evolutionarily conserved function associated with a global epigenetic reprogramming event during dedifferentiation in mammals and frogs.

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Chromosome higher order structure has long remained unclear since the discovery of chromosome. So far, chromosome structure has been studied using electron microscopy because of its superior resolution and magnification. Recently, researchers started using focused ion beam/scanning electron microscopy (FIB/SEM) to obtain chromosome interior by simultaneous dissection and direct observation of the sections. To minimize distortion of ultrastructure of samples caused by rapid water evaporation under vacuum condition, a critical point drying (CPD) method including pre-dehydration has been extensively used. However, shrinkage or other artifacts in biological samples have also been reported using this method. On the other hand, the ionic liquid (IL) method has been developed to observe biological samples without dehydration, drying and metal/carbon coating by covering samples with a nonvolatile salt. In this study, the inner structure of isolated human chromosomes prepared using CPD and the IL method was observed by FIB/SEM. As a result, it became clear that the cavities appeared in chromosome only when CPD was applied during the preparation steps, and other steps such as fixation and dehydration may have less effect on the appearance of cavities compared to CPD. In conclusion, CPD method should be carefully used when the target is a very small biological sample such as chromosome.

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A chromosome preparation method using young leaves of pear (Pyrus spp.) was developed. Young leaves, 1-2 cm long, of grafted Japanese pear ‘Kosui’ (Pyrus pyrifolia Nakai) were used as materials. The leaves were cut into approximately 2 mm² for enzymatic maceration/air-drying (EMA). For EMA, enzyme mixture containing 4% Cellulase Onozuka RS, 1.5% Macerozyme R200 (Yakult), 0.3% Pectolyase Y-23 (Seishin Pharmaceutical Co., Ltd.), and 1 mM EDTA, pH 4.2, at 37°C for 60-75 min was optimum for chromosome preparation because a large num-ber of good preparations, with all 34 chromosomes relatively extended and well spread without cytoplasm, were observed. The 18S-5.8S-25S ribosomal RNA gene (rDNA) site was detected in telomeric positions of six chromosomes in fluorescent in situ hybridization (FISH). The number and positions of rDNA sites were the same as in the results using root tips as materials (Yamamoto, et al. 2010, 2012). The method developed in the present study is considered to be promising for further cytogenetic studies in pear since true-to-type chromosome samples are obtained from young leaf.

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