To generate the ump1 mutant with the split-marker approach63 (link), the flanking sequences of UMP1 were amplified and linked to the hph cassette amplified from pCX6364 (link) by overlapping PCR with the primers listed in Supplementary Table 2 . The resulting gene replacement fragments were transformed into strain JL3 by PEG-mediated protoplast transformation. Transformants resistant to 30 µg/ml hygromycin B (Sigma-Aldrich) were isolated and screened by PCR with primer pairs 5F/6R, H850/H852, 7F/HY-R, and YG-F/8R (Fig. S6b ) to identify ump1 deletion mutants.
For complementation assays, a 2574-bp fragment containing the entire UMP1 coding region (except the TAG stop codon) and its promoter was cloned between the KpnI and HindIII sites on pKNTG that carries the geneticin resistance marker and GFP65 (link) to generate the in-frame UMP1-GFP fusion construct pUmp1GFP. A 382-bp fragment containing the 3’-UTR and terminator sequences UMP1 was then cloned into the BamHI site on pUmp1GFP. The resulting UMP1-GFP construct was transformed into the ump1 mutant as described9 (link). Transformants resistant to both 30 µg/ml hygromycin B and 30 µg/ml geneticin (Sigma-Aldrich) were isolated and confirmed by PCR analysis.
For complementation assays, a 2574-bp fragment containing the entire UMP1 coding region (except the TAG stop codon) and its promoter was cloned between the KpnI and HindIII sites on pKNTG that carries the geneticin resistance marker and GFP65 (link) to generate the in-frame UMP1-GFP fusion construct pUmp1GFP. A 382-bp fragment containing the 3’-UTR and terminator sequences UMP1 was then cloned into the BamHI site on pUmp1GFP. The resulting UMP1-GFP construct was transformed into the ump1 mutant as described9 (link). Transformants resistant to both 30 µg/ml hygromycin B and 30 µg/ml geneticin (Sigma-Aldrich) were isolated and confirmed by PCR analysis.
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