Construction of various ALA1-lexA or GRS1-lexA fusion constructs for the Western blot analyses was as previously
described . Briefly, an initiator mutant of lexA was OSI-906 in vivo amplified by PCR as an SpeI-XhoI fragment and cloned in the pADH high-copy-number yeast shuttle vector. A wild-type (WT) or mutant ALA1 sequence containing base pairs -105 to -24 relative to ATG1 was amplified by PCR as a PstI-SpeI fragment and was cloned in-frame into the 5′ end of lexA, resulting in various ALA1-lexA fusion constructs. Construction of GRS1-lexA fusion constructs followed a similar strategy. The expression of these lexA fusion constructs was under the control of a constitutive ADH promoter . The Western blot analysis was as previously described . Complementation assays for the Selleckchem eFT508 cytoplasmic and mitochondrial functions of ALA1 The yeast ALA1 knockout strain, TRY11 (MATa, his3Δ200, leu2Δ1, lys2-801, trp1Δ101, ura3-52, and ala1Δ::TRP1) GS-1101 chemical structure was maintained by a plasmid carrying the WT ALA1 gene
and a URA3 marker . Complementation assays for the cytoplasmic function of plasmid-borne ALA1 and its derivatives were carried out by introducing a test plasmid (with a LEU2 marker) into TRY11 and determining the ability of transformants to grow in the presence of 5-fluoroorotic acid (5-FOA). Cultures were incubated at 30°C for 3~5 days or until colonies appeared. The transformants evicted the maintenance plasmid that carries the URA3 marker in the presence of 5-FOA. Thus, only an enzyme with cytoplasmic AlaRS activity encoded by the test plasmid could rescue the growth defect. Following 5-FOA selection, a single colony of transformants was selected and grown to the stationary phase in synthetic medium lacking leucine. Starting from a cell density of 1.0 A 600, cultures were 5-fold serially diluted, and 5-μl aliquots of each dilution were spotted onto the designated YPG plates. The plates were incubated at 30°C
for 3~5 days. Photos were taken of the complementation assays on day 3 following incubation. Because yeast cells cannot survive on glycerol without functional mitochondria, the transformants did not grow on YPG plates unless a functional mitochondrial AlaRS was generated by the test plasmid. Assays of the cytoplasmic and mitochondrial GlyRS activities PAK5 followed a similar protocol . Reverse-transcription (RT)-PCR To determine the relative levels of specific ALA1-lexA mRNAs derived from the fusion constructs, a semiquantitative RT-PCR experiment was carried out following the protocols provided by the manufacturer (Invitrogen). Briefly, total RNA was first isolated from the transformants, and aliquots (~1 μg) of RNA were then reverse-transcribed into single-stranded complementary (c)DNA using an oligo-dT primer. After RNase H treatment, the single-stranded cDNA products were amplified by a PCR using a pair of specific primers.