For the transformation of the yeast Hansenula polymorpha we have constructed a set of hybrid plasmids carrying the LEU2 gene of Saccharomyces cerevisiae as a selective marker and fragments of mitochondrial DNA of Candida utilis and H. polymorpha or chromosomal DNA fragments of H. polymorpha as replicator sequences. The replication properties of chimeric plasmids in the yeast H. polymorpha were investigated. We showed that for plasmids propagated autonomously in this yeast the plasmid monomers could be detected in the transformants only during the immediate time after the transformation event. Further growth under selective conditions led to the selection of polymeric forms of plasmid DNA as it was clearly shown for transformants carrying cosmid pL2 with mtDNA fragment of C. utilis. Such transformants carrying polymerized plasmids showed a remarkably increased stability of the transformed phenotype. Cosmid pL2 was able to shuttle between Escherichia coli, S. cerevisiae and H. polymorpha, whereas plasmids with DNA fragments from H. polymorpha did not transform S. cerevisiae effectively.
We developed a host-vector system for transformation and gene cloning experiments using the methylotrophic yeast Hansenula polymorpha. Regeneration of protoplasts in a medium containing polyethylene glycol before plating made transformation more efficient and reproducible (2 to 3 x 10(4) micrograms DNA). The frequency of transformation was significantly lower when dominant resistance marker Cup1r was used for transformant selection. The transformation system developed was used to clone the DNA fragment which complements functionally the defect in the dihydroxyacetone kinase (DHAK*) activity of a H. polymorpha mutant strain. The DNA insert isolated was shown to increase by up to ten times the activity of DHAK in transformants carrying recombinant plasmids. When recombinant plasmids were introduced into S. cerevisiae, the transformants obtained acquired the ability to grow on the medium with dihydroxyacetone as a sole carbon source and the activity of DHAK was observed.
ВВЕДЕНИЕ О микоплазменной инфекции за прошедшие деся-тилетия написано несколько монографий и множе-ство статей. На протяжении многих лет и в России, и за рубежом интерес к этой проблеме не ослабева-ет. Это прежде всего связано с тем, что существуют периоды подъема (эпидемии) заболеваемости мико-плазменной инфекцией (один раз в 5-8 лет), которые влекут за собой увеличение числа больных с пневмо-нией, бронхитами, острыми респираторными забо-леваниями.Однако, несмотря на определенные успехи, достигну-тые в диагностике этого заболевания, открытии важных патогенетических звеньев, получении данных о фор-мировании аутоиммунных заболеваний, до сих пор существуют недостаточно изученные вопросы о продол-жительности курса лечения, в особенности серопозитив-ных лиц, и т. д.
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