♥ Alamat korespondensi: Jl. Pabelan, Sukoharjo 57102 Tel. +62-271-717417 Fax. +62-271715448 e-mail: biology@mipa.uns.ac.id (ed.). Konstruksi Strain Haploid Saccharomyces cerevisiae Pembawa Kromosom XII yang Terpotong pada Sisi Kanan Lokus rDNA Construction of Saccharomyces cerevisiae haploid strain carrying splitted chromosome XII at right region of rDNA locus TRIASTUTI RAHAYU 1, ♥ , DONNY WIDIANTO 2,3 , TRIWIBOWO YUWONO 2,3 1 Jurusan Pendidikan Biologi FKIP Universitas Muhammadiyah Surakarta (UMS) Sukoharjo 57102 2 Program Studi Bioteknologi Program Pascasarjana Universitas Gadjah Mada (UGM) Yogyakarta 55281. 3 Fakultas Pertanian Universitas Gadjah Mada (UGM) Yogyakarta 55281. Diterima: 15 Maret 2004. Disetujui: 17 Mei 2004. 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. Keywords: Saccharomyces cerevisiae, Chromosome XII and rDNA. PENDAHULUAN Saccharomyces cerevisiae merupakan khamir yang banyak digunakan dalam industri fermentasi dan sebagai jasad model dalam penelitian-penelitian biologi molekular. Oleh karena itu, banyak dilakukan usaha perbaikan strain-strain S. cerevisiae untuk meningkatkan produktivitas maupun kualitas produk yang dihasilkan dengan biaya produksi yang lebih rendah. Pendekatan dalam perbaikan strain khamir tersebut dapat dicapai melalui metode- metode genetika, baik yang konvensional seperti: mutasi, hibridisasi seksual dan seleksi, maupun teknik-teknik yang baru dikembangkan seperti: fusi protoplas, transformasi, dan DNA rekombinan, atau kombinasi dari kedua kategori metode genetika tersebut (Shimizu, 1994). Bioteknologi 2 (1): 9-13, Mei 2005, ISSN: 0216-6887 '2, ELRWHNF