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.