Fig 1.
Structures of natural TRE5-A elements and the cloned TRE5-Absr.
(A) The autonomous retrotransposition-competent TRE5-A.1 element is shown schematically. This element consists of two open reading frames (ORF1 and ORF2) that are flanked by untranslated regions. ORF2 contains an apurinic or apyrimidinic site DNA repair endonuclease (APE), a reverse transcriptase domain (RT), and a zinc finger-like motif (ZF). The A-module has promoter activity for plus strand transcription [20] but can be replaced by a heterologous promoter [21]. The C-module is essential for retrotransposition [21]. The TRE5-A.2 element is a non-autonomous derivative of TRE5-A.1. It is apparently mobilized in trans by the ORF2 protein provided by TRE5-A.1 elements [18]. TRE5-A.2 served as a blueprint for the design of TRE5-Absr elements. (B) Outline of the TRE5-Absr retrotransposition assay. In the cloned TRE5-Absr element, the A-module promoter is replaced by the stronger actin6 promoter (A6P). TRE5-Absr further contains the ORF1 gene and the C-module of TRE5-A.1. Downstream of ORF1 is the mbsrI gene, which is inserted in the retrotransposon’s minus strand direction. The mbsrI gene is transcribed from the actin15 promoter (A15P). The TRE5-Absr element is transformed into D. discoideum cells together with a vector that confers G418 resistance to transformants. The transformed TRE5-Absr is inserted at random positions in the D. discoideum genome. When the transformed element is transcribed, the intron is removed and restores mbsr ORF function after completion of reverse transcription and integration. Cells affected by at least one retrotransposition obtain blasticidin resistance due to the expression of the functional mbsr gene. In a typical retrotransposition assay, a pool of G418-resistant cells was subjected to selection in medium containing blasticidin. Apparent clones were counted and pooled for genomic DNA preparation. UTR: untranslated region.
Fig 2.
Outline of TRE5-Absr retrotransposition profiling.
Note that TRE5-A always integrates in an orientation-specific manner with the 5’ end of the retrotransposon facing the 5’ end of the targeted tRNA gene. LAM-PCR was performed on genomic DNA prepared from a pool of blasticidin-resistant clones. The 5'-biotinylated primer bound selectively to the codon-adapted ORF1 gene of the TRE5-Absr element and did not recognize ORF1 genes of endogenous TRE5-A elements. The resulting linear, single-stranded LAM-PCR products were immobilized on streptavidin beads and washed extensively. To perform profiling of TRE5-Absr insertions at tRNA genes, exponential PCRs were performed in parallel reactions with primers specific for selected tRNA gene families (“tDNA primer library”). To profile TRE5-Absr insertions at any position in the genome, second-strand synthesis was initiated with a random hexamer primer linked to a unique adapter oligonucleotide (adapter-N6). Next, exponential PCR was performed with the adapter primer and an ORF1-specific primer to yield an “adapter primer library”. SD, splice donor site; SA, splice acceptor site.
Fig 3.
TRE5-Absr integrations at the LysCUU-2 gene.
(A) Gene map around the LysCUU-2 gene (coordinates 2688000 to 2706000 on chromosome 1). Note that the gene is associated with a full-length TRE5-A.1 element, of which only the annotated ORF1 and ORF1 regions are shown. In the sequenced example shown, TRE5-Absr integrated 46 bp upstream of the LysCUU-2 gene, thus forming a tandem with the endogenous TRE5-A.1 element. Note that TRE5-Absr left most of the act6 promoter behind, and the retrotransposed copy contained 42 bp of the 5' untranslated region (5'-UTR) upstream of the ORF1 gene. The tRNA gene sequence is boxed. The +1 G nucleotide, A box, and B box are shown with a red background. A non-template extra C nucleotide is indicated, and the integration junction upstream of the +1 G is shown in lowercase letters. “DDB_G” refers to gene ID numbers listed in dictyBase [34]. (B) Individual integrations of TRE5-Absr were cloned from PCR products obtained with a LysCUU-specific and TRE5-Absr ORF1-specific primer. The upper lane shows the sequence of the TRE5-Absr 5’-UTR (i.e., the act6 promoter) in the transformed TRE5-Absr element. Below is the sequence upstream of the LysCUU-2 gene (upstream sequence in lowercase letters). The sequences of six integration junctions are shown with the +1 G complement of the LysCUU2 with a red background. The integration junction sequence is shown in lowercase letters. Note the insertion of non-templated extra-nucleotides (bold uppercase letters), which help distinguish insertions that occurred at exactly the same position.
