Construction of Saccharomyces cerevisiae haploid strain carrying splitted chromosome XII at right region of rDNA locus

Abstract

Saccharomyces cerevisiae has a great potential for fermentation industry and molecular biology research. Many works have been conducted to increase S. cerevisiae growth rate. The objective of this research was to obtain a rDNA fragment of chromosome XII of S. cerevisiae with splitting at right region of rDNA locus. Chromosome XII was splitted by using a chromosome-splitting vector (pDW49) that carried a cloned DNA fragment of rDNA locus. An E. coli strain DH5α was used as a host for pDW49 amplification. The pDW49 plasmid was digested with BamHI to eliminate the HIS3 DNA stuffer. The larger BamHI fragment was isolated by agarose gel electrophoresis. The BamHI fragment was subsequently self-ligated, resulting in the Tr ends being joined head-to-head. The recircularized DNA molecule was linearized by cleavage at the homologous sequence (at the right region of rDNA locus) using BglII. The linearized DNA molecule was introduced into S. cerevisiae strain W303-1A by lithium acetate method. Confirmation of chromosome XII splitting was analyzed by PFGE (Pulsed Field Gel Electrophoresis) for 24 h with switching interval of 45 sec followed by 6 h run with switching interval of 15 sec. The result of PFGE showed an additional chromosomal band (611 kbp), suggesting that the chromosome XII has been splitted.