We have mapped transcription termination sites for RNA polymerase I in the yeast strains used were W3031a (ade2-1 trp1-1 may1-100 leu2-3,112 his3-11,15 ura3-1leu2-3,112 ura3-52 trp1 his3 lys2 pep4-3 prb1 prc1 rnt1tRNA per mlC1,000 U of S1 nuclease per ml. Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2S)4, 2 mM MgSO4, 0.1% Triton X-100, and 0.1 mg of bovine serum albumin per ml. Samples had been layered with paraffin essential oil and put through amplification cycles comprising 94C for 1 min, 55C for 1 min, and 72C for 2 min. Aliquots of 10 l had been taken out every five cycles for evaluation by gel electrophoresis. For quantitative-competitive PCR, multiple reactions that contains equal levels of cDNA and different quantities of an interior regular DNA template (illustrated in Fig. ?Fig.3A)3A) were create. Amplification was permitted to proceed before earliest point of which products could possibly be detected, and samples had been after that gel electrophoresed. Gels had been dried, and band intensities had been quantitated on a PhosphorImager. Open in another window FIG. 3 Quantitation of transcript abundance by RT-PCR. (A) Places of primers utilized for RT-PCR analysis Dovitinib of PolI termination. Also illustrated is the location of a 75-bp place used to make an internal standard DNA HES7 for quantitative PCR. (B) Qualitative RT-PCR analysis of PolI transcript abundance. As explained in Materials and Methods, primers 1 through 4 were individually used to make cDNA from equal aliquots of total yeast RNA. A fifth mock cDNA reaction in which no primer was added was run. An equal aliquot of each cDNA was then amplified by using the relevant primer pair (i.e., P1-P5 was used to amplify P1 cDNA, etc.). An sample was removed from each PCR every five cycles to measure the progress of amplification. As a control to show that every primer pair was equally active, each primer pair was also used to amplify rDNA. (C) Quantitative RT-PCR Dovitinib analysis of PolI transcript abundance. Aliquots of cDNA made with primers P1 through P4 (plus a blank cDNA in which no primer was added) were mixed with known amounts Dovitinib of an internal standard DNA and amplified with the appropriate primer pair. Each aliquot of cDNA corresponded to the amount of cDNA made from 0.15 g of total yeast RNA. After separation by gel electrophoresis, amplification products were quantitated on a PhosphorImager. Extraction of yeast RNA. For a typical RNA planning, yeast cells were grown in 25 ml to an optical density of 0.5 to 1 1.0. NaN3 was added to 100 mM, and pelleted cells were frozen in liquid nitrogen for storage. For RNA isolation, a pellet of cells from a 25-ml tradition was suspended in 0.5 ml of 10 mM Tris-HCl (pH 7.5)C10 mM EDTAC0.5% sodium dodecyl sulfate to which was added 0.5 ml of glass beads and 0.5 ml of water saturated phenol. The combination was heated to 65C, vortexed for 2 min and then incubated a further 60 min at 65C. Samples were chilled on ice and centrifuged, and the aqueous coating was reextracted with 0.5 ml of phenol plus 0.5 ml of CHCl3. After ethanol precipitation, RNA was digested with DNase I (1 g/ml) for 30 min at 37C, phenol extracted, precipitated, and dissolved in water. RESULTS S1 nuclease safety analysis of RNA 3 end formation on the chromosomal ribosomal genes. A common laboratory strain of yeast (W3031a) was grown to mid-log phase in rich medium, and total RNA was extracted. Aliquots of RNA were then hybridized with a single-stranded, end-labeled DNA probe which has a labeled 3 terminus at ?72 relative to the 3 end of 25S rRNA and extends to +179, where its sequence diverges from the sequence of the rDNA in strain W3031a (probe 1; termini illustrated in Fig. ?Fig.1).1). The hybrids were digested with S1 nuclease, and safeguarded probe fragments were displayed on a denaturing urea-acrylamide gel as demonstrated in Fig. ?Fig.2A,2A, lane 1. Open in a separate window FIG. 2 (A) S1 nuclease analysis of RNA 3 ends created on the chromosomal rDNA of strain W3031a. In lane 1, probe 1 was hybridized to total cellular RNA and digested with S1 nuclease, and the safeguarded fragments were displayed on a denaturing acrylamide gel. The most prominent band is definitely a cluster corresponding to the mature 3 end of 25S rRNA (?1). The next most prominent bands are in a cluster extending from +93 downward to about +72. These bands are consistent with PolI termination at +93 followed by 3 end processing of the released transcripts. A minor band at +179 gives an estimate of the amount of transcription that reads past the Reb1p terminator. Lane 2 is the same as lane 1 except that tRNA was added instead of yeast.