Results: 4

1.
Fig. 2

Fig. 2. From: A Functional Role for Transposases in a Large Eukaryotic Genome.

Silencing of TBE transposases leads to accumulation of aberrantly processed DNA. MDS segments (white boxes) and IESs (hatched boxes) are shown schematically and not to scale. Shown are the sequences of PCR products for TEBPβ between segments 2 and 7 in the progeny of triply silenced cells treated 12 to 15 hours after mixing; shown are all 32 examples of aberrantly rearranged chromosomes found among 37 sequenced clones [redundancy is indicated by the number after an x (table S2)]. Open triangles indicate locations of cryptic junctions between neighboring (pointing up) and nonneighboring (pointing down) segments on the basis of precursor micronuclear order. MIC, nonrearranged micronuclear gene sequences; MAC, expected rearranged macronuclear gene sequences.

Mariusz Nowacki, et al. Science. 2009 May 15;324(5929):935-938.
2.
Fig. 3

Fig. 3. From: A Functional Role for Transposases in a Large Eukaryotic Genome.

RNA interference (RNAi) against TBE transposases leads to accumulation ofnonprocessedDNA.PCR amplification of gene regions between two distant MDSs is shown (pol-α between MDSs 3 to 31; actin I between MDSs 3 to 8; TEBPβ between MDSs 1 to 7; and TEBPα between MDSs 5 to 17) (23). Samples that were triply silenced (12 to 15 hours after mixing) against all three TBE transposases (lanes 4, 14, 24, and 34) show accumulation of nonrearranged (MIC) gene versions. Controls were non-treated cells (lanes 2, 12, 22, and 32) and cells injected with 184-nucleotide vector polylinker dsRNA (lanes 3, 13, 23, and 33); MAC, macronuclear; MIC, micronuclear. Quantitative PCR (QPCR) with TEBPα primers shows 6.55-fold more nonrearranged DNA as compared with that of the untreated control in the triple silencing example (bottom).

Mariusz Nowacki, et al. Science. 2009 May 15;324(5929):935-938.
3.
Fig. 1

Fig. 1. From: A Functional Role for Transposases in a Large Eukaryotic Genome.

Expression of TBE transposase genes. (A to F) Northern hybridization against total RNA extracted from vegetative cells (V) as well as 11-, 24-, and 34-hour time points (T) after mixing of mating types. TBE transposase transcripts are most abundant 24 hours after mixing, when DNA rearrangements occur (22). 10 μg of RNA was loaded per lane and hybridized to ~1-kb DNA probes (23) for (A) TBE1, (B) TBE3, (D) TBE1, -2, and -3 mixed together (1:1:1 ratio), (E) TBE2 transposase, and (C and F) small-subunit ribosomal RNA (rRNA); (C) is the control for (A) and (B), and (F) is the control for (D) and (E). (G) Reverse transcription PCR of TBE1 (lanes 17 to 26) and TBE2 (lanes 27 to 36) transposase polyadenylate-containing mRNA from vegetative cells (V) and three time points (T) during conjugation (23). Plus and minus symbols indicate the presence or absence of reverse transcriptase (RT), respectively. Arrowheads indicate the size of relevant markers [1-kb ladder (Invitrogen, Carlsbad, CA)].

Mariusz Nowacki, et al. Science. 2009 May 15;324(5929):935-938.
4.
Fig. 4

Fig. 4. From: A Functional Role for Transposases in a Large Eukaryotic Genome.

Transposase silencing leads to increased levels of nonprocessed high–molecular weight DNA and transposon retention. (A) 1 μg total DNA from vegetative progeny of cells treated with RNAi against TBE transposases (lanes 4 to 10) as well as untreated and control cells (23); ethidium bromide staining was used. (C) DNA from (A) hybridized to a TBE1 transposase DNA probe (23). Samples that were treated with RNAi (12 to 15 hours after mixing) against all three transposases or TBE1 and -2 together (lanes 4 and 8) show increased levels of (A) high–molecular weight DNA and (C) TBE1 hybridization to high–molecular weight DNA as well as decreased levels of transposase mRNA (F). (B and D) Gray bars indicate relative amount of ethidium bromide (B) and TBE1 probe signals (D) in high–molecular weight DNA. (E) Control hybridization with a small-subunit rDNA probe. Arrowheadsindicate size of relevant markers (1-kb ladder). (F) Relative transposase mRNA levels for TBE1, TBE2, and TBE3 in injected (12 to 15 hours after mixing) versus noninjected cells in an independent experiment (23). Absolute values for each transcript were obtained with QPCR (in femtograms) and normalized against the transcript level for the mitochondrial large-subunit rRNA. Transcript levels of the control injected sample were set to 1.

Mariusz Nowacki, et al. Science. 2009 May 15;324(5929):935-938.

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