In Vitro Measures of MDR-Transporter Function and Whole-Hive Exposure Dynamics Using Fluorescent Dyes

We need to study Apis mellifera both in vivo and ex vivo to better understand honey bee biology. In vivo synergism of chemicals can occur when xenobiotic transporters are inhibited by one chemical, allowing a second chemical to accumulate and become toxic. I have conducted assays between 2010 and 2013 that demonstrated RhB dye- a xenobiotic transporter substrate, is fed in the presence of the xenobiotic inhibitor verapamil, it is found in higher levels in the hemolymph of the Apis mellifera Two types of bee food combined with two dyes were tested in 2012 for the impact of food type, and the impact of dye type on the fate of the dye in a Apis mellifera hive. Slightly hydrophobic RhB and slightly hydrophilic UrO were used. Dyed syrup persisted longer in hives than dyed pollen patties, and dyes did not spread uniformly throughout the hive

RPA and PCNA suppress formation of large deletion errors by yeast DNA polymerase δ

In fulfilling its biosynthetic roles in nuclear replication and in several types of repair, DNA polymerase δ (pol δ) is assisted by replication protein A (RPA), the single-stranded DNA-binding protein complex, and by the processivity clamp proliferating cell nuclear antigen (PCNA). Here we report the effects of these accessory proteins on the fidelity of DNA synthesis in vitro by yeast pol δ. We show that when RPA and PCNA are included in reactions containing pol δ, rates for single base errors are similar to those generated by pol δ alone, indicating that pol δ itself is by far the prime determinant of fidelity for single base errors. However, the rate of deleting multiple nucleotides between directly repeated sequences is reduced by ∼10-fold in the presence of either RPA or PCNA, and by ≥90-fold when both proteins are present. We suggest that PCNA and RPA suppress large deletion errors by preventing the primer terminus at a repeat from fraying and/or from relocating and annealing to a downstream repeat. Strong suppression of deletions by PCNA and RPA suggests that they may contribute to the high replication fidelity needed to stably maintain eukaryotic genomes that contain abundant repetitive sequences

The fidelity of DNA synthesis by yeast DNA polymerase zeta alone and with accessory proteins

DNA polymerase zeta (pol z) participates in several DNA transactions in eukaryotic cells that increase spontaneous and damage-induced mutagenesis. To better understand this central role in mutagenesis in vivo, here we report the fidelity of DNA synthesis in vitro by yeast pol z alone and with RFC, PCNA and RPA. Overall, the accessory proteins have little effect on the fidelity of pol z. Pol z is relatively accurate for single base insertion/deletion errors. However, the average base substitution fidelity of pol z is substantially lower than that of homologous B family pols a, d and «. Pol z is particularly error prone for substitutions in specific sequence con-texts and generates multiple single base errors clustered in short patches at a rate that is unprece-dented in comparison with other polymerases. The unique error specificity of pol z in vitro is consistent with Pol z-dependent mutagenic specificity reported in vivo. This fact, combined with the high rate of single base substitution errors and complex muta-tions observed here, indicates that pol z contributes to mutagenesis in vivo not only by extending mismatches made by other polymerases, but also by directly generating its own mismatches and then extending them

Ultra-fast Au(III)-mediated Arylation of Cysteine

Through mechanistic work and rational design, we have developed the fastest organometallic abiotic Cys bioconjugation. As a result, the developed organometallic Au(III) bioconjugation reagents enable selective labeling of Cys moieties down to pM concentrations and allow for the rapid construction of complex heterostructures from peptides, proteins, and oligonucleo-tides. This work showcases how organometallic chemistry can be interfaced with biomolecules and lead to the range of reac-tivities that are largely unmatched by classical organic chemistry tools

An Acidic Motif Retains Vesicular Monoamine Transporter 2 on Large Dense Core Vesicles

The release of biogenic amines from large dense core vesicles (LDCVs) depends on localization of the vesicular monoamine transporter VMAT2 to LDCVs. We now find that a cluster of acidic residues including two serines phosphorylated by casein kinase 2 is required for the localization of VMAT2 to LDCVs. Deletion of the acidic cluster promotes the removal of VMAT2 from LDCVs during their maturation. The motif thus acts as a signal for retention on LDCVs. In addition, replacement of the serines by glutamate to mimic phosphorylation promotes the removal of VMAT2 from LDCVs, whereas replacement by alanine to prevent phosphorylation decreases removal. Phosphorylation of the acidic cluster thus appears to reduce the localization of VMAT2 to LDCVs by inactivating a retention mechanism

Low-fidelity DNA synthesis by the L979F mutator derivative of Saccharomyces cerevisiae DNA polymerase ζ

To probe Pol ζ functions in vivo via its error signature, here we report the properties of Saccharomyces cerevisiae Pol ζ in which phenyalanine was substituted for the conserved Leu-979 in the catalytic (Rev3) subunit. We show that purified L979F Pol ζ is 30% as active as wild-type Pol ζ when replicating undamaged DNA. L979F Pol ζ shares with wild-type Pol ζ the ability to perform moderately processive DNA synthesis. When copying undamaged DNA, L979F Pol ζ is error-prone compared to wild-type Pol ζ, providing a biochemical rationale for the observed mutator phenotype of rev3-L979F yeast strains. Errors generated by L979F Pol ζ in vitro include single-base insertions, deletions and substitutions, with the highest error rates involving stable misincorporation of dAMP and dGMP. L979F Pol ζ also generates multiple errors in close proximity to each other. The frequency of these events far exceeds that expected for independent single changes, indicating that the first error increases the probability of additional errors within 10 nucleotides. Thus L979F Pol ζ, and perhaps wild-type Pol ζ, which also generates clustered mutations at a lower but significant rate, performs short patches of processive, error-prone DNA synthesis. This may explain the origin of some multiple clustered mutations observed in vivo

Profiling of Genes Related to Cross Protection and Competition for NbTOM1 by HLSV and TMV

10.1371/journal.pone.0073725PLoS ONE89-POLN