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. 2001 Aug;21(16):5541–5553. doi: 10.1128/MCB.21.16.5541-5553.2001

FIG. 1.

FIG. 1

(A) Structure of rDNA repeats in S. cerevisiae. One repeat unit of rDNA is 9,137 bp and is numbered with respect to the Pol I transcription start site (+1). By convention, one repeat unit is shown as the fragment obtained after digestion of rDNA with SmaI, between positions 8931 and 8932. The locations of the 35S and 5S rRNA genes (black bars; the direction of transcription indicated by arrows), the two nontranscribed spacer regions (NTS1 and NTS2), and enhancer and RFB regions are shown. The orientation with respect to the centromere (CEN) and telomere (TEL) is indicated. Although the enhancer studied by Elion and Warner (5, 6) is the ∼190-bp EcoRI (6743)-HindIII (6931) region, subsequent studies on the HOT1 system showed that the ∼130-bp HindIII (6931)-HpaI (7060) region (called the RFB region [see the text]) is also required for efficient Pol I transcription in this system, and the 320-bp EcoRI-HpaI region was called the E element. Following this nomenclature, we define the enhancer and the E element as shown in the figure, even though the entire E element may represent an enhancer. The three restriction sites used to define these regions are indicated. Other EcoRI, HindIII, and HpaI sites are not shown. The I element required for the HOT1 activity is also indicated. In addition, restriction sites for XbaI (X) and the fragment used as a probe (P) (thin black bar) relevant to experiments shown in Fig. 2B are indicated. (B) Structures of pNOY373 (pPol I), pNOY454 (an E deletion [ΔE] derivative of pNOY373), and pNOY130 (pPol II). (C) PCR analysis of genomic DNA showing that the E element was deleted in rdnΔΔ strain NOY906. DNA was isolated from strains NOY505 (wild type [WT]), NOY903 (ΔΔpPol I), NOY906 (ΔΔpPol IΔE), and NOY892 (ΔΔpPol II) and subjected to PCR analysis using two primers outside the E element as indicated by arrowheads 1 and 2 in panel A. The NOY505 and NOY903 genomic DNA samples (lanes 1 and 2) yielded the 1.1-kb PCR product. The NOY906 sample (lane 3) yielded the 0.8-kb PCR product, confirming the absence of the E element. As references, plasmids pNOY373 (pPol I), pNOY454 (pPol IΔE), and pNOY130 (pPol II) were also subjected to PCR analysis (lanes 6, 7, and 8, respectively). As expected, pPol II plasmid (lane 8) and rdnΔΔ strain NOY892 (lane 4) did not yield any PCR product, because they did not contain DNA sequence corresponding to primer 2. Lane 5 contains size markers (M).