Transfonnation of Saccharomyces cerevisiae. Spheroplasts were prepared as described by Hutchinson and Hartwell (9). A fresh logarithmic phase culture (80 ml; 2 X 107 cells per ml) was concentrated to l/1o volume by centrifugation and treated with 1% Glusulase (Endo Laboratories) in 1 M sorbitol for 1 hr at 300. Spheroplasts were washed three times with 1 M sorbitol and resuspended in 0.5 ml of 1 M sorbitol/10 mM Tris-HCl/10 mM CaCl2, pH 7.5. Plasmid DNA was added to a final concentration of [10][11][12][13][14][15][16][17][18][19][20] ,ug/ml and incubated for 5 min at room temperature. Then, 5 ml of 40% polyethylene glycol 4000 (Baker Chemical Co., Phillipsburg, NJ)/10 mM Tris-HCI/10 mM CaCl2, pH 7.5, was added as recently described by van Solingen and van der Plaat (10). After 10 min the spheroplasts were sedimented by centrifugation and resuspended in 5 ml of the sorbitol/Tris/CaCl2 mixture; 0.2-ml aliquots were added to 10 ml of regeneration agar and poured on minimal agar plates [regeneration agar is Difco yeast nitrogen base without amino acids, supplemented with 1 M sorbitol, 2% glucose, 2% YEPD, and 3% agar (10)].Hybridization Analysis of Restriction Digests. Total yeast DNA was digested with restriction endonuclease HindIll (New England Biolaboratories, Beverly, MA). Restriction digests were separated on 0.8% agarose gels and transferred to nitrocellulose filters (Millipore HAWP) according to the method of Southern (11). Details of the blotting procedures and hybridization conditions have been described (5). 32P-Labeled plasmid DNA was prepared by nick translation with E. coli DNA polymerase I (Worthington Biochemical Co.) (12).Yeast Colony Hybridization. Colony hybridization was performed as described by Grunstein and Hogness (13) with

The chromatin fine structure in the promoter region of PHO5, the structural gene for a strongly regulated acid phosphatase in yeast, was analyzed. An upstream activating sequence 367 bp away from the start of the coding sequence that is essential for gene induction was found to reside in the center of a hypersensitive region under conditions of PHO5 repression. Under these conditions three related elements at positions ‐469, ‐245 and ‐185 are contained within precisely positioned nucleosomes located on both sides of the hypersensitive region. Upon PHO5 induction the chromatin structure of the promoter undergoes a defined transition, in the course of which two nucleosomes upstream and two nucleosomes downstream of the hypersensitive site are selectively removed. In this way approximately 600 bp upstream of the PHO5 coding sequence become highly accessible and all four elements are free to interact with putative regulatory proteins. These findings suggest a mechanism by which the chromatin structure participates in the functioning of a regulated promoter.

The repressible acid phosphatase gene PHOS of Saccharomyces cerevisiae requires the two positively acting regulatory proteins PHO2 and PHO4 for expression. pho2 or pho4 mutants are not able to derepress the PHOS gene under low-Pi conditions. Here we show that both PHO2 and PHO4 bind specifically to the PHOS promoter in vitro. Gel retardation assays using promoter deletions revealed two regions involved in PH04 binding. Further characterization by DNase I footprinting showed two protected areas, one located at -347 to -373 (relative to the ATG initiator codon) (UASpl) and the other located at -239 to -262 (UASp2). Exonuclease m footprint experiments revealed stops at -349 and -368 (UASpl) as well as at -245 and -260 (UASp2). Gel retardation assays with the PH02 protein revealed a binding region that lay between the two PH04-binding sites. DNase I footprint analysis suggested a PH02-binding site covering the region between -277 and -296.The promoter region of the strongly regulated acid phosphatase gene PH05 (4) has been recently analyzed by deletion mapping (6,19,24). In addition to the TATA box, two regions were found (24) to be essential for activation of the PHOS promoter. Sequence comparisons defined four elements from which a 19-base-pair dyad consensus sequence was deduced. Three of the elements, called UASp, were located within deletions showing reduced transcription of PHOS (24), at -367, -245, and -185 relative to the translational start.Not much is known about the physical interaction of regulatory proteins with the PHOS promoter. Since mutations defective in PH02 or PH04 are epistatic to all other regulatory mutations and show a negative phenotype, it is likely that the PHO2 and PHO4 proteins are positive transcription factors that interact with the PHOS promoter DNA. The finding that a pho2 mutation can be complemented by overexpression of the PHO4 protein (20) is a further indication that the PHO2 and PHO4 proteins exert their functions at the same late hierarchical level of PH05 activation.Extensive studies of the chromatin fine structure of the PH05 gene (1, 2, 5, 7) have been done. Interestingly, the element at -367, which is essential for gene induction (24), resides in the center of a hypersensitive region under conditions of PH05 repression (1, 2).In press). Interestingly, the pho2 mutation showed no influence on the basal level of TRP4 expression but rather modulated the general control response of TRP4.MATERIALS AND METHODS Escherichia coli vector for PH04 and PH02 expression. The PH04 gene (13, 15) was reisolated from a S288C background by screening an ordered centromere library (25). An NcoI site at the ATG of PH04 was introduced by site-specific mutagenesis (14). The PH04 gene (NcoI-BamHI fragment) was placed under control of the inducible PL promoter by using the vector pPLmu, a derivative of PLc24 (23) containing the phage Mu Ner gene ribosome-binding site with an NcoI site at its ATG start codon (obtained from Biogen).The PH02 gene (25) was fused to the same vector but carrying a differ...

In a systematic approach to the study of Saccharomyces cerevisiae genes of unknown function, 150 deletion mutants were constructed (1 double, 149 single mutants) and phenotypically analysed. Twenty percent of all genes examined were essential. The viable deletion mutants were subjected to 20 different test systems, ranging from high throughput to highly specific test systems. Phenotypes were obtained for two-thirds of the mutants tested. During the course of this investigation, mutants for 26 of the genes were described by others. For 18 of these the reported data were in accordance with our results. Surprisingly, for seven genes, additional, unexpected phenotypes were found in our tests. This suggests that the type of analysis presented here provides a more complete description of gene function.

A new centromere vector for the construction of a Saccharomyces cerevisiae gene library, allowing direct selection for DNA insert, will be described. From that library the gene for the regulatory protein PHO2 involved in PHO5 induction has been cloned by complementation of a pho2 mutation. The complementing activity was shown to be located on a 3.6 kb HindIII fragment. This fragment was used to evict the genomic copy and with appropriate genetic crosses we proved, that the cloned gene is PHO2. The DNA sequence of PHO2 was determined. Analysis of the sequence data uncovered striking homology regions with PHO4, another protein necessary for the induction of PHO5. The relevance of the observed homology will be discussed.