Highly tolerated amino acid substitutions increase the fidelity of Escherichia coli DNA polymerase I

Fidelity of DNA synthesis, catalyzed by DNA polymerases, is critical for the maintenance of the integrity of the genome. Mutant polymerases with elevated accuracy (antimutators) have been observed, but these mainly involve increased exonuclease proofreading or large decreases in polymerase activity. We have determined the tolerance of DNA polymerase for amino acid substitutions in the active site and in different segments of E. coli DNA polymerase I and have determined the effects of these substitutions on the fidelity of DNA synthesis. We established a DNA polymerase I mutant library, with random substitutions throughout the polymerase domain. This random library was first selected for activity. The essentiality of DNA polymerases and their sequence and structural conservation suggests that few amino acid substitutions would be tolerated. However, we report that two-thirds of single base substitutions were tolerated without loss of activity, and plasticity often occurs at evolutionarily conserved regions. We screened 408 members of the active library for alterations in fidelity of DNA synthesis in Escherichia coli expressing the mutant polymerases and carrying a second plasmid containing a beta-lactamase reporter. Mutation frequencies varied from 1/1000- to 1000-fold greater compared with wild type. Mutations that produced an antimutator phenotype were distributed throughout the polymerase domain, with 12% clustered in the M-helix. We confirmed that a single mutation in this segment results in increased base discrimination. Thus, this work identifies the M-helix as a determinant of fidelity and suggests that polymerases can tolerate many substitutions that alter fidelity without incurring major changes in activity.     

Analysis of S-glutathionylated proteins during adipocyte differentiation using eosin-glutathione and glutaredoxin 1

Protein S-glutathionylation is a reversible post-translational modification on cysteine residues forming a mixed disulfide with glutathione. S-glutathionylation, not only protects proteins from oxidation but also regulates the functions of proteins involved in various cellular signaling pathways. In this study, we developed a method for the detection of S-glutathionylated proteins (ProSSG) using eosin-glutathione (E-GSH) and mouse glutaredoxin 1 (mGrx1). ProSSG was efficiently and specifically labeled with E-GSH to form ProSSG-E via thiol-disulfide exchange. ProSSG-E was readily luminescent allowing the detection of ProSSG with semi-quantitative determination. In addition, a deglutathionylation enzyme mGrx1 specifically released E-GSH from ProSSG-E, which increased fluorescence allowing a sensitive determination of ProSSG levels. Application of the method to the adipocyte differentiation of 3T3-L1 cells showed specific detection of ProSSG and its increase upon differentiation induction, which was consistent with the result obtained by conventional immunoblot analysis, but with greater specificity and sensitivity.     

Overexpression of Newcastle disease virus (NDV) V protein enhances NDV production kinetics in chicken embryo fibroblasts

Newcastle disease virus (NDV) is not only one of the most economically important pathogen of poultry but also has a potential as anticancer virotherapy. The role of NDV V protein in virus-production kinetics was investigated using DF-1 cell-based production system. The presence of an anti-interferon (IFN)-alpha antibody resulted in enhanced NDV production kinetics in a dose-dependent manner by blocking binding of NDV-induced IFN to its receptor. To prepare DF-1 cell whose cellular IFN signaling is blocked efficiently, stable cell lines expressing either lentogenic or velogenic NDV V protein known as an IFN antagonist were established. The overexpression of NDV V protein enhanced NDV production kinetics and expedited the rate of NDV production, while it had no effect on Japanese encephalitis virus production. NDV V protein functions as an IFN antagonist by inhibiting the increase in type I IFNs by NDV infection. The IFN signals in cells expressing NDV V protein were weakened by decreased activation or expression of the dsRNA-activated enzymes. These IFN antagonist activities enhance rapid virus replication and spread in the early phase of viral infection and will be useful in improving the production of viral vaccine strains.     

Expression of protein phosphatase 2A mutants and silencing of the regulatory B alpha subunit induce a selective loss of acetylated and detyrosinated microtubules

Carboxymethylation and phosphorylation of protein phosphatase 2A (PP2A) catalytic C subunit are evolutionary conserved mechanisms that critically control PP2A holoenzyme assembly and substrate specificity. Down-regulation of PP2A methylation and PP2A enzymes containing the B alpha regulatory subunit occur in Alzheimer's disease. In this study, we show that expressed wild-type and methylation- (L309 Delta) and phosphorylation- (T304D, T304A, Y307F, and Y307E) site mutants of PP2A C subunit differentially bind to B, B', and B'-type regulatory subunits in NIH 3T3 fibroblasts and neuro-2a (N2a) neuroblastoma cells. They also display distinct binding affinity for microtubules (MTs). Relative to controls, expression of the wild-type, T304A and Y307F C subunits in N2a cells promotes the accumulation of acetylated and detyrosinated MTs. However, expression of the Y307E, L309 Delta, and T304D mutants, which are impaired in their ability to associate with the B alpha subunit, induces their loss. Silencing of B alpha subunit in N2a and NIH 3T3 cells is sufficient to induce a similar breakdown of acetylated and detyrosinated MTs. It also confers increased sensitivity to nocodazole-induced MT depolymerization. Our findings suggest that changes in intracellular PP2A subunit composition can modulate MT dynamics. They support the hypothesis that reduced amounts of neuronal B alpha-containing PP2A heterotrimers contribute to MT destabilization in Alzheimer's disease.     

