The expression patterns of zLPTS in zebrafish tissues and embryos. (A) RT-PCR analysis of zLPTS transcript in 11 zebrafish tissues as indicated. (B) Northern blotting analysis of zLPTS transcript in 9 zebrafish tissues as indicated. Hybridization was performed with a ∼ 800 bp, α-³²P-labeled DNA fragment of zLPTS. The below 28S and 18S rRNA, stained by methylene blue, was used as a loading control. (C) RT-PCR analysis of zLPTS transcript in embryos at different times after fertilization (hour postfertilization, hpf). Zebrafish β-actin was served as a control. DL2000 (DNA Marker, TaKaRa) was loaded as a marker.

Whole mount in situ hybridization of zLPTS at early stages of zebrafish embryos. Embryos were showed at different developmental stages such as 1-cell (0 hpf) (A and B), 2-cell (0.75 hpf) (C and D), 8-cell (1.25 hpf) (E and F), 30% epiboly (4.3 hpf) (G and H), ring stage (5.25 hpf) (I and J), 70% epiboly (7 hpf) (K and L), 90% epiboly (8.5 hpf) (M and N), 5 somites (12 hpf) (O and P). Hybridization was performed with a digoxigenin-labeled zLPTS antisense probe, and the purple reaction products were visualized by bright-field microscopy. Each pair is viewed from the side (A, C, E, G, I, K, M, O), top (B, D, F, H, J), or bottom (L, M and P).

Subcellular localization of zLPTS in the cells. The Bel-7404 hepatoma cells, CHO Cells or zebrafish BRF41 cells were transfected with pEGFP-C1 control vector or pEGFP-C1-zLPTS plasmids, respectively. The cells were stained with Hoechst 33258 and visualized by fluorescence microscope at the appropriate wavelength. Two images (GFP and Hoechst 33258) were overlaid (merge). The arrows indicate the punctate telomere signals.

Recombinant GST- zLPTS protein interacts with zTERT. (A) Scheme of zebrafish TERT protein structures. Three fragments that corresponding to the N-terminal (zTERT-N), RNA-binding domain (zTERT-M), RT and C-terminal (zTERT-C) of full-length protein were indicated. Motifs of CP, QFP, T, 1 and 2 were further mapped in RNA-binding domain. (B) 10% SDS-PAGE analysis of recombinant GST-zLPTS fusion protein. Lane1, Cell lysate of E. coli BL21(DE3) containing the pGEX-4T2-zLPTS plasmid. Lane2, Cell lysate of IPTG-induced E. coli BL21(DE3) containing the pGEX-4T2-zLPTS plasmid. Lane3, GST-zLPTS fusion protein purified by affinity chromatography. Lane 4, the GST protein purified from the cells harboring the pGEX-4T2 vector that used as a control. The GST-zLPTS or GST protein was indicated by arrow. (C) Western blotting analysis of the in vitro translated three N-terminal Myc-tagged zTERT protein fragments (zTERT-N, zTERT-M and zTERT-C) by using anti-myc antibody. (D) Interactions between zLPTS and zTERT protein fragments by GST pulldown assay. GST or GST-zLPTS fusion protein was used to incubate with Myc-tagged zTERT-N, M or C protein fragment. Western blotting was performed with anti-myc antibody. (E) Interactions between zLPTS and zTERT-M sub-fragments by GST pulldown assay. The in vitro translated six N-terminal Myc-tagged zTERT-M protein fragments including zTERT-M, M1, M2, M3, M1 + M2 and M2 + M3 as indicated in (A) were used to incubate with GST or GST-zLPTS fusion protein. Interaction result was examined with anti-myc antibody.

Recombinant GST- zLPTS protein inhibits telomerase activity in vitro. Various concentrations of GST-zLPTS protein as indicated were incubated with 1 μl of telomerase extracts from Bel-7404 cells or 24 hpf embryos for 10 min prior to a TRAP assay. The GST protein was used as a negative control. RNase A was also included in one assay as a negative control. The arrow indicates the 36-bp internal control (IC).