Do genetic structure and landscape heterogeneity impact color morph frequency in a polymorphic salamander?

Landscape heterogeneity plays an important role in population structure and divergence, particularly for species with limited vagility. Here, we used a landscape genetic approach to identify how landscape and environmental variables affect genetic structure and color morph frequency in a polymorphic salamander. The eastern red- backed salamander, Plethodon cinereus, is widely distributed in northeastern North America and contains two common color morphs, striped and unstriped, that are divergent in ecology, behavior, and physiology. To quantify population structure, rates of gene flow, and genetic drift, we amplified 10 microsatellite loci from 648 individuals across 28 sampling localities. This study was conducted in northern Ohio, where populations of P. cinereus exhibit an unusually wide range of morph frequency variation. To test whether genetic distance was more correlated with morph frequency, elevation, canopy cover, waterways, ecological niche or geographic distance, we used resistance distance and least cost path analyses. We then examined whether landscape and environmental variables, genetic distance or geographic distance were correlated with variation in morph frequency. Tests for population structure revealed three genetic clusters across our sampling range, with one cluster monomorphic for the striped morph. Rates of gene flow and genetic drift were low to moderate across sites. Genetic distance was most correlated with ecological niche, elevation and a combination of landscape and environmental variables. In contrast, morph frequency variation was correlated with waterways and geographic distance. Thus, our results suggest that selection is also an important evolutionary force across our sites, and a balance between gene flow, genetic drift and selection interact to maintain the two color morphs

Exploration of factors driving incorporation of unnatural dNTPS into DNA by Klenow fragment (DNA polymerase I) and DNA polymerase α

In order to further understand how DNA polymerases discriminate against incorrect dNTPs, we synthesized two sets of dNTP analogues and tested them as substrates for DNA polymerase α (pol α) and Klenow fragment (exo(−)) of DNA polymerase I (Escherichia coli). One set of analogues was designed to test the importance of the electronic nature of the base. The bases consisted of a benzimidazole ring with one or two exocyclic substituent(s) that are either electron-donating (methyl and methoxy) or electron-withdrawing (trifluoromethyl and dinitro). Both pol α and Klenow fragment exhibit a remarkable inability to discriminate against these analogues as compared to their ability to discriminate against incorrect natural dNTPs. Neither polymerase shows any distinct electronic or steric preferences for analogue incorporation. The other set of analogues, designed to examine the importance of hydrophobicity in dNTP incorporation, consists of a set of four regioisomers of trifluoromethyl benzimidazole. Whereas pol α and Klenow fragment exhibited minimal discrimination against the 5- and 6-regioisomers, they discriminated much more effectively against the 4- and 7-regioisomers. Since all four of these analogues will have similar hydrophobicity and stacking ability, these data indicate that hydrophobicity and stacking ability alone cannot account for the inability of pol α and Klenow fragment to discriminate against unnatural bases. After incorporation, however, both sets of analogues were not efficiently elongated. These results suggest that factors other than hydrophobicity, sterics and electronics govern the incorporation of dNTPs into DNA by pol α and Klenow fragment

Largazole and Its Derivatives Selectively Inhibit Ubiquitin Activating Enzyme (E1)

Protein ubiquitination plays an important role in the regulation of almost every aspect of eukaryotic cellular function; therefore, its destabilization is often observed in most human diseases and cancers. Consequently, developing inhibitors of the ubiquitination system for the treatment of cancer has been a recent area of interest. Currently, only a few classes of compounds have been discovered to inhibit the ubiquitin-activating enzyme (E1) and only one class is relatively selective in E1 inhibition in cells. We now report that Largazole and its ester and ketone analogs selectively inhibit ubiquitin conjugation to p27Kip1 and TRF1 in vitro. The inhibitory activity of these small molecules on ubiquitin conjugation has been traced to their inhibition of the ubiquitin E1 enzyme. To further dissect the mechanism of E1 inhibition, we analyzed the effects of these inhibitors on each of the two steps of E1 activation. We show that Largazole and its derivatives specifically inhibit the adenylation step of the E1 reaction while having no effect on thioester bond formation between ubiquitin and E1. E1 inhibition appears to be specific to human E1 as Largazole ketone fails to inhibit the activation of Uba1p, a homolog of E1 in Schizosaccharomyces pombe. Moreover, Largazole analogs do not significantly inhibit SUMO E1. Thus, Largazole and select analogs are a novel class of ubiquitin E1 inhibitors and valuable tools for studying ubiquitination in vitro. This class of compounds could be further developed and potentially be a useful tool in cells

Effectiveness of Wildlife Mitigation Treatments Along the Nelsonville Bypass

SJN 135024The Nelsonville Bypass is a 9 mile stretch of U.S. Route 33 that runs through the Wayne National Forest, an area high in species diversity and home to several threatened and endangered species. The motorist safety, economic and conservation values of building effective mitigation features that reduce vehicle-wildlife collisions along the bypass have been nationally recognized. Mitigation features include: high and low fencing to reduce wildlife trespass into the right-of-way (ROW), uni-directional jump outs for wildlife exit from the ROW, underpasses and ecopassages to maintain habitat connectivity across the highway, high-mast lighting to lure bats above traffic flow, and replacement of wetlands and bat roosting habitat. Our two-year study employed road surveys, continuous monitoring of jump outs and wildlife passages, population estimations, detailed mapping of fence structures and breaches, and radio telemetry of an endangered target species. Road surveys of the bypass and control highways revealed that the mitigation structures reduced deer-vehicle collisions, but collisions still occurred on the bypass. Although, generally well-constructed, we identified several ways in which the mitigation features could be made more effective. Placement of fencing near the outer boundary of the ROW made it vulnerable to damage from erosion and tree falls, and isolated high-quality habitats within the ROW. Placement of the fence within 30-50 ft. of the roadway on less rugged terrain away from the forest would likely reduce costs of construction and maintenance while allowing wildlife access to habitat within the ROW. We also recommended regular maintenance inspections and mowing on both sides of the fencing. Jump outs were effective uni-directional exits, but wildlife, particularly deer, were not compelled to exit the expansive area within the ROW fencing. Placement of the fence with jump outs closer to the road would reduce habitat within the fence and combined with traffic noise may increase jump out use. Large wildlife underpasses and crossings were well used by a variety of mammal species. Smaller mammals used the small wildlife ecopassages. Reptiles and amphibians avoided the use of underpasses and road mortality rates of amphibians were high on Ohio State Route 78 (tributary road) near wetlands. Placement and passage design were contributing factors to high amphibian mortality. Radio-tracking of rattlesnakes discovered that snakes easily trespassed the small wildlife fencing and used the habitat within the ROW, likely because it was warmer than the surrounding forested habitat. No road mortality or attempted road crossings by rattlesnakes were detected. Finally, while bats foraged near the lights, most species were detected with equal frequency at different heights under the lighting. Our report details these findings and provides additional recommendations to improve design and construction of wildlife mitigation features both along the Nelsonville Bypass, and for future design of mitigation features for roadways in high-density wildlife areas

Protein Displacement by Herpes Helicase-Primase and the Key Role of UL42 during Helicase-Coupled DNA Synthesis by the Herpes Polymerase

The herpes helicase-primase (UL5-UL8-UL52) very inefficiently unwinds double-stranded DNA. To better understand the mechanistic consequences of this inefficiency, we investigated protein displacement activity by UL5-UL8-UL52, as well as the impact of coupling DNA synthesis by the herpes polymerase with helicase activity. While the helicase can displace proteins bound to the lagging strand template, bound proteins significantly impede helicase activity. Remarkably, UL5-UL8-UL52, an extremely inefficient helicase, disrupts the exceptionally tight interaction between streptavidin and biotin on the lagging strand template. It also unwinds DNA containing streptavidin bound to the leading strand template, although it does not displace the streptavidin. These data suggest that the helicase may largely or completely wrap around the lagging strand template, with minimal interactions with the leading strand template. We utilized synthetic DNA minicircles to study helicase activity coupled with the herpes polymerase-processivity factor (UL30-UL42). Coupling greatly enhances unwinding of DNA, although bound proteins still inhibit helicase activity. Surprisingly, while UL30-UL42 and two noncognate polymerases (Klenow Fragment and T4 DNA polymerase) all stimulate unwinding of DNA by the helicase, the isolated UL30 polymerase (i.e., no UL42 processivity factor) binds to the replication fork but in a manner that is incompetent in terms of coupled helicase-polymerase activity