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... 1998), and detailed protocols can be found at the author’s web site: http://web.wi.mit.edu/young/ expression/. Briefly, total RNA was first extracted from frozen cell pellets by a hot phenol method (=-=Schmitt et al., 1990-=-). Cells were thawed on ice and then mixed with 3 ml of acid phenol/chloroform (5:1; prewarmed for 10 min at 65°C) and 3 ml of TES (10 mM Tris, pH 7.5/10 mM EDTA/0.5% SDS) per 200 OD600 U. The mixture...

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...D METHODS Strains. The strains and plasmids used in these experiments are described in Table 1. RNase protections. RNA was extracted from Saccharomyces cerevisiae cells as described by Schmitt et al. =-=(34)-=-, and RNase protection analysis (25) was adapted to the use of RNase T2 (22). The probe was constructed by PCR. It includes 35 nucleotides (nt) of the RPL32 intron and 39 splice site and 128 nt of the...

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...ins were analyzed on 12% polyacrylamide gels. Proteins were visualized by Western blotting or by autoradiography of the gels after drying. Northern blotting. RNA was extracted as described previously =-=(36)-=- and separated by using 1.2% agarose-formaldehyde gels for the large rRNA species and 6% polyacrylamide–7 M urea gels for the small rRNA species. RNA was transferred to nylon membranes (Zetaprobe; Bio...

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...1.76 � 10 9 ) were harvested by centrifugation (4,000 � g, 5 min at 4�C) and washed three times with diethyl pyrocarbonate-treated water (5), and the RNA was extracted by the method of Schmitt et al. =-=(46)-=-. Serial dilutions of RNA were vacuum blotted onto nylon membranes and then hybridized under high-stringency conditions with [ 32 P]dCTP-labelled PCR-generated CDR1 (38), BEN r (12), ERG16 (L1A1 [28])...

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... (including TFASTA and LFASTA) and BLAST (2) sequence comparison software packages. RNA preparation and Northern (RNA) analysis. Preparation of total yeast RNA was done as described by Schmitt et al. =-=(54)-=-. Briefly, samples were centrifuged and resuspended in 350 ,ul of AE buffer (50 mM Na acetate [pH 5.2], 10 mM EDTA). Forty microliters of 10% sodium dodecyl sulfate (SDS) and 400 ,lI of phenol (phenol...

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... grown in YPD to an optical density at 600 nm between 1.3 and 1.9 or they were induced in 2.5 mM GlcNAc as described by Holmes and Shepherd (18). Total RNA was prepared as described by Schmitt et al. =-=(33)-=-. RNA blotting was performed as described previously using the 1.5-kb ClaI-SalI ACT1 fragment (9), a 1.5-kb BamHI-HincII fragment carrying a portion of the PMT1 coding region (41), and the 1.5-kb PstI...

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.../vol), 2 ml aliquots were stored at �80°C. Working stocks were maintained on YPE agar slants. Molecular biology techniques. DNA and RNA were prepared and manipulated according to published procedures =-=(22, 23, 25)-=-. Transformation of yeast cells was carried out by the freeze method (10). Escherichia coli JM101, DH5�F�, and SURE (Stratagene GmbH) were transformed by electroporation. p426MET25 (18) served as a ve...

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...r Northern analyses were grown in minimal medium to an OD 600 of 1.0, and then cycloheximide at a final concentration of 0.3 �g/ml was added. Total RNA was isolated from cells as described previously =-=(32)-=- at different times after cycloheximide addition. Thirty micrograms of RNA was separated on agarose gel containing 6% formaldehyde, blotted to a nylon membrane (Hybond N�; Amersham), and hybridized to...

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...e used to screen for adr1 disruption mutants. RNA procedures. Fungal cells were grown on charcoal-containing double complete medium agar for 48 h, and RNA was isolated essentially as described before =-=(47)-=-. Standard molecular techniques were followed for gel electrophoresis, RNA blotting, and hybridization (46). A 680-bp EcoRV fragment was used to probe for mfa1 (6). The ras2 transcript was identified ...