Behavioral responsiveness of young chimpanzees (Pan troglodytes) to a novel environment

Behavioral responsiveness to a novel environment was documented in 22 chimpanzees grouped according to age; 6-months, 1-year, 2-years and 5-years. An attachment figure, a human caretaker, accompanied each subject during the 15-min test sessions so as to preclude confounding of responses to novelty with separation responses. Extreme distress reported previously for chimpanzees and human children when tested alone in a novel situation was rarely observed in these tests when an attachment figure was present. Stereotyped rocking, an indication of mild distress occurred more frequently in the younger animals. Younger animals engaged in distal visual exploration of the environment while remaining close to the attachment figure, whereas the older animals locomoted more frequently and explored the environment directly with their hands. Repeated exposure to the environment reduced the differences among the 6-month, 1-year and 2-year groups. The 6-month group, however, continued to locomote least and least frequently engaged in tactile exploration. These data on chimpanzees resemble data on human children which suggest that an attachment figure: attenuates the distress exhibited by young individuals of these species when exposed to novel stimuli, and thereby provides a secure base which supports the exploration of novel stimuli, a prerequisite to behavioral adaptation.     

Do flood pulses structure amphibian communities in floodplain environments?

Beta diversity can provide insights into the processes that regulate communities subjected to frequent disturbances, such as flood pulses, which control biodiversity in floodplains. However, little is known about which processes structure beta diversity of amphibians in floodplains. Here, we tested the influence of flood pulses on the richness, composition, and beta diversity of amphibians in Amazonian floodplain environments. We also evaluated indicator species for each environment. We established linear transects in three environments: low várzea, high várzea, and macrophyte rafts. Species richness decreased and beta diversity increased according to the susceptibility of habitats to flood pulses. Indicator species differed among environments according to forest succession promoted by the flood pulse. The decrease in species richness between high and low várzea is due to non‐random extinctions. The higher rates of species turnover between várzeas and macrophyte rafts are driven by the colonization of species adapted to open areas. Our results highlight that the maintenance of complex environments is needed to protect biodiversity in floodplains.     

Adipogenic function of tetranectin mediated by enhancing mitotic clonal expansion via ERK signaling

Tetranectin (TN), an adipogenic serum protein, enhances adipocyte differentiation, however, its functional mechanism has yet to be elucidated. In the present study, we investigated the adipogenic function of TN by using medium containing TN-depleted fetal bovine serum (TN-del-FBS) and recombinant mouse TN (mTN). The adipocyte differentiation of 3T3-L1 cells was significantly enhanced by mTN supplementation essentially at differentiation induction, which indicated a potential role of the protein in the early differentiation phase. The adipogenic effect of mTN was more significant with insulin in the differentiation induction cocktail, implicating their close functional relationship. mTN enhanced not only the proliferation of growing cells, but also mitotic clonal expansion (MCE) that is a prerequisite for adipocyte differentiation in the early phase. Consistently, mTN increased the phosphorylation of ERK in the early phase of adipocyte differentiation. Results of this study demonstrate that the adipogenic function of mTN is mediated by enhancing MCE via ERK signaling.     

Colorimetric pH measurement of animal cell culture media

Most animal cell culture media can be buffered using bicarbonate and high pressure CO₂ in a closed system. However, in an open system, the pH of the culture media increases continuously due to the marked difference in CO₂ pressure between the culture media and the atmosphere. Therefore, it is important to measure the exact pH of the culture media in an intact closed system. In this study, a pH measurement method was developed using visible light. The pH was calculated from light absorbance by the cells and by the culture media. This method was successfully applied to both suspension and anchorage-dependent cell cultures.     

A dual-fluorescence reporter system for high-throughput clone characterization and selection by cell sorting

Molecular biology critically depends upon the isolation of desired DNA sequences. Flow cytometry, with its capacity to interrogate and sort more than 50000 cells/s, shows great potential to expedite clone characterization and isolation. Intrinsic heterogeneity of protein expression levels in cells limits the utility of single fluorescent reporters for cell-sorting. Here, we report a novel dual-fluorescence strategy that overcomes the inherent limitations of single reporter systems by controlling for expression variability. We demonstrate a dual-reporter system using the green fluorescent protein (GFP) gene fused to the Discosoma red fluorescent protein (DsRed) gene. The system reports the successful insertion of foreign DNA with the loss of DsRed fluorescence and the maintenance of GFP fluorescence. Single cells containing inserts are readily recognized by their altered ratios of green to red fluorescence and separated using a high-speed cell-sorter for further processing. This novel reporter system and vector were successfully validated by shotgun library construction, cloned sequence isolation, PCR amplification and DNA sequencing of cloned inserts from bacteria after cell-sorting. This simple, robust system can also be adapted for diverse biosensor assays and is amenable to miniaturization. We demonstrated that dual-fluorescence reporting coupled with high-speed cell-sorting provides a more efficient alternative to traditional methods of clone isolation.