Advantages of genome sequencing by long-read sequencer using SMRT technology in medical area

PacBio RS II is the first commercialized third-generation DNA sequencer able to sequence a single molecule DNA in real-time without amplification. PacBio RS II’s sequencing technology is novel and unique, enabling the direct observation of DNA synthesis by DNA polymerase. PacBio RS II confers four major advantages compared to other sequencing technologies: long read lengths, high consensus accuracy, a low degree of bias, and simultaneous capability of epigenetic characterization. These advantages surmount the obstacle of sequencing genomic regions such as high/low G+C, tandem repeat, and interspersed repeat regions. Moreover, PacBio RS II is ideal for whole genome sequencing, targeted sequencing, complex population analysis, RNA sequencing, and epigenetics characterization. With PacBio RS II, we have sequenced and analyzed the genomes of many species, from viruses to humans. Herein, we summarize and review some of our key genome sequencing projects, including full-length viral sequencing, complete bacterial genome and almost-complete plant genome assemblies, and long amplicon sequencing of a disease-associated gene region. We believe that PacBio RS II is not only an effective tool for use in the basic biological sciences but also in the medical/clinical setting.     

Studies of the fine structure of β-d-glucans of barleys extracted at different temperatures

Enzymic analyses of aqueous extracts of barley obtained at 25–100° demonstrated an exponential relationship between β-d-glucan content and temperature. Purified β-d-glucans, in particular those from extracts made at 40° and 100°, were compared by the Smith-degradation method followed by g.l.c. analysis of the methyl and the trimethylsilyl ethers of the products. All extracts yielded qualitatively the same products, including oligosaccharides which were characteristic of sequences of adjacent (1→3)-linkages. The material extracted at 100° contained relatively more of these sequences, and also showed a much higher specific viscosity, than did the material extracted at low temperatures. The structural implications of these findings are discussed.     

Mode of expression of muscle-type enolase isozyme in the developing limb bud of human embryos

The appearance of beta-enolase, a glycolytic enzyme, was studied immunohistochemically using the upper limb bud of human embryos at Carnegie stages from 13 to 21. beta-Enolase-immunoreactive cells first appeared at stage 15 in the proximal portion of the upper limb bud. It was evidenced that glycogen granules first appear at the same stage. These results may suggest that changes in energy metabolism might be one of the earliest events in the differentiating steps of the skeletal muscles because this stage is earlier than the stages of cell fusion, myofilament formation and innervation of the muscle cells.     

Immunohistochemical study of creatine kinase isozymes in the human upper limb bud

Creatine kinase (CK) is involved in the production of ATP and is composed of two immunologically distinct subunits, B (CK-B) and M (CK-M). In the differentiation of myoblasts, the isozyme of CK changes from CK-B to CK-M. In the present study, the expression of CK subunits was studied immunohistochemically in the upper limb bud of human embryos (Carnegie stages 13-21). It was found that CK-B and CK-M immunoreactive cells appeared at stage 15 and at stage 18, respectively.     

Development of near-infrared firefly luciferin analogue reacted with wild-type and mutant luciferases

Interestingly, only the D-form of firefly luciferin produces light by luciferin–luciferase (L–L) reaction. Certain firefly luciferin analogues with modified structures maintain bioluminescence (BL) activity; however, all L-form luciferin analogues show no BL activity. To this date, our group has developed luciferin analogues with moderate BL activity that produce light of various wavelengths. For in vivo bioluminescence imaging, one of the important factors for detection sensitivity is tissue permeability of the number of photons emitted by L–L reaction, and the wavelengths of light in the near-infrared (NIR) range (700–900 nm) are most appropriate for the purpose. Some NIR luciferin analogues by us had performance for in vivo experiments to make it possible to detect photons from deep target tissues in mice with high sensitivity, whereas only a few of them can produce NIR light by the L–L reactions with wild-type luciferase and/or mutant luciferase. Based on the structure–activity relationships, we designed and synthesized here a luciferin analogue with the 5-allyl-6-dimethylamino-2-naphthylethenyl moiety. This analogue exhibited NIR BL emissions with wild-type luciferase (λ_max = 705 nm) and mutant luciferase AlaLuc (λ_max = 655 nm).     

Separation of abscission zone cells in detached Azolla roots depends on apoplastic pH

In studies on the mechanism of cell separation during abscission, little attention has been paid to the apoplastic environment. We found that the apoplastic pH surrounding abscission zone cells in detached roots of the water fern Azolla plays a major role in cell separation. Abscission zone cells of detached Azolla roots were separated rapidly in a buffer at neutral pH and slowly in a buffer at pH below 4.0. However, cell separation rarely occurred at pH 5.0–5.5. Light and electron microscopy revealed that cell separation was caused by a degradation of the middle lamella between abscission zone cells at both pH values, neutral and below 4.0. Low temperature and papain treatment inhibited cell separation. Enzyme(s) in the cell wall of the abscission zone cells might be involved in the degradation of the pectin of the middle lamella and the resultant, pH-dependent cell separation. By contrast, in Phaseolus leaf petioles, unlike Azolla roots, cell separation was slow and increased only at acidic pH. The rapid cell separation, as observed in Azolla roots at neutral pH, did not occur. Indirect immunofluorescence microscopy, using anti-pectin monoclonal antibodies, revealed that the cell wall pectins of the abscission zone cells of Azolla roots and Phaseolus leaf petioles looked similar and changed similarly during cell separation. Thus, the pH-related differences in cell separation mechanisms of Azolla and Phaseolus might not be due to differences in cell wall pectin, but to differences in cell wall-located enzymatic activities responsible for the degradation of pectic substances. A possible enzyme system is discussed.