Fig 4.
Visualization of TRE5-Absr integrations at tRNA genes.
The 42 tRNA gene families and their individual copy numbers are shown as white squares. The colors indicate the association of a distinct tRNA gene copy with a TRE5-A or TRE5-Absr element. See S1 Fig for the distribution of individual tRNA genes on the six chromosomes. Gray squares indicate mitochondrial tRNA genes. (A) Display of tRNA gene loci associated with a TRE5-A element. (B) TRE5-Absr-targeted tRNA genes identified in the tDNA primer library prepared after 20 generations (20G) or 100 generations (100G) of cell culture. Light red and dark red squares indicate tRNA genes identified only in the 20G or 100G library, respectively. Bright red squares indicate tRNA genes found in both libraries. (C) TRE5-Absr-targeted tRNA genes found in the adapter primer library prepared after 100 generations of cell culture (100G).
Table 1.
Summary of Illumina sequence mapping results.
Fig 5.
Distances of TRE5-Absr integrations to targeted tRNA genes.
The analysis is based on 303 targeted tRNA gene locations and 1,003 TRE5-Absr individual integrations that could be distinguished due to different distances to the inserted TRE5-Absr element to the target. Note that 38 of 1,003 integrations were detected outside the window of 34–62 bp shown here.
Fig 6.
B-boxes on the extrachromosomal rDNA palindrome.
(A) Scheme of the left arm (~39 kb) of the mirror-symmetric rDNA element. The rRNA genes are indicated as arrows. The B box positions are indicated by numbers according to the rDNA palindrome core sequence as published in GenBank entry AY171066. CRA: central region of asymmetry. The asterisks indicate the TRE5-Absr integration targets determined in this study. (B) Sequences of individual B box loci on the D. discoideum rDNA palindrome. WebLogo presentations of A box and B box motifs of D. discoideum tRNA genes are provided for comparison with the respective sequences on the rDNA palindrome. The most conserved nucleotide positions in the B boxes and putative A boxes are highlighted in red. The asterisks indicate the positions where TRE5-Absr integrations were identified in this study.
Fig 7.
Determination of TRE5-Absr integrations sites on the rDNA palindrome.
(A) The genomic DNA from a 15,000-clone pool of blasticidin-resistant cells was used for LAM-PCR-based enrichment of TRE5-Absr integrations. Exponential PCR amplification of the insertion junctions between TRE5-Absr ORF1 and any of the indicated B box positions was performed using locus-specific forward and ORF1-specific reverse primers. Note that a faint signal was reproducibly obtained at the 27726 locus that is barely visible in this reproduction (B) Example of an TRE5-Absr integration 47 bp upstream of the +1 G at position 18638/22168. The +1 G nucleotide, A box, and B box are indicated in red boxes. The integration junctions upstream of the +1 G are shown in lowercase letters. Note that the rDNA palindrome contains stretches of nearly identical sequences and thus B box positions 18638/22168 cannot be distinguished by PCR. (C) Integration of cellular TRE5-A.1 on the rDNA palindrome. The presence of a natural TRE5-A.1 element upstream of positions 18638/22168 on the rDNA palindrome was detected by nested PCR on genomic DNA of untransformed D. discoideum cells using primers specific for TRE5-A ORF1 and the 18638/22168 region. The +1 G nucleotide, A box, and B box are indicated in red boxes. The integration junctions upstream of the +1 G are shown in lowercase letters.