Using Yeast Transformation

We describe a general method for analyzing the genetic fine structure of plasmid-borne genes in yeast. Previously we had reported that a linearized plasmid is efficiently rescued by recombination with a homologous restriction fragment when these are co-introduced by DNA-mediated transformation of yeast. Here, we show that a mutation can be localized to a small DNA interval when members of a deletion series of wild-type restriction fragments are used in the rescue of a linearized mutant plasmid. The resolution of this method is to at least 30 base pairs and is limited by the loss of a wild-type marker with proximity to a free DNA end. As a means for establishing the nonidentity of two mutations, we determined the resolution of two-point crosses with a mutant linearized plasmid and a mutant homologous restriction fragment. Recombination between mutations separated by as little as 100 base pairs was detected. Moreover, the results indicate that exchange within a marked interval results primarily from one of two single crossovers that repair the linearized plasmid. These approaches to mapping the genetic fine structure of plasmids should join existing methods in a robust approach to the mutational analysis of gene structure in yeast. ECOMBINANT DNA technology currently apR plied to the yeast, Saccharomyces cerevisiae, includes convenient methods that facilitate a mutational analysis of gene structure and function (reviewed in BOTSTEIN and DAVIS 1982). To isolate new mutations, a cloned gene residing on a plasmid can be mutagenized and introduced via transformation into an appropriate yeast host to screen for altered gene function. T h e plasmid of a transformant displaying a mutant phenotype can be returned to an Escherichia coli host, permitting isolation of the mutant gene for direct analysis and further manipulation. This basic methodology lacks a simple, sensitive method for mapping to a high resolution a mutation on a plasmid in yeast. Even with the use of in vitro mutagenesis, new mutations need to be localized unambiguously to the gene of interest, to be distinguished from silent or modifying lesions, and to be localized to DNA segments small enough to be convenient for sequence analysis. Furthermore, localizing genetic regions of substantial importance to gene function is of immediate interest in a mutational analysis. To devise an approach to the high resolution mapping of plasmids, we utilized the known property of free DNA ends as efficient substrates for homologous recombination in yeast (ORR-WEAVER, SZOSTAK and ROTHSTEIN 198 1 ; reviewed in ORR-WEAVER and SZOSTAK 1985). We have reported (KUNES, BOTSTEIN Present address: Biotechnica International Inc., 85 Bolton Street, Cambridge, Massachusetts 02 140. Genetics 115: 73-81 (January, 1987) and Fox 1984, 1985) that a plasmid DNA broken in a sequence absent from the yeast genome is efficiently rescued by recombination with a homologous restriction fragment included during yeast transformation. Here, we describe some features of recombination with these substrates and utilize this reaction in two approaches to plasmid fine structure analysis, twopoint heteroallelic crosses and deletion mapping. MATERIALS AND METHODS Strains and media: Yeast strains DBY1226 (MATa h i d 5 1 9 met8-1 leu2-3,112 ura3-3) and DBY1227 (MATa h i d 5 1 9 met8-1 leu2-3,112 ura3-18) were constructed in this laboratory by standard methods (SHERMAN, FINK and LAWRENCE 1979). Yeast strain DBY2052 (MATa 1ys2-801 1eu.23, I I 2 ura3-52 hxk1::LEUZ hxk2-202) is a derivative of DBY 13 15 constructed by disruption of HXKl with the cloned LEU2 gene (RATZKIN and CARBON 1977; H. MA and D. BOTSTEIN, unpublished data) and deletion of the HXKP locus. The hxk2 deletion allele, hxk2-202, is a derivative of the cloned HXK2 gene (see below) constructed in vitro by removing the HXKZ coding region from between the flanking sites Sac1 and XbaI (see pRB313 in Figure 5 for the positions of these sites). The genomic HXKZ locus was replaced with this deletion allele by recombination (see BOTSTEIN and DAVIS 1982 for details), yielding strain DBY2052. As a result of the presence of the hxkl and hxk2 mutations, this strain cannot grow on fructose as a sole carbon source, but can grow on glucose due to the presence of glucokinase. Yeast was grown in YEPD (complete) or SD (minimal) medium (SHERMAN, FINK and LAWRENCE 1979). When appropriate, 2% fructose was substituted for 2% glucose as carbon source. E. coli was grown in LB (complete) medium