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Silencing in Yeast rDNA Chromatin
Francesco Cioci, Loan Vu, Kristilyn Eliason, Melanie Oakes, Imran N. Siddiqi, Masayasu Nomura Molecular Cell Volume 12, Issue 1, Pages (July 2003) DOI: /S (03)
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Figure 1 Structures of Plasmids Used for Integration of Reporter Genes into rDNA Repeats at MluI in the 25S rRNA Coding Region and at SphI in the Spacer One repeat unit of rDNA is 9,137 bp and is numbered with respect to the Pol I transcription start site. The regions coding for the mature rRNAs are lightly filled. The promoter region (P), the region containing the E element (“E”; ∼320 bp EcoRI-HpaI fragment), and the region (“T/E”) containing both the E element and the Pol I termination site (immediately upstream of the E region) are indicated. Orientation of the Pol I transcription and Pol II transcription of reporter genes is shown by arrows. Molecular Cell , DOI: ( /S (03) )
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Figure 2 The Absence of Silencing of mURA3 in rrn9Δ PSW Strains
Three independent Leu+ transformants obtained from PSW strain (NOY902) and control strain (W303-1a; “WT”) were grown and tested for Leu+ and Ura+ phenotypes by spotting aliquots of 10-fold serial dilutions (left panel) and for copy numbers of the integrated mURA3 by Southern analysis (right panel). For Southern hybridization analyses, DNA was isolated and digested with EcoRI, and detection was done using a 32P-labeled URA3 probe. By comparing with marker DNA fragments (lane M), two radioactive bands of the expected sizes, one corresponding to chromosomal URA3 (“G”) and the other corresponding to integrated mURA3 (“I”), were detected. Copy numbers of the integrated mURA3 were calculated by comparing intensities of the two bands and are shown at the bottom of each lane. Molecular Cell , DOI: ( /S (03) )
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Figure 3 Silencing of Both Reporter mURA3 Integrated in rDNA and Pol II Transcription of Chromosomal rRNA Genes in rpa12 ts Mutant Strain Decreases at Elevated Temperatures (A) Both the rpa12 mutant (NOY506) and the control (W303-1a) strains were grown at 25°C and tested for His+ and Ura+ phenotypes at 25°C and 30°C by spot test and for copy numbers of integrated mURA3 by Southern analysis (for G and I, and copy numbers, see Figure 2). (B) The two strains used in (A) were grown at 25°C. At cell density of A600 ∼0.2, cultures were divided into three aliquots and each was incubated at temperatures indicated, respectively, for three hours. Cells were harvested and the 5′-ends of rRNA precursors were quantified by primer extension using equal amounts of total RNA. The sum of radioactivity in the bands indicated by dots, which correspond to Pol II initiated transcripts, was divided by radioactivity in the band corresponding to the Pol I initiated transcript, and the values obtained are shown below the lane numbers (as %). PSW, RNA isolated from a PSW strain (NOY897), was analyzed in parallel as a reference. (C) The two strains used in (A) and an rpa190 ts strain (NOY259) were grown as in (B). RNA samples obtained from cells harvested at 3 hr after temperature shift from 25° to 37° were analyzed by primer extension, and an autoradiogram is shown. Molecular Cell , DOI: ( /S (03) )
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Figure 4 Comparison of Degrees of Silencing of Pol II Reporter Genes in the ∼190 Copy and ∼25 Copy Strains (A) The sizes of chromosome XII analyzed by CHEF. A gel stained with ethidium bromide (left; chromosome XII is indicated by a dot) and an autoradiogram of the same gel obtained after hybridization with an rDNA probe (right) are shown. Using the size markers in another lane (not shown), chromosome XII size estimated for NOY886, NOY1051, NOY1071, and NOY1064 (lanes 1 to 4, respectively) was 37, 112, 16, and 189, respectively. (B) Southern analysis to estimate rDNA copy numbers relative to the previously characterized ∼140 copy (NOY1051) and ∼40 copy (NOY886) strains (French et al., 2003). An autoradiogram is shown. The values for chromosomal rDNA were first normalized to the values for reference LEU2 DNA, and then divided by the value obtained for the ∼40 copy strain. Averages of the ratios calculated in this way from two experiments are shown below each lane. (NOY886 and NOY1051 carry an additional LEU2 gene used for disruption of chromosomal RPA135 [indicated as rpa135].) Using the reliable value of 40 for rDNA copy number for NOY886 (French et al., 2003), rDNA repeat numbers for NOY1071 and NOY1064 were calculated to be ∼25 and ∼190, respectively. In view of inherent errors for values obtained from CHEF analysis, we consider the values obtained by Southern analysis more reliable. (C) Four independent Leu+ transformants carrying integrated pJSS60-2 (1 to 4) were analyzed for expression of mURA3 by spot test, for mURA3 copy numbers by Southern analysis (given on the right), and for correct integration into chromosomal rDNA repeats (not shown). In separate control experiments, pJSS60-2 was integrated into the LEU2 locus of NOY1064 and NOY1071 by cleavage of the plasmid at BstXI and analyzed in the same way. Expression of mURA3 by spot test and copy numbers are shown (indicated as C). All the spot test plates were incubated at 30°C for 3 days. Molecular Cell , DOI: ( /S (03) )
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Figure 5 Silencing of Reporter mURA3 Integrated into the rRNA Coding Region in W303-1a and Its Dependence on the Presence of the Upstream Pol I Promoter (A) Structure of rDNA repeats carrying integrated pNOY572 (“+P”) and pNOY573 (“ΔP”). (B) Four independent Leu+ transformants obtained after integration of pNOY572 (+P) or pNOY573 (ΔP) at the SphI site of rDNA spacers were analyzed for mURA3 expression by spot test, for mURA3 copy numbers by Southern analysis using NcoI (right panel; numbers indicated), and for integration into correct chromosomes (not shown). Two independent control clones carrying pNOY572 (integrated after cleavage with BstXI) at the LEU2 locus were also analyzed (shown as clones C1 and C2). The Southern analysis of the control clones was done by digesting DNA with BamHI, which produces the 5.6 kb fragment for genomic URA3 (“G”; lane 10, W303-1a without integration) and a unique 6.8 kb fragment for a single integration of the plasmid at LEU2. If there is a tandem integration, we expect to see 9.0 kb fragment. The control clones (lanes 11 and 12) showed only the two bands expected for a single copy integration at LEU2. Molecular Cell , DOI: ( /S (03) )
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