Fission yeast retrotransposon Tf1 integration is targeted to 5' ends of open reading frames

Abstract

Target site selection of transposable elements is usually not random but involves some specificity for a DNA sequence or a DNA binding host factor. We have investigated the target site selection of the long terminal repeat-containing retrotransposon Tf1 from the fission yeast Schizosaccharomyces pombe. By monitoring induced transposition events we found that Tf1 integration sites were distributed throughout the genome. Mapping these insertions revealed that Tf1 did not integrate into open reading frames, but occurred preferentially in longer intergenic regions with integration biased towards a region 100-420 bp upstream of the translation start site. Northern blot analysis showed that transcription of genes adjacent to Tf1 insertions was not significantly changed.

Figure 1

Determination of genomic Tf1-neo insertions. (A) The transposition assay plasmid pHL414 contained the URA3 gene of S.cerevisiae and the Tf1-neo element fused to an inducible nmt promoter. The neo gene inserted in Tf1 confers G418 resistance. The large white box represents the single full-length ORF of Tf1. The positions of the protein domains Gag, protease (PR), reverse transcriptase (RT) and integrase (IN) are indicated. Triangles within grey boxes represent LTRs. The plasmid contains an autonomous replication sequence of S.pombe (ars). (B) Southern blot analysis of Tf1 transposition clones. DNA from G418-resistant clones was cut with restriction enzymes as indicated, blotted, and hybridised to a Tf1-specific probe shown in the schematic presentation of the nmt–Tf1-neo fusion construct. As controls, DNA from an isogenic strain without Tf1 insertion (wt) and from the same strain carrying the pHL414 plasmid (pHL414) in the presence of thiamine were used. Of 40 analysed transposition clones, 10 representative clones are shown here. Molecular weights are shown to the left of the gel. (C) The genomic locations of induced Tf1-neo insertion events were determined using IPCR and the S.pombe genome database. IPCR primers (see Materials and Methods; RB30, RB32: primer set 1; RB20, RB39: primer set 2) and sequencing primers (SQ) are shown as arrows. HindIII restriction sites are indicated, and genes are represented as black boxes.

Figure 2

Analysis of Tf1 integration sites within intergenic regions. (A) The location of all mapped Tf1 insertion sites with regard to the closest ORF (5′ or 3′ end) is shown. Bars indicate single Tf1 insertions. Long bars are used to represent two Tf1 insertions which occurred at about the same distance to the closest ORF (clone 8, 141 bp and clone 39, 144 bp; clone 2, 258 bp and clone 29, 261 bp; clone 21, 307 bp and clone 26, 308 bp). The arrow indicates the 5′ end of an ORF (grey box). (B) Composition of 5 bp Tf1 target site duplication. The total and average base usage at the 5 bp duplicated target site is indicated. The total number of analysed target sites was 27 and the orientation of the target site is 5′ to 3′ and parallel to the Tf1 element.

Figure 3

Northern blot analysis of genes adjacent to Tf1 insertions. The transcription level and the size of the transcript of the two genes adjacent to a Tf1 insertion were analysed by northern blot. The names of the gene transcripts detected from transposition (m) and control clones (wt) are shown to the right of the gels. No transcript was detected for SPCC777.07 (clone 21). As a loading control, rRNA was stained with methylene blue for the 3.5 and 1.8 kb band, or the his3 transcript level was monitored.

Figure 4

Screen for Tf1 insertion into the wee1 gene. The pHL414 plasmid was transformed into a cdc25 temperature-sensitive strain and transposition was induced at the permissive temperature. G418-resistant revertants which grew at the restrictive temperature were analysed for the disruption of the wee1 gene. Genomic DNA from such clones (m1, m2) was digested with AlwNI and analysed by Southern blotting using wee1- and Tf1-specific (see Fig. 1B) probes. The blot shows that the Tf1 insertions were not on the same genomic fragment as the wee1 gene. As control, DNA from a strain without Tf1 insertion was included (wt). Molecular weights are shown to the left of the gel. The wee1 ORF is presented as a box with an arrow indicating the translation start site, and the AlwNI restriction sites in the vicinity of the wee1 gene are indicated.