It remains to be determined experimentally whether increasing fitness is related to positive selection, while stationary fitness is related to neutral evolution. Long-term laboratory evolution in Escherichia coli was performed under conditions of thermal stress under defined laboratory conditions. The complete cell growth data showed common continuous fitness recovery to every 2°C or 4°C stepwise temperature upshift, finally resulting in an evolved E. coli strain with an improved upper temperature limit as high as 45.9°C after 523 days of serial transfer, equivalent to 7,560 generations, in minimal medium. Two-phase fitness dynamics, a rapid growth recovery phase followed by a gradual increasing growth phase, was clearly observed at diverse temperatures throughout the entire evolutionary process. Whole-genome sequence analysis revealed the transition from positive to neutral in mutation fixation, accompanied with a considerable escalation of spontaneous substitution rate in the late fitness recovery phase. It suggested that continually increasing fitness not always resulted in the reduction of genetic diversity due to the sequential takeovers by fit mutants, but caused the accumulation of a considerable number of mutations that facilitated the neutral evolution.
The molecular clock of neutral mutations, which represents linear mutation fixation over generations, is theoretically explained by genetic drift in fitness-steady evolution or hitchhiking in adaptive evolution. The present study is the first experimental demonstration for the molecular clock of neutral mutations in a fitness-increasing evolutionary process. The dynamics of genome mutation fixation in the thermal adaptive evolution of Escherichia coli were evaluated in a prolonged evolution experiment in duplicated lineages. The cells from the continuously fitness-increasing evolutionary process were subjected to genome sequencing and analyzed at both the population and single-colony levels. Although the dynamics of genome mutation fixation were complicated by the combination of the stochastic appearance of adaptive mutations and clonal interference, the mutation fixation in the population was simply linear over generations. Each genome in the population accumulated 1.6 synonymous and 3.1 non-synonymous neutral mutations, on average, by the spontaneous mutation accumulation rate, while only a single genome in the population occasionally acquired an adaptive mutation. The neutral mutations that preexisted on the single genome hitchhiked on the domination of the adaptive mutation. The successive fixation processes of the 128 mutations demonstrated that hitchhiking and not genetic drift were responsible for the coincidence of the spontaneous mutation accumulation rate in the genome with the fixation rate of neutral mutations in the population. The molecular clock of neutral mutations to the fitness-increasing evolution suggests that the numerous neutral mutations observed in molecular phylogenetic trees may not always have been fixed in fitness-steady evolution but in adaptive evolution.
Protein kinase C-e (ePKC) induces neurite outgrowth in neuroblastoma cells but molecular mechanism of the ePKCinduced neurite outgrowth is not fully understood. Therefore, we investigated the ability of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) binding of ePKC and its correlation with the neurite extension. We found that full length ePKC bound to PIP 2 in a 12-o-tetradecanoylphorbol-13-acetate dependent manner, while the regulatory domain of ePKC (eRD) bound to PIP 2 without any stimulation. To identify the PIP 2 binding region, we made mutants lacking several regions from eRD, and examined their PIP 2 binding activity. The mutants lacking variable region 1 (V1) bound to PIP 2 stronger than intact eRD, while the mutants lacking pseudo-substrate or common region 1 (C1) lost the binding. The PIP 2 binding ability of the V3-deleted mutant was weakened. Those PIP 2 bindings of ePKC, eRD and the mutants well correlated to their neurite induction ability. In addition, a chimera of pleckstrin homology domain of phospholipase Cd and the V3 region of ePKC revealed that PIP 2 binding domain and the V3 region are sufficient for the neurite induction, and a first 16 amino acids in the V3 region was important for neurite extension. In conclusion, ePKC directly binds to PIP 2 mainly through pseudo-substrate and common region 1, contributing to the neurite induction activity. Keywords: actin, neurite outgrowth, neuroblastoma, phosphatidylinositol 4,5-bisphosphate, protein kinase C. Protein kinase C (PKC) plays pivotal roles in proliferation, differentiation, and apoptosis etc. The PKC family consists of at least 10 subtypes that are classified into three groups based on the structure of their regulatory domain (RD) (Nishizuka 1992;Shirai and Saito 2002;Newton 2006). Conventional PKCs (a, b1, b2, and c) have two common regions, C1 domain and C2 domain, in the RD. The former is responsible for diacylglycerol (DAG) and phorbol ester binding, the latter binds to calcium. Thus, calcium and DAG are required for the activation of conventional PKCs. On the other hand, novel PKCs (e, d, g, and h) are activated by DAG, but not by Ca 2+ because novel PKCs lack the C2 domain. Atypical PKCs (f and k/i) are insensitive to both Ca 2+ and DAG because of lack of the C2 domain and one of the C1 domains. Each subtype shows different enzymatic properties and distinct tissue and cellular distribution, suggesting specific functions of each PKC subtype (Ohno 1997), but the individual functions have not been fully understood.Among them, ePKC is abundant in the central nervous system and is thought to play important roles in nervous system (Tanaka and Nishizuka 1994;Akita 2002). Specifically, ePKC is localized at nerve terminus and seems to mediate synaptic function (Saito et al. 1993; Prekeris et al. Re-use of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.5, which does not permit commercial exploitation.Abbreviations used: ABS, actin-binding site; C1, common region 1; C1A, first half of C1 domain; ...
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