Fig. 8

Mini-chromosome loss assay of duplicated mini-chromosome. a Represents transformants harboring Chr2-6 (dup + rep) and b represents transformant containing Chr11-2 (dup + rep), respectively. After serial dilution of full growth culture in YPDA liquid medium, cells were plated on YPDA plate. This YPDA plate was treated as a master plate for subsequent replica-plating experiment. After colonies appeared on YPDA plate, the YPDA plate was replica-plated on Leu minus, Ura minus, Leu and Ura minus media, 5-FOA and YPDA plates as a control. After incubation, we found that cells plated on YPDA master plate formed three types of colonies for Chr2-6 (dup + rep) and two types of colonies for Chr11-2 (dup + rep) cases. In both cases, one type of colonies were supposed to lose mini-chromosome. As a consequence, they showed Ura⁻ and 5-FOA⁺ phenotype. Two representatives of this type of colonies were marked with red-colored arrow. On the other hand, most of other colonies of both cases were supposed to keep mini-chromosome for which they showed Ura⁺ phenotype but they also formed colonies in 5-FOA medium. We think that these colonies consist of cells lost mini-chromosome and therefore they showed 5-FOA⁺ phenotype. Two of this type of colonies were marked with blue-colored arrow. Besides, in only Chr2-6 (dup + rep) case, we found very few colonies which were supposed to hold mini-chromosome and therefore showed Ura⁺ phenotype but which, by unknown reason, did not lose mini-chromosome and consequently showed 5-FOA⁻ phenotype. Two representatives of this type of colonies were marked with green-colored arrow