All eukaryotic genomes thus far examined contain simple sequence repeats. A particularly common simple sequence in many organisms (including humans) consists of tracts of alternating GT residues on one strand.Allelic poly(GT) tracts are often of different lengths in different individuals, indicating that they are likely to be unstable. We examined the instability of poly(GT) and poly (G) cerevisiae. We find that the poly(GT) tracts in S. cerevisiae are unstable enough to account for the polymorphisms observed in higher eukaryotes.
MATERIALS AND METHODSMedia and growth conditions. Media for yeast growth were prepared as described by Sherman et al. (40). 5-Bromo-4-chloro-3-indolyl-13-D-galactopyranoside (X-Gal) plates for S. cerevisiae were prepared as described by Rose and Botstein (35). Medium containing 5-fluoroorotic acid (5-FOA) was used to select for ura3 mutants (4), and medium with DL-aminoadipate was used to select for strains containing lys2 mutations (6). Yeast strains were grown at 32°C. E. coli strains were grown at 37°C in LB broth or in X-Gal medium (26). E. coli HB101, DH5a, and DK1 were used for cloning and plasmid rescues and were handled by following standard molecular techniques (20, 26).Plasmid constructions. (i) pSH31, pSH36, pSH40, and pJA4. These plasmids were derived from plasmid pHT259 (provided by H. Tu, University of Chicago), which has the LEU2 promoter and the first 12 codons of the yeast LEU2 protein fused to the eighth codon of the E. coli ,B-galactosidase gene (Fig. 1). We annealed various oligonucleotides containing simple repetitive DNAs into the BamHI site near the beginning of the ,B-galactosidase gene (Table 1) as follows: (i) 200 ng of each pair of complementary oligonucleotides was suspended in 25 ,ll of 1 mM Tris-HCl (pH 7.5)-100 mM NaCl; (ii) the mixture was heated to 100°C for 4 min, cooled to 50°C, and incubated 1 h at 50°C; and (iii) the sample was reheated to 75°C for 4 min, cooled to 50°C for 30 min, and then slowly cooled to room temperature. These double-stranded oligonucleotides were treated with T4 polynucleotide kinase (26). For construction of the plasmids pSH31, pSH36, and pJA4, pHT259 was digested with BamHI, the oligonucleotides were added with a molar excess of 100:1, and the mixture was treated at 12°C with T4 DNA ligase. E. coli transformations were done with strain DH5a. Since the LEU2 promoter functions in S. cerevisiae and in E. coli, transformants were screened for the loss of f-galactosidase activity on X-Gal plates, and the junctions were then confirmed by DNA sequencing.The oligonucleotides used to construct plasmid pSH40 had cohesive ends compatible with XhoI, not BamHI. Consequently, we inserted a 10-bp XhoI linker (New England Biolabs) into a filled-in BamHI site in pHT259, creating 2749 on May 11, 2018 by guest