Experimental Analysis of Functional Variation within Protein Families: Receiver Domain Autodephosphorylation Kinetics

ABSTRACT Plants and microorganisms use two-component signal transduction systems (TCSs) to mediate responses to environmental stimuli. TCSs mediate responses through phosphotransfer from a conserved histidine on a sensor kinase to a conserved aspartate on the receiver domain of a response regulator. Typically, signal termination occurs through dephosphorylation of the receiver domain, which can catalyze its own dephosphorylation. Despite strong structural conservation between receiver domains, reported autodephosphorylation rate constants ( k dephos ) span a millionfold range. Variable receiver domain active-site residues D + 2 and T + 2 (two amino acids C terminal to conserved phosphorylation site and Thr/Ser, respectively) influence k dephos values, but the extent and mechanism of influence are unclear. We used sequence analysis of a large database of naturally occurring receiver domains to design mutant receiver domains for experimental analysis of autodephosphorylation kinetics. When combined with previous analyses, k dephos values were obtained for CheY variants that contained D + 2/T + 2 pairs found in 54% of receiver domain sequences. Tested pairs of amino acids at D + 2/T + 2 generally had similar effects on k dephos in CheY, PhoB N , or Spo0F. Acid or amide residues at D + 2/T + 2 enhanced k dephos . CheY variants altered at D + 2/T + 2 exhibited rate constants for autophosphorylation with phosphoramidates and autodephosphorylation that were inversely correlated, suggesting that D + 2/T + 2 residues interact with aspects of the ground or transition states that differ between the two reactions. k dephos of CheY variants altered at D + 2/T + 2 correlated significantly with k dephos of wild-type receiver domains containing the same D + 2/T + 2 pair. Additionally, particular D + 2/T + 2 pairs were enriched in different response regulator subfamilies, suggesting functional significance. IMPORTANCE One protein family, defined by a conserved domain, can include hundreds of thousands of known members. Characterizing conserved residues within a conserved domain can identify functions shared by all family members. However, a general strategy to assess features that differ between members of a family is lacking. Fully exploring the impact of just two variable positions within a conserved domain could require assessment of 400 (i.e., 20 × 20) variants. Instead, we created and analyzed a nonredundant database of receiver domain sequences. Five percent of D + 2/T + 2 pairs were sufficient to represent 50% of receiver domain sequences. Using protein sequence analysis to prioritize mutant choice made it experimentally feasible to extensively probe the influence of positions D + 2 and T + 2 on receiver domain autodephosphorylation kinetics

Assessment of intra and interregional genetic variation in the Eastern Red-backed Salamander, Plethodon cinereus, via analysis of novel microsatellite markers

The red-backed salamander (Plethodon cinereus) has long-served as a model system in ecology, evolution, and behavior, and studies surveying molecular variation in this species have become increasingly common over the past decade. However, difficulties are commonly encountered when extending microsatellite markers to populations that are unstudied from a genetic perspective due to high levels of genetic differentiation across this species’ range. To ameliorate this issue, we used 454 pyrosequencing to identify hundreds of microsatellite loci. We then screened 40 of our top candidate loci in populations in Virginia, Pennsylvania, and Ohio—including an isolated island population ~ 4.5 km off the shore of Lake Erie (South Bass Island). We identified 25 loci that are polymorphic in a well-studied region of Virginia and 11 of these loci were polymorphic in populations located in the genetically unstudied regions of Ohio and Pennsylvania. Use of these loci to examine patterns of variation within populations revealed that South Bass Island has low diversity in comparison to other sites. However, neither South Bass Island nor isolated populations around Cleveland are inbred. Assessment of variation between populations revealed three well defined genetic clusters corresponding to Virginia, mainland Ohio/Pennsylvania, and South Bass Island. Comparisons of our results to those of others working in various parts of the range are consistent with the idea that differentiation is lower in regions that were once glaciated. However, these comparisons also suggest that well differentiated isolated populations in the formerly glaciated portion of the range are not uncommon. This work provides novel genetic resources that will facilitate population genetic studies in a part of the red-backed salamander’s range that has not previously been studied in this manner. Moreover, this work refines our understanding of how neutral variation is distributed in this ecologically important organism

Models of Models: The Symbiotic Relationship between Models and Wargames

Military planning uses wargames to model the processes and decisions of an operation. As these operations become increasingly complex, the wargames similarly become more complex. Complex wargames are difficult to design and execute. As such, computer-based modeling and simulation can aid the wargame development, ensuring smooth execution. In particular, computer-based modeling and simulation can develop and validate the processes, determine initial conditions, evaluate the rules, and aid in validation. In turn, the wargame can provide useful data that can be fed into detailed models that can provide quantitative analysis to decision-makers

Genomics of a metamorphic timing QTL: \u3ci\u3emet1\u3c/i\u3e maps to a unique genomic position and regulates morph and species-specific patterns of brain transcription

Very little is known about genetic factors that regulate life history transitions during ontogeny. Closely related tiger salamanders (Ambystoma species complex) show extreme variation in metamorphic timing, with some species foregoing metamorphosis altogether, an adaptive trait called paedomorphosis. Previous studies identified a major effect QTL (met1) for metamorphic timing and expression of paedomorphosis in hybrid crosses between the biphasic Eastern tiger salamander (Ambystoma tigrinum tigrinum) and the paedomorphic Mexican axolotl (Ambystoma mexicanum). We used existing hybrid mapping panels and a newly created hybrid cross to map the met1 genomic region and determine the effect of met1 on larval growth, metamorphic timing, and gene expression in the brain. We show that met1 maps to the position of a urodele specific chromosome rearrangement on linkage group 2 that uniquely brought functionally-associated genes into linkage. Further, we found that \u3e 200 genes were differentially expressed during larval development as a function of met1 genotype. This list of differentially expressed genes is enriched for proteins that function in the mitochondria, providing evidence of a link between met1, thyroid hormone signaling, and mitochondrial energetics associated with metamorphosis. Finally, we found that met1 significantly affected metamorphic timing in hybrids, but not early larval growth rate. Collectively, our results show that met1 regulates species and morph-specific patterns of brain transcription and life history variation

Genomics of a Metamorphic Timing QTL: Met1 Maps to a Unique Genomic Position and Regulates Morph and Species-Specific Patterns of Brain Transcription

Very little is known about genetic factors that regulate life history transitions during ontogeny. Closely related tiger salamanders (Ambystoma species complex) show extreme variation in metamorphic timing, with some species foregoing metamorphosis altogether, an adaptive trait called paedomorphosis. Previous studies identified a major effect quantitative trait locus (met1) for metamorphic timing and expression of paedomorphosis in hybrid crosses between the biphasic Eastern tiger salamander (Ambystoma tigrinum tigrinum) and the paedomorphic Mexican axolotl (Ambystoma mexicanum). We used existing hybrid mapping panels and a newly created hybrid cross to map the met1 genomic region and determine the effect of met1 on larval growth, metamorphic timing, and gene expression in the brain. We show that met1 maps to the position of a urodele-specific chromosome rearrangement on linkage group 2 that uniquely brought functionally associated genes into linkage. Furthermore, we found that more than 200 genes were differentially expressed during larval development as a function of met1 genotype. This list of differentially expressed genes is enriched for proteins that function in the mitochondria, providing evidence of a link between met1, thyroid hormone signaling, and mitochondrial energetics associated with metamorphosis. Finally, we found that met1 significantly affected metamorphic timing in hybrids, but not early larval growth rate. Collectively, our results show that met1 regulates species and morph-specific patterns of brain transcription and life history variation

Genomics of a Metamorphic Timing QT:: Met1 Maps to a Unique Genomic Position and Regulates Morph and Species-Specific Patterns of Brain Transcription

Very little is known about genetic factors that regulate life history transitions during ontogeny. Closely related tiger salamanders (Ambystoma species complex) show extreme variation in metamorphic timing, with some species foregoing metamorphosis altogether, an adaptive trait called paedomorphosis. Previous studies identified a major effect quantitative trait locus (met1) for metamorphic timing and expression of paedomorphosis in hybrid crosses between the biphasic Eastern tiger salamander (Ambystoma tigrinum tigrinum) and the paedomorphic Mexican axolotl (Ambystoma mexicanum).We used existing hybrid mapping panels and a newly created hybrid cross to map the met1 genomic region and determine the effect of met1 on larval growth, metamorphic timing, and gene expression in the brain. We show that met1 maps to the position of a urodele-specific chromosome rearrangement on linkage group 2 that uniquely brought functionally associated genes into linkage. Further more, we found that more than 200 genes were differentially expressed during larval development as a function of met1 genotype. This list of differentially expressed genes is enriched for proteins that function in the mitochondria, providing evidence of a link between met1, thyroid hormone signaling, and mitochondrial energetics associated with metamorphosis. Finally, we found that met1 significantly affected metamorphic timing in hybrids, but not early larval growth rate. Collectively, our results show that met1 regulates species and morph-specific patterns of brain transcription and life history variation

Transcriptional response of Mexican axolotls to \u3ci\u3eAmbystoma tigrinum\u3c/i\u3e virus (ATV) infection

Background Very little is known about the immunological responses of amphibians to pathogens that are causing global population declines. We used a custom microarray gene chip to characterize gene expression responses of axolotls (Ambystoma mexicanum) to an emerging viral pathogen, Ambystoma tigrinum virus (ATV). Result At 0, 24, 72, and 144 hours post-infection, spleen and lung samples were removed for estimation of host mRNA abundance and viral load. A total of 158 up-regulated and 105 down-regulated genes were identified across all time points using statistical and fold level criteria. The presumptive functions of these genes suggest a robust innate immune and antiviral gene expression response is initiated by A. mexicanum as early as 24 hours after ATV infection. At 24 hours, we observed transcript abundance changes for genes that are associated with phagocytosis and cytokine signaling, