site to amplify DermC, giving an amplicon that was cloned as
a HindIII-EcoRI fragment into the pYH250 vector. For some
constructs, a mutagenic oligonucleotide that contained the HpaI
site consisting of codons 17 and 18 was used in conjunction with
a primer that contained the XbaI site located about 600 base pairs
upstream of DermC. For construction of the NotI and ClaI
mutants (Fig. 2C), complementary mutagenic oligonucleotides
containing the mutated sequence were used in separate PCR
reactions with an oligonucleotide containing either the upstream
XbaI sequence or the downstream EcoRI sequence. The two PCR
amplicons were annealed to each other and were amplified in
a second round with the upstream and downstream primers. The
mutated fragment was digested with XbaI and EcoRI and used to
replace the small XbaI-EcoRI fragment of pYH250.
Supplemented minimal medium for RNA isolation, Northern blot
analysis, and 5S rRNA probing as a quantitation control were as
described (Oussenko et al. 2005). For expression of RNase J1 in the
conditional mutant strain, IPTG was added to 1 mM. For the
analysis of steady-state RNA shown in Figure 2A,B, strains were
grown in 23 YT medium (1% yeast extract, 2% tryptone, 1% NaCl),
in the absence of IPTG. For induction of ribosome stalling (Fig. 3B),
Em was added to a concentration of 5.0 mg/mL. Quantitation of full-
length DermC mRNA and RNA processing products was done with
a Storm 860 PhosphorImager (Molecular Dynamics) or a Typhoon
TRIO variable mode imager (GE Healthcare).
This work was supported by Public Health Service grant GM-
48804 from the National Institutes of Health.
Received May 22, 2009; accepted August 24, 2009.
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RNase J1-dependent decay of DermC mRNA