In the yeast Saccharomyces cerevisiae, the yeast episomal plasmid (YEp), containing a partial sequence from a natural 2-μm plasmid, has been frequently used to induce high levels of gene expression. In this study, we used Japanese sake yeast natural cir 0 strain as a host for constructing an entire 2-μm plasmid with an expression construct using the three-fragment gap-repair method without Escherichia coli manipulation. The 2-μm plasmid contains two long inverted repeats, which is problematic for the amplification by polymerase chain reaction. Therefore, we amplified it by dividing into two fragments, each containing a single repeat together with an overlapping sequence for homologous recombination. TDH3 promoter-driven yEmRFP (TDH3p-yEmRFP) and the URA3 were used as a reporter gene and a selection marker, respectively, and inserted at the 3′ end of the RAF1 gene on the 2-μm plasmid. The three fragments were combined and used for the transformation of sake yeast cir 0 ura3strain. The resulting transformant colonies showed a red or purple coloration, which was significantly stronger than that of the cells transformed with YEp-TDH3p-yEmRFP. The 2-μm transformants were cultured in YPD medium and observed by fluorescence microscopy. Almost all cells showed strong fluorescence, suggesting that the plasmid was preserved during nonselective culture conditions. The constructed plasmid maintained a high copy state similar to that of the natural 2-μm plasmid, and the red fluorescent protein expression was 54 fold compared with the chromosomal integrant. This vector is named YHp, the Yeast Hyper expression plasmid.

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Various surgical procedures have been used to eradicate cholesteatoma. The advantages and disadvantages of the open and closed methods are discussed. Having used both methods to treat cholesteatoma in children, we present the results obtained from each of these methods.Thirty-three patients with middle ear cholesteatoma were treated surgically between January 1982 and December 1990 at the Department of Otolaryngology, Okayama University. All subjects were under the age of 15. All children were treated by either the closed method or the open method. Residual disease was found in 52.2% of the cases treated by the closed method and 30.0% of those treated by the open method, the overall rate of residual disease was 45.5%. The rates of recurrence were 26.1% and 0%, respectively, with a combined rate of 18.2% for all patients.Overall, 60.9% of the patients treated by the closed method and 30.0% of those treated by the open method had residual and/or recurrent cholesteatoma. The combined rate of residual and recurrent disease for all patients was 51.5%. The hearing results obtained by the two methods were nearly the same. In the open method, the incidence of postoperative problems was insignificant and there were fewer surgical procedures per ear. From our results, we conclude that the open method is more useful in treating cholesteatoma in children.