Journal: PLoS Biology

Article Title: Cellular labeling of endogenous retrovirus replication (CLEVR) reveals de novo insertions of the gypsy retrotransposable element in cell culture and in both neurons and glial cells of aging fruit flies

doi: 10.1371/journal.pbio.3000278

Figure Lengend Snippet: (A) Schematic showing the replication cycle of the gypsy-CLEVR reporter. (i) gypsy-CLEVR contains 5XUAS and the Watermelon reporter that are inserted into the U5 region of 5′LTR and U3 region within 3′LTR, respectively, with reverse orientation to gypsy element. (ii) During normal replication of LTR-RTEs and ERVs, a series of template switches transfers DNA sequence information from between subregions of the 3′ and 5′LTRs. These two template switches cause a rearrangement in the replicated cDNA such that the WM reporter is placed downstream to the UAS regulatory element. (iii) The full-length cDNA reintegrates into the host genome at a distinct location. If that cell expresses Gal4, it will trigger expression of WM by binding to UAS regulatory element. The wild-type (WT), deletion (ΔPBS), and mutations (PBSm1 and PBSm2) of the primer binding site (PBS) variants of gypsy-CLEVR are shown, along with sequence traces confirming their fidelity. (B) Fluorescent images showing that WM positive cells are detected for gypsy-CLEVR with tub-Gal4, but not for deleted (gypsy-CLEVR ΔPBS ) or mutated (gypsy-CLEVR PBSm1 and gypsy-CLEVR PBSm2 ) gypsy-CLEVR variants. A higher magnification image from the tub-Gal4 and gypsy-CLEVR co-transfection group displays membranous GFP, nuclear mCherry, and nuclear DAPI. Scale bar = 20 μm. (C) Quantification of the numbers of WM-labeled cells in gypsy-CLEVR, gypsy-CLEVR ΔPBS , gypsy-CLEVR PBSm1 , and gypsy-CLEVR PBSm2 with and without tub-Gal4. Significance is determined by using a Fisher exact test comparing each genotype to gypsy-CLEVR with tub-Gal4. (D) An RT-qPCR approach confirms that WM transcript levels correspond to fluorescence detection from each gypsy-CLEVR variant (gypsy-CLEVR, gypsy-CLEVR ΔPBS , gypsy-CLEVR PBSm1 , and gypsy-CLEVR PBSm2 ) co-transfected with tub-Gal4. Each value from these experimental groups was normalized to the tub>gypsy-CLEVR group in order to yield relative fold change. Relative fold changes of both of GFP (tub>gypsy-CLEVR: 1.95 ± 0.25, tub>gypsy-CLEVR ΔPBS : 7.52 × 10 −5 ± 3.39 × 10 −5 , tub>gypsy-CLEVR PBSm1 : 0.28 ± 0.04, tub>gypsy-CLEVR PBSm2 : 6.21 × 10 −3 ± 3.76 × 10 −3 , tub-Gal4: under detectable level) and mCherry (tub>gypsy-CLEVR: 1.99 ± 0.35, tub>gypsy-CLEVR ΔPBS : 7.05 × 10 −5 ± 5.63 × 10 −5 , tub>gypsy-CLEVR PBSm1 : 0.22 ± 0.03, tub>gypsy-CLEVR PBSm2 : 1.78 × 10 −4 ± 4.05 × 10 −5 , tub-Gal4: under detectable level) derived from WM are compared (see ). n = 3 biological replicates. (*** p < 0.001, **** p < 0.0001, unpaired t test). CLEVR, cellular labeling of endogenous retrovirus replication; ERV, endogenous retrovirus; GFP, green fluorescent protein; LTR, long terminal repeat; PBS, primer binding site; PBSm1, PBS mutant1; PBSm2, PBS mutant2; PPT, polypurine tract; RT-qPCR, reverse-transcription quantitative PCR; RTE, retrotransposable element; tub-Gal4, tubulin promoter driven Gal4; UAS, upstream activating sequence; WM, Watermelon; WT, wild-type; 5XUAS, 5 UAS elements.

Article Snippet: Cell culture data were analyzed using a Fisher exact test variant of the chi-squared analysis in order to obtain a p -value for significance.

Techniques: Sequencing, Expressing, Binding Assay, Cotransfection, Labeling, Quantitative RT-PCR, Fluorescence, Variant Assay, Transfection, Derivative Assay, Reverse Transcription, Real-time Polymerase Chain Reaction