Variant 2 and variant 4 Ogta proteins are active in vitro and in vivo. (A) in vitro Ogt activity assay of bacterially expressed, HIS-tagged variant 2 Ogta and variant 4 Ogta proteins. Both enzymes incorporate significant amounts of [3H]UDP-GlcNAc onto the synthetic CKII peptide identical to the human sequence, although variant 4 Ogta is more active than variant 2. A western blot of the bacterial lysates reveals that equivalent amounts of enzyme were added to the reactions. (B-G) Images of live 24 hpf embryos injected with 500 pg β-galactosidase mRNA (B, C), variant 4 ogta mRNA (D, E), or variant 2 ogt (F, G). Embryos injected with β-galactosidase mRNA developed normally (B, C). Overexpression of variant 4 ogta mRNA resulted in three phenotypic classes of embryos at this stage. Class I embryos had a visible, but shortened, body axis and reduced brain, including reduced or absent eyes (D). The body axis in Class II embryos was strongly reduced and in many cases, not discernable (E). Class III embryos are indistinguishable from wild type (not shown). Expression of variant 2 ogta mRNA produced the same range of phenotypes, including Class I (F) and Class II (G) embryos. (H, I) Western blot of protein extracts of 18 hpf embryos injected with 500 pg mRNA encoding β-galactosidase (lane 1), variant 2 ogta (lane 2), variant 4 ogta (lane 3). The blot was probed with the RL2 antibody, which recognizes the O-GlcNAc modification. This experiment was performed in biological duplicate (I) Quantification of the blot shown in (H, set 1) and a technical duplicate (set 2). B and C are duplicate images of the same control embryo.