Picture from Gibson, Sophia B et al. (2021) MicroPubl Biol

Figure 1. Caenorhabditis elegans and Haemonchus contortus beta-tubulin genes cannot substitute for loss of the sole Saccharomyces cerevisiae beta-tubulin TUB2: A) A phylogenetic tree of the beta-tubulin orthogroup for C. elegans, H. contortus, and S. cerevisiae is shown. The tree was created using MAFFT (Katoh et al.., 2002; Katoh & Standley, 2013) and IQ-TREE with ultrafast bootstrapping (Hoang et al., 2018; Minh et al., 2020). The tree was constructed using a LG+R3 substitution model selected by the highest Bayesian Information Criterion score using ModelFinder (Kalyaanamoorthy et al., 2017). Visualization was performed using iTOL (Letunic & Bork, 2021). B) The experimental design for replacement of S. cerevisiae TUB2 with ben-1 is shown. For C through F, PCR products were separated on a 1% agarose gel with the sizes of each band shown on the left. C) Colony PCR confirmed replacement of TUB2 with a NAT cassette. Lane 1 contained a fragment amplified from one primer downstream of TUB2 and another internal to the NAT cassette. Lane 2 is a product amplified from a sequence internal to the MAT-alpha locus, which serves as a positive control. D) Colony PCR confirmed codon-optimized ben-1 replacement at the TUB2 locus. Lane 1 corresponded to primers for an internal ben-1 fragment. Lane 2 corresponded to primers for an internal MAT-alpha sequence. E) Tetrad dissection results of ben-1 replacement diploids are shown. Six tetrads (rows 1-6) were separated into four haploid cells each (lanes A-D). F) RT-PCR results confirmed ben-1 expression in S. cerevisiae. An endpoint PCR product was amplified from cDNA reverse transcribed from RNA extracted from the ben-1 replacement sample. Lane 1 corresponded to PCR products generated using primers for an internal ben-1 fragment. Lane 2 corresponded to PCR products generated using primers for an internal MAT-alpha sequence. This figure was created using BioRender.com.

Picture from Lall S et al. (2005) Mol Biol Cell "Caenorhabditis elegans decapping proteins: localization and functional ...."

Figure 1. C. elegans Dcp2 is found in cytoplasmic bodies from early embryogenesis. (A) Western blots using the anti-Dcp2 antisera on C. elegans mixed stage embryo extract reveal a dominant band close to 90 kDa. A second lower band was observed upon Western blotting of C. elegans embryonic extracts with two different antisera raised to Dcp2 protein. This lower band may represent a breakdown product of Dcp2, as a similar but slower migrating band is frequently observed upon purification of recombinant Dcp2 (Cohen et al., 2005). (B) dcp2 RNAi depletes the particular staining, indicating that these foci are due to specific staining of Dcp2. Punctate Dcp2 staining is observed in embryos treated with dsluciferase RNA (top panel), but is absent in the majority of embryos treated with dsdcp2 RNA (bottom panel). In addition the disappearance of these particles upon dsdcp2 treatment suggests that this RNAi treatment diminishes levels of the protein. This shows that the immunostaining is specific and that Dcp2 RNAi is depleting the protein recognized by the antibody. Embryos shown are at the division following the four-cell stage when particles are easily visualized. (C) Immunolocalization of Dcp2 shows that punctate staining is first observed before the pronuclei fuse (top row). Cytoplasmic Dcp2 foci are seen in both the somatic cells and the germ line lineage in the 2-cell (second row), and 8-cell (third row) embryo. The fourth row shows a closer view of punctate Dcp2 staining in an embryonic cell in C. The intensity of foci typically appears to increase during the initial divisions in development up to the 8-cell stage, with strong labeling of somatic foci then observable throughout embryogenesis. This is consistent with an increase in Dcp2 foci as maternal transcripts are turned over or as zygotic transcription initiates. (D) CGH-1 and Dcp2 completely colocalize in cytoplasmic foci in early embryos, with CGH-1 becoming difficult to detect subsequently in development.