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We isolated and purified yeast chromosome DNA molecules using pulse field gel electrophoresis (PFG). The isolated DNA had nearly the same size as the native chromosomal DNA on PFG. We could directly transform Saccharomyces cerevisiae yeasts with it, and obtain transformants that were selected by complementation of several markers. They had new chromosome DNA bands observed on PFG. The new chromosome was very stable during mitosis and mating processes, and each of the three homologous chromosomes in the derivative zygotes of transformants was separated equally in daughter cells.