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. 2010 May 5;38(17):5833–5843. doi: 10.1093/nar/gkq345

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© The Author(s) 2010. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 4. — Ectopic expression of the trypanosomal tRNA^Sec gene requires an external promoter. (A) Predicted secondary structure of the tagged tRNA^Sec and tRNA^Met−i. The 2-nt changes introduced as tags are indicated. The tags allow the specific detection of the two tRNA variants by oligonucleotide hybridizations. (B) Cassette used for ectopic expression of the tRNA^Sec (the one encoded on the shorter intergenic region) and tRNA^Met−i, respectively. It contains the Pol-I procyclin promoter followed by two tetracycline operators and a splice acceptor site (SAS). The tagged tRNA^Sec was expressed in its own genomic context, whereas the tagged tRNA^Met−i was fused to the 5′-flanking region of a trypanosomal tRNA^Leu but retained its own 3′-flanking region (31). (C) Northern analyses of total RNA isolated from cell lines expressing the tetracycline repressor and transfected with the constructs shown in (A). tRNA^Sec, cell line expressing the tagged tRNA^Sec. tRNA^Met−i cell line expressing the tagged tRNA^Met−i. Top panel, hybridization with oligonucleotides that specifically recognize the tagged tRNA^Sec and the tagged tRNA^Met−i, respectively. Middle panel, same blot as above but reprobed with an oligonucleotide recognizing both the tagged and the endogenous tRNA^Sec. Bottom panel, ethidium bromide stained tRNA region of the gel used for the northern analyses. (D) To scale drawing of the wild-type Tb-VDAC locus (41) and the situation after homologous recombination leading to replacement of one allele by a tRNA^Sec/G418 resistance cassette. Relevant AvaII (A) restriction sites are indicated. The jagged line marks the probe used in the Southern analysis. Left panel: Southern analysis of genomic DNA isolated from the parental cell line (Tb-VDAC, +/+) and from a single knock out Tb-VDAC cell line (Tb-VDAC, +/−) containing the tRNA^Sec/G418 resistance cassette. Right panel, northern blot of total RNA isolated from the same cell lines that were analyzed by the Southern blot hybridized with a probe specific for the tagged tRNA^Sec (top panel). The same blot was reprobed with a probe recognizing both the tagged and the endogenous tRNA^Sec (middle panel). Bottom panel, ethidium bromide stained tRNA region of the gel used for the northern analyses.