Scheme of the yeast genomic DNA region containing JEN1 and Southern blot analysis of its disruption. (a) Schematic restriction map of the yeast genomic DNA fragment contained in the plasmid pT12. This plasmid, which contains the complete sequence of JEN1 (YKL 217w), was isolated from the original YEp13 genomic library. (b) Disruption of JEN1 was carried out as described in the text. The 2.7-kb SacI-XbaI fragment was used to replace the genomic wild-type copy. (c) Southern blot analysis of the JEN1 disruption. Genomic DNA was digested with HindIII, subjected to electrophoresis in a 0.8% agarose gel, transferred to Hybond-N membrane, and hybridized with the 855-bp probe indicated in panels a and b. Lanes: 1, DNA from wild type; 2, DNA from BLC 203 strain carrying the jen1::HIS3 disruption. Sizes of the fragments are indicated at left.

Eadie-Hoffstee plots of the initial uptake rates of lactate by the wild type and a jen1::HIS3 disrupted strain. Uptake studies were done at pH 5.0 as described in the text. (a) Cells growing exponentially in YNB-glucose were harvested by centrifugation, washed twice with deionized water, and incubated in YP-lactate for 4 h. (b) Cells growing exponentially in YP-lactate. Symbols: ■, wild type; ●, mutant BLC 203; ○, mutant BLC 203 transformed with plasmid pT12.

Northern blot analysis of JEN1 transcripts from strains differing in locus state. JEN1, wild type; Δjen1, disruptant mutant BLC 203; Δjen1(pT12), BLC 203 strain transformed with pT12 plasmid. Total cellular mRNA was prepared from cells growing exponentially in YNB-glucose (lane 1) and subsequently incubated in YP-lactate for 4 h (lanes 2, 3, and 4) (14). Total RNA (20 μg) was separated on 1.5% agarose morpholinepropanesulfonic acid (MOPS)-formaldehyde gels and blotted to nylon membranes by vacuum transference. Hybridization was carried out using the 844-bp NcoI-PstI fragment as a probe for JEN1 (Fig. 1a), and PDA1 (22) served as internal standard.