Ty1 mRNA levels increase under conditions of adenine starvation in mutants that block the de novo AMP biosynthesis pathway. (A) Schematic of the de novo AMP biosynthesis pathway and of the pathway that metabolizes the extracellular adenine. For the metabolism of extracellular adenine, the thickness of the arrow indicates which pathway is preferentially used. (B) Northern blot analysis of RNA isolated from cells grown under adenine limitation. Wild type, strain FYBL1-23D; ade2Δ, strain LV679; bas1Δ, strain LV426. Cultures were grown at 22°C in SDc minimum medium supplemented with 0.3 mM (high) or 0.025 mM (low) adenine. Ty1 mRNA was normalized to the ACT1 signal. The mRNA ratios are given relative to the wild-type strain grown in SDc medium supplemented with 0.3 mM adenine (high). The activation is normalized to the “high-adenine” condition for each strain and represents the average of at least two independent analyses. (C) Northern blot analysis of RNA isolated from cells grown under conditions of adenine limitation. Wild type, strain 350; ade13-52, strain 206. Growth conditions and mRNA normalization are as described for panel B.

Ty1 cDNA accumulates in bas1Δ cells starved for adenine. (A) Diagrams of unintegrated Ty1 cDNA and a genomic Ty1 element, indicating the location of the TYB1 hybridization probe (hatched box) and relevant PvuII cleavage sites. The TYB1 probe hybridizes to a 2-kb PvuII fragment of unintegrated Ty1 cDNA and variably sized >2-kb fragments containing the junction of Ty1 elements with chromosomal DNA at different locations. (B) Southern blot analysis of PvuII-digested total cellular DNA. WT, strain FYBL1-23D; bas1Δ, strain LV426; fus3Δ, strain LV243; spt3-101, strain LV47. Cultures were grown at 22°C in HC medium supplemented (+ade) or not (−ade) with 0.3 mM adenine. Ty1 cDNA levels were determined in wild-type and bas1Δ cells by quantifying the intensity of the 2.0-kb Ty1 cDNA band relative to the intensities of two genomic Ty1 junction bands (G1 and G2). The ratios are given relative to the BAS1 strain grown in HC medium supplemented with adenine.

Activation of Ty1 transcription by adenine starvation is abolished in hta1-htb1Δ and snf2Δ mutants. Strains are FYBL1-23D derivatives. Cultures were grown at 22°C in HC medium supplemented (+) or not (−) with 0.3 mM adenine, except for the experiment shown in panel D, for which strains were grown in SDc medium supplemented or not with adenine. (A) Northern blot analysis of RNA isolated from BAS1 HTA1-HTB1 (wild type), bas1Δ, hta1-htb1Δ, and bas1Δ hta1-htb1Δ cells containing the TY1A(DR1)-lacZ fusion. For each sample, 10 μg of total RNA was loaded on the gel. Ty1 mRNA was normalized to the ACT1 signal. The mRNA ratios are given relative to the wild-type strain grown in HC medium supplemented with 0.3 mM adenine (+). The activation was normalized to the “+adenine” condition for each strain. The right panel was more exposed than the left panel. (B) β-Galactosidase activities of the TY1A(DR1)-lacZ fusion in BAS1 HTA1-HTB1 (WT), hta1-htb1Δ, bas1Δ, and hta1-htb1Δ bas1Δ cells. White bars, +adenine; gray bars, −adenine. The relative activation levels observed under conditions of adenine limitation are indicated above the gray bars. (C) β-Galactosidase activities of the TY1A(DR1)-lacZ fusion in BAS1 HTA1-HTB1(WT) and hta1-htb1Δ cells transformed with p11-4/HIS3 (STE11-4 HIS3 CEN) or pRS313 (HIS3 CEN). White bars, +adenine; gray bars, −adenine. The relative activation levels observed under adenine limitation are indicated above the gray bars. (D) β-Galactosidase activities of the TY1A(DR1)-lacZ fusion in BAS1 SNF2 (WT), bas1Δ, snf2Δ, and bas1Δ snf2Δ cells. White bars, +adenine; gray bars, −adenine. The relative activation levels observed under adenine limitation are indicated above the gray bars.

Northern blot analysis of RNA isolated from wild-type (BAS1), bas1Δ, and bas1Δ gcn4Δ cells grown under adenine starvation. Adenine (0.3 mM) was added (+) or not (−) to cultures grown at 22°C in SDc minimal medium. (A) BAS1, Y329 strain transformed with p79 (BAS1 URA3 CEN) and pAM19 (GCN4 HIS3 CEN); bas1Δ, Y329 transformed with YCp50 (URA3 CEN) and pAM19 (GCN4 HIS3 CEN). Ty1 mRNA was normalized to the ACT1 signal. The mRNA ratios are given relative to the BAS1 strain grown in SDc medium supplemented with adenine. The activation was normalized to the “+ adenine” condition for each strain and represents the average of at least two independent analyses. The ADE1 gene, which is activated by BAS1, serves as a positive control. (B) gcn4Δ, Y329 transformed with p79 (BAS1 URA3 CEN) and pRS313 (HIS3 CEN); gcn4Δ bas1Δ, Y329 transformed with YCp50 (URA3 CEN) and pRS313 (HIS3 CEN). Ty1 mRNA was normalized to the ACT1 signal. The mRNA ratios are given relative to the gcn4Δ bas1Δ strain grown in SDc medium supplemented with adenine.

Adenine starvation preferentially stimulates Ty1 elements expressed at low levels. Strains are FYBL1-23D derivatives. Ty1 elements are named according to the Ty1 sequence annotation by the Munich Information Center for Protein Sequences (http://mips.gsf.de/genre/proj/yeast/). (A) β-Galactosidase activities of weakly and highly expressed TY1A-lacZ fusions at native Ty1 elements in BAS1 and bas1Δ cells. Cultures were grown at 22°C in SDc minimum medium supplemented (+ade, white bars) or not (-ade, gray bars) with 0.3 mM adenine. The relative activation levels observed in bas1Δ cells grown in the absence of adenine are indicated above the gray bars. (B) β-Galactosidase activities of TY1A(ML1)-lacZ and TY1A(ML2)-lacZ fusions in BAS1 ADE2, bas1Δ, ade2Δ, and bas1Δ ade2Δ cells. Cultures were grown at 22°C in SDc minimum medium supplemented with 0.3 mM (high; white bars) or 0.025 mM (low; gray bars) adenine. The relative activation levels of TY1A(ML1)-lacZ observed under conditions of adenine limitation are indicated above the gray bars. (C) Northern blot analysis of RNA isolated from BAS1 and bas1Δ cells containing either the weakly expressed TY1A(ML1)-lacZ fusion or the highly expressed TY1A(ML2)-lacZ fusion. mRNA samples were extracted from the cultures described for panel A. Ty1 mRNA was normalized to the ACT1 signal. The mRNA ratios are given relative to the BAS1 strain grown in SDc medium supplemented with 0.3 mM adenine and represent the average of at least two independent analyses. TY1A-lacZ and ADE1 mRNA signals were too faint to be normalized. ADE1 served as a positive control.

Ty1 sequences necessary for the response to adenine starvation are located in TY1A. (A) Schematic of the Ty1-lacZ fusions. In the TY1A-lacZ fusion, lacZ is fused at coordinate 1571 of Ty1H3 (3). This construct contains all of the Ty1 transcriptional control sequences. In the TY1A^F-lacZ fusion, lacZ is fused at coordinate 425 of Ty1H3, adjacent to the 3′ side of the FRE (3). In the TY1A^mir-lacZ fusion, lacZ is fused at coordinate 1571 of Ty1H3 (3) and the sequences recognized by Mcm1, Rap1, and Tea1 have been replaced by three Gal4 binding sites. (B) β-Galactosidase activities of TY1A^F-lacZ fusions at Ty1(DR3), Ty1(ML1), Ty1(ML2), and Ty1(PR1) in BAS1 and bas1Δ cells. Cultures were grown at 22°C in SDc minimum medium supplemented (+ade; white bars) or not (−ade; gray bars) with 0.3 mM adenine. The relative activation levels observed in bas1Δ cells grown in the absence of adenine are indicated above the gray bars. (C) β-Galactosidase activities of TY1A-lacZ fusions at the weakly expressed Ty1(DR3) and the highly expressed Ty1(ML2) elements in BAS1 STE12 (wild type), bas1Δ, ste12Δ, and bas1Δ ste12Δ cells. Growth conditions were as described for panel B. White bars, +adenine; gray bars, −adenine. The relative activation levels observed in bas1Δ and bas1Δ ste12Δ cells grown in the absence of adenine are indicated above the gray bars. (D) β-Galactosidase activities of TY1A-lacZ and TY1A^mir-lacZ fusions at the weakly expressed Ty1(DR3) and the highly expressed Ty1(ML2) elements in bas1Δ cells. Strains carrying TY1A^mir-lacZ fusions at Ty1(DR3) and at Ty1(ML2), LV726 and LV502, respectively, lack GAL4 to avoid activation of the fusion, since the MIR sequence has been replaced by three Gal4 binding sites in these fusions. Growth conditions were as described for panel B. White bars, +adenine; gray bars, −adenine. The relative activation levels observed in the absence of adenine are indicated above the gray bars. (E) β-Galactosidase activities of the weakly expressed TY1A(DR3)-lacZ fusion in BAS1 TEA1 (wild-type), bas1Δ, tea1Δ, and bas1Δ tea1Δ cells. Growth conditions are as described for panel B. White bars, +adenine; gray bars, −adenine. The relative activation levels observed in cells grown in the absence of adenine are indicated above the gray bars. Strains are FYBL1-23D derivatives.