PMC

Impact of the PLE on translation in vitro. (A) The mRNAs shown in Table 1 were translated in a HeLa cytoplasmic extract for 1 h together with a control Renilla luciferase mRNA. A0 and A98 mRNA have 5′ ApppG, and mRNAs labeled “cap” have 5′ ⁷mGpppG. The fold translation shown in the upper panel represents firefly luciferase activity normalized to Renilla luciferase activity, and each bar consists of the mean ± SD for triplicate determinations. To facilitate comparison between the individual mRNAs, the value for capped A0 mRNA was arbitrarily set to 1. (B) RNA extracted from each pooled set of reactions was analyzed by Northern blot as in Figure 1 using a mixed probe for firefly (Fluc) and Renilla (Rluc) luciferase. (C) The PLE identified in HIV-EP2 mRNA (HIVEP2) was inserted into A20 or A98 firefly luciferase mRNA in place of PLE B from albumin mRNA. These capped mRNAs were translated in triplicate and compared to translation of control A20 and A98 mRNA together with Renilla luciferase control. In this experiment the translation of capped A20 mRNA normalized to translation of Renilla luciferase mRNA was arbitrarily set to 1 and results with the other mRNAs were normalized to this. The bars represent the mean ± SD for triplicate determinations.

Possible helical wheel structure of α H2 of IntDOT, spanning amino acid residues 91 to 99. The wheel is predicted by the method of Gibrat et al. (6). Proteins with substitutions V95M, K94E, and K96E lack HJ resolution activity. The A98, V95, F99, and A92 residues could form a hydrophobic cluster that affects resolution by IntDOT.

Patterns of variation in developmental timing among strains were not found to reflect patterns of phylogenetic relatedness (Fig. 3). For example, the fast developing isolates A66, A75 and A98 do not cluster together in a common clade, but are distributed across the phylogeny, as are the two slowest developing strains, GJV1 and A94. Ancestral character states for rate of development are difficult to infer for several nodes.

Western blot with the selected mouse monoclonal antibodies on extracts of membrane proteins prepared from embryonic mouse brain. The purified antibodies (A74, A79, A98, and 30H12) were diluted at 5 μg/ml, and the ascitic fluid anti-MenB was diluted 1/400. The mouse MAb IgM anti-MenB and IgG2a 30H12 were used as positive controls. Anti-MenB (lane 1), 30H12 (lane 5), and A79 (lane 3) precipitated a large high-molecular-weight band characteristic of PSA-NCAM.