Transposon Invasion of the Paramecium Germline Genome Countered by a Domesticated PiggyBac Transposase and the NHEJ Pathway
Figure 4
PiggyMac: a domesticated PiggyBac transposase in Paramecium. (a) Conserved domains in ciliate domesticated transposases. PiggyMac and the related Tpb2 protein from Tetrahymena thermophila were aligned with the transposase of the piggyBac transposon isolated from Trichoplusia ni, using the MUSCLE software (http://www.ebi.ac.uk/Tools/msa/muscle/). The conserved catalytic core domain is represented in orange, and the putative DDD catalytic residues are indicated for each protein (numbers refer to amino acid positions in the primary sequence). A β strand-rich module (yellow box) can be predicted between the second and third catalytic residues using the PSIPRED package (http://bioinf.cs.ucl.ac.uk/psipred/); in ciliate proteins, additional residues inserted within this module are represented by a purple bar. The cysteine-rich domain is drawn as a light green box at the C-terminus of PiggyBac, and corresponding variant domains in Pgm and Tpb2 as hatched boxes. The C-terminal coiled-coil extensions of ciliate domesticated proteins are not drawn to scale (light beige boxes). The divergent N-terminal domains of the three proteins are represented in different shades of blue. (b) A PiggyMac-GFP fusion localizes to developing new MACs. A transgene encoding a C-terminal GFP fusion expressed under the control of endogenous PGM transcription signals was microinjected into the MAC of vegetative cells (Dubois, unpublished). During autogamy, cells were fixed and nuclei were stained with DAPI and observed with a Zeiss epifluorescence microscope (magnification 630x). No GFP fluorescence was observed in vegetative cells (V) or at early stages during autogamy, when the old MAC starts its fragmentation (F). The GFP fusion protein was detected specifically in the two developing MACs of autogamous cells (arrowheads in A and A′) and GFP fluorescence disappeared from the new MAC after karyonidal division (C).
