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. 2015 Apr 6;43(8):4249–4261. doi: 10.1093/nar/gkv280

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© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 3. — Different introns alleviate the expression defects caused by THO complex inactivation. (A) Schematic representation of the LacZ constructs used in panels (B) and (C). LacZ-based reporter genes bear either the small synthetic intron used previously (RP51A* intron), or natural introns (PRE3, ACT1 and RPL35A introns) downstream of the initiation codon. As for the ‘splicing’ reporter, only the spliced mRNA encodes ß-galactosidase (see Supplementary Table S2, pRS426 constructs). (B) Expression of the LacZ reporter genes was monitored by measurement of β-gal activities (RLU/OD, see Material and Methods; mean ± SD; n = 4) in the indicated strains. Values were set to 1 for wt cells with the intronless reporter. Fold decreases in THO mutants relative to wt are indicated by numbers. (C) β-gal activities (mean ± SD; n = 4) obtained in panel (B) from intron-containing reporters were normalized to the values obtained from the intronless reporter in the same mutants. (D) Schematic representation of the YAT1 constructs used in panels (E) and (F). The natural YAT1 gene was expressed under the control of a galactose-inducible promoter as previously reported (40). The small synthetic RP51A* intron was introduced downstream of the initiation codon (see Supplementary Table S2). (E) Expression of YAT1 reporters was monitored by quantifying YAT1 mRNA levels by RT-qPCR (normalized to ACT1 mRNA values; mean ± SD; n = 4) upon extraction of total mRNAs from the indicated strains. YAT1 mRNA was detected using YAT1–3′ primers (see Supplementary Table S3); similar results were obtained with YAT1–5′ primers (our unpublished data). Values were set to 1 for wt cells carrying the intronless reporter. Fold decreases in tho mutants relative to wt are indicated by numbers. Note that similar data were obtained when normalizing YAT1 mRNA levels to the 25S rRNA standard (our unpublished results). (F) mRNA levels (mean ± SD; n = 4) obtained in panel (E) from the intron-containing construct were normalized to the values obtained from the intronless construct in the same mutants.