β-Galactosidase activity of LTR-lacZ and TYA-lacZ fusions. (A) β-Galactosidase activity of LTR-lacZ and TYA-lacZ fusions at Ty1-DR3, Ty1-ML1, and Ty1-PR1 in bas1Δ cells. Cultures were grown at 22°C in SDc minimum medium supplemented (+) or not (−) with 0.3 mM adenine. AF, activation factor (versus adenine). Data represent the average with standard error from three independent cultures. Exact average β-galactosidase specific activities are given above the bars and are expressed in nanomoles of 2-nitrophenyl-β-d-galactopyranoside hydrolyzed per minute per milligram of protein. (B) β-Galactosidase activity of LTR-lacZ and TYA-lacZ fusions carried on high-copy-number plasmids in bas1Δ cells. Growth conditions and data representations are described in the legend to panel A. (C) β-Galactosidase activity of LTR-lacZ fusions at Ty1-DR3 and Ty1-PR1 in bas1Δ, snf2Δ, and bas1Δ snf2Δ cells. Growth conditions and data representations are described in the legend to panel A.

β-Galactosidase activity of TDH3-lacZ fusions expressed from Ty1 sequences. (A) Schematic of the TDH3-lacZ reporter gene. (B) β-Galactosidase activity in bas1Δ cells of TDH3-lacZ fusions carried on centromeric plasmids. The structures of the different TDH3-lacZ fusions and the names of the plasmids are indicated. Gray and dotted boxes indicate the MIR and the FRE regions. The arrow indicates the LTR sequences. Cultures were grown at 22°C in SDc minimum medium supplemented (+ade) or not (−ade) with 0.3 mM adenine. β-Galactosidase specific activities are expressed in nanomoles of 2-nitrophenyl-β-d-galactopyranoside hydrolyzed per minute per milligram of protein. Data represent the average with standard error from three independent cultures.

The ESF1 gene is under the control of the Ty1-DR6 endogenous element. RNA extracted from cultures of BAS1 STE12 (wild type [WT]), bas1Δ, ste12Δ, and bas1Δ ste12Δ cells was subjected to Northern blotting (A) and primer extension analysis (B). Cultures were grown at 22°C in SC medium supplemented with (+) or without (−) 0.3 mM adenine. (A) For each sample, approximately 15 μg of RNA was loaded onto the gel. The blot was sequentially hybridized to ESF1, ACT1, and Ty1 probes. The sizes of the mRNA molecules are 5.6 kb (Ty1), 1.9 kb (ESF1), and 1.3 kb (ACT1). Lanes are numbered for clarity as cited in the text. (B) One hundred micrograms of RNA was analyzed by primer extension using a ³²P-end-labeled O-GS71 primer. A sequencing ladder obtained with the same primer is shown (TGCA). The 5′ ends of the different products are indicated with respect to ESF1 +1 and LTR +1. Black circles indicate specific 5′ ends. Lanes are numbered for clarity as cited in the text. (C) Sequences encompassing the Ty1 LTR and ESF1, with the positions of the different RT stops observed by primer extension with the O-GS71 primer. ESF1 and Ty1 LTR sequences are indicated in boldface. Three short ORFs present in the sequences between Ty1 LTR and ESF1 are underlined. The ESF1 ATG initiation codon is indicated by an arrow pointing to the right, and the +1 coordinate of Ty1 LTR is indicated by an arrow pointing to the left. TATA-rich sequences upstream of LTR coordinates +15 and +45 are indicated by shaded boxes.

(A) Ty1 structure and promoter. Ty1 transcription regulatory sequences are located within the first kilobase of the retrotransposon. Ty1 transcription starts in the 5′ LTR (symbolized by an arrow pointing to the right) and terminates in the 3′ LTR (symbolized by a circle on top of a vertical line). A, B, C, D, and E indicate Gcn4 binding sites. FRE and MIR binding sites are also shown. The ATG initiation codon is located in the LTR, upstream of Gcn4^E binding site. Gcn4, Gcr1, Ste12, Tec1, and Tea1 are activators of Ty1 transcription, while Mot3 is a repressor. Although the MIR region contributes positively to Ty1 transcription, the exact roles of Mcm1 and Rap1 in Ty1 transcription have not been clearly defined. The presence of the five Gcn4 binding sites is a signature of highly expressed Ty1 elements. (B) Multiple alignment of the 5′ LTR sequences of Ty1-PR1, Ty-DR3, and Ty1-PR1. Ty1-PR1 is a highly expressed element (H) and contains five Gcn4 binding sites in its LTR (in boldface). Ty1-DR3 and Ty1-ML1 are weakly expressed Ty1 elements (W). Ty1-DR3 lacks the Gcn4^E binding site, while Ty1-ML1 lacks the full set of Gcn4 binding sites and TATA box 1.

Severe adenine starvation activates Ty1-his3Δ4 expression. (A) BAS1 STE12 (wild type [WT]), bas1Δ, ste12Δ, and bas1Δ ste12Δ cells containing either his3Δ4, Ty1-his3Δ4, or LTR-his3Δ4 alleles were spotted onto plates in a series of 10-fold dilutions. All plates were incubated at 30°C for 4 days.

Pattern of transcription of Ty1-his3Δ4 and LTR-his3Δ4 alleles. RNA extracted from BAS1 STE12 (wild type [WT]), bas1Δ, ste12Δ, and bas1Δ ste12Δ cells containing either his3Δ4, Ty1-his3Δ4, or LTR-his3Δ4 alleles and from BAS1 STE12 HIS3 (wild type [HIS3]) cells was subjected to Northern blotting (A) and primer extension analysis (B). Cultures were grown in HC medium supplemented with (+) or without (−) 0.3 mM adenine at 22°C. (A) For each sample, approximately 15 μg of RNA was loaded onto the gel. The blot was sequentially hybridized to HIS3, ACT1, and Ty1 probes. The sizes of the mRNA molecules are 5.6 kb (Ty1), 0.7 kb (HIS3), and 1.3 kb (ACT1). Lanes are numbered for clarity as cited in the text. The HIS3 signal in the right panel has been overexposed to permit detection of weak bands. (B) For each sample, 100 μg of RNA was analyzed by primer extension using ³²P-end-labeled O-GS73 primer. A sequencing ladder obtained with O-GS73 is shown (TGCA). The 5′ ends of the different products are indicated with respect to the +1 nucleotide of the HIS3 ATG codon and the +1 nucleotide of the 5′ LTR. Black circles indicate specific RT stops. Open circles indicate nonspecific arrests. Lanes are numbered for clarity as cited in the text. (C) Sequences encompassing the Ty1 LTR and his3Δ4 with the position of the different RT stops observed by primer extension with the O-GS73 primer. HIS3 and Ty1 LTR sequences are indicated in boldface. Sequences upstream of the HIS3 ORF, which are conserved in his3Δ4, are in capital letters. Two ORFs present in the sequences between the Ty1 LTR and HIS3 are underlined. The HIS3 ATG initiation codon is indicated by an arrow pointing to the right, and the +1 coordinate of the Ty1 LTR is indicated by an arrow pointing to the left. TATA-rich sequences upstream of LTR coordinates +15 and +45 are indicated by shaded boxes.