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First published online 22 May 2007
doi: 10.1242/jcs.006502

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Identification of novel chromatin-associated proteins involved in programmed genome rearrangements in Tetrahymena

Meng-Chao Yao^1,*, Ching-Ho Yao¹, Lia M. Halasz¹, Patrick Fuller¹, Charles H. Rexer², Sidney H. Wang², Rajat Jain², Robert S. Coyne^1, and Douglas L. Chalker^2,

¹ Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
² Department of Biology, Washington University, St. Louis, MO 63130, USA

View larger version (91K): [in this window] [in a new window]   Fig. 1. Pdd1p shows dynamic localization during development. C-terminal CFP (A-D) and N-terminal GFP (E-H) Pdd1p fusion protein (constructs shown) localization was visualized by fluorescence microscopy of live cells, co-stained with 4',6-Diamidino-2-phenylindole (DAPI). (DAPI accumulation in vacuoles can produce significant cytoplasmic fluorescence in addition to nuclear staining.) Differential interference contrast images (DIC) of cells are paired with GFP or CFP and DAPI fluorescence. The observed nuclear Pdd1p localization is illustrated in the chronological progression through conjugation in the top diagram as gray shading. Image panels corresponding to the illustrations are: (A) prophase Meiosis I; (B) metaphase Meiosis I; (C) nuclear exchange (data not shown for post-zygotic divisions and new macronuclei emergence – Mac I); (D,E) early macronuclear differentiation (Mac II); (F-H) macronuclear development after pair separation (exconjugants Mac II). Selected nuclei are labeled in the illustration and on the DIC images as: om, old macronuclei; mic, micronuclei; gmi, gametic micronuclei; relic, discarded meiotic products; zmi, zygotic micronuclei; NM, new macronuclear anlagen.

View larger version (77K): [in this window] [in a new window]   Fig. 2. GFP-Pdd1p localizes to the conjusome and DNA rearrangement foci. GFP-Pdd1p was visualized in live cells between 6 and 7 hours (A-D) or 12 and 14 hours (E-G). DIC and corresponding views of GFP fluorescence are shown. F and G show a threefold enlargement of the region boxed in E. White arrowheads highlight cytoplasmic structures (A-D) or nuclear (E-G) DNA rearrangement foci.

View larger version (50K): [in this window] [in a new window]   Fig. 3. GFP localization identifies candidate genes involved in nuclear differentiation. (A) A developmental cDNA library, inserted into GFP expression vector pCGF1, was transformed into Tetrahymena to generate panels of transformants that were selected in 96-well tissue culture plates. Replicates of these panels were crossed to wild-type strain CU428 and visualized for GFP-nuclear localization using an inverted fluorescence microscope. (B) Representative images of mating cells expressing four different GFP fusions to coding sequences identified as histone H2B, histone H4, micronuclear linker histone (MLH), PDD2 and one novel gene, LIA3, are shown. For each pair or exconjugant, a series of DIC, green fluorescence and DAPI-stained images is presented. White arrowheads indicate the different nuclei for positional reference between images.

View larger version (30K): [in this window] [in a new window]   Fig. 4. LIA genes encode novel proteins expressed during nuclear differentiation. (A) Each lane contains 20 µg total RNA isolated from vegetatively growing (gr), starved (st) or conjugating cells at the indicated time after mixing of complementary mating types. The estimated transcript sizes are given to the left of each scanned autoradiogram. (B) The gene structures of LIA1 to LIA5 are depicted: open boxes, coding regions; gaps spanned with carets, introns; solid black bars, transcribed non-translated regions. The size is given above each intron. The divisions of the scale bar represent 250 bp. All non-translated sequences, introns and the LIA4 polyadenylation site (pA) were verified by our cloned cDNAs or database cDNA sequence. Gray bars underline the sequences found in the original cDNAs recovered in the screen, which were radiolabeled as probes for the northern blot analysis shown in A.

View larger version (91K): [in this window] [in a new window]   Fig. 5. Lia proteins exhibit dynamic localization during conjugation. N-terminal GFP fusion proteins were expressed from the pIGF-LIA expression cassette shown at the top. Representative DIC, GFP and DAPI-stained image series are shown for LIA1, LIA3, LIA4 and LIA5 as indicated. (A) Early macronuclear development 7-8 hours into conjugation. (B) Late macronuclear development 12-14 hours into conjugation. White arrows indicate conjusome (C) localization. White arrowheads denote representative punctate foci that are similar to Pdd1p-containing foci.

View larger version (99K): [in this window] [in a new window]   Fig. 6. Lia proteins co-localize with Pdd1 in DNA rearrangement foci. Conjugating cells expressing the indicated GFP-Lia fusion proteins were fixed with paraformaldehyde to preserve GFP fluorescence and incubated with Pdd1p-specific antibodies, detected with Rhodamine-conjugated anti-rabbit secondary antibodies. Images of GFP or immunofluorescence are shown separately and merged with the corresponding DAPI-stained images and pseudocolored (GFP-Lia, green; Pdd1p, red; DAPI, blue) to highlight overlapping localization (yellow). White arrows are given for positional reference between corresponding images.

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