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Kuniyo Ohtoko

Publications and source records attributed to Kuniyo Ohtoko.

3 recordsLinked to original sources

Molecular phylogeny of parabasalids inferred from small subunit rRNA sequences, with emphasis on the Hypermastigea.

Small subunit rRNA gene sequences were identified without cultivation from parabasalid symbionts of termites belonging to the hypermastigid orders Trichonymphida (the genera Hoplonympha, Staurojoenina, Teranympha, and Eucomonympha) and Spirotrichonymphida (Spirotrichonymphella), and from four yet-unidentified parabasalid symbionts of the termite Incisitermes minor. All these new sequences were analyzed by Bayesian, likelihood, and parsimony methods in a broad phylogeny including all identified parabasalid sequences available in databases and some as yet unidentified sequences probably derived from hypermastigids. A salient point of our study focused on hypermastigids was the polyphyly of this class. We also noted a clear dichotomy between Trichonymphida and the other parabasalid taxa. However, this hypermastigid order was apparently polyphyletic, probably reflecting its morphological diversity. Among Trichonymphida, Teranympha (Teranymphidae) grouped together with the members of the family Eucomonymphidae, suggesting that its family status is ambiguous. The monophyletic lineage composed by Spirotrichonymphida exhibited a narrower branching pattern than Trichonymphida. The root of parabasalids was examined but could not be discerned accurately.

Animals↗

Vector-capping: a simple method for preparing a high-quality full-length cDNA library.

Full-length cDNAs play an essential role in identifying genes and determining their promoter regions. Here we describe a simple method for constructing a full-length cDNA library, which has the following advantages: (i) it consists of only three steps including direct ligation between a vector and a cDNA strand using T4 RNA ligase, (ii) it contains neither a PCR process generating mutations nor restriction enzyme treatment causing truncation of cDNA, (iii) the intactness of cDNA is assured due to the presence of an additional dGMP at its 5' end, (iv) approximately 95% of cDNA clones are full-length when cultured cells or fresh tissues are used, (v) several micrograms of total RNA without mRNA purification is sufficient for preparation of a library containing >10(5) independent clones, and (vi) a long-sized full-length cDNA up to 9.5 kbp can be cloned. This method will accelerate comprehensive gene analysis in a variety of eukaryotes.

Animals↗

Determination of the capped site sequence of mRNA based on the detection of cap-dependent nucleotide addition using an anchor ligation method.

The sequence analysis of the 5' ends of cDNAs prepared using the anchor ligation method has revealed that most of the full-length cDNAs have an additional dGMP at their 5' end that is absent in the corresponding genome sequence. Using model RNA transcripts with cap analogues possessing 7-methylguanosine and adenosine, the base of the added nucleotide has been shown to be complementary to the base of the cap analogue, suggesting that the cDNAs possessing an additional dGMP are derived from intact mRNAs with the cap structure. On the other hand, cap-free RNA did not produce cDNA with an extra dGMP. These findings suggest that we can determine whether or not the cDNA starts from the capped site sequence of mRNA based on the presence or absence of an additional dGMP at the 5' end of the cDNA synthesized using the anchor ligation method. This approach will be useful to determine the capped site sequence of mRNA, thus, to identify transcription start sites.

DNA, Complementary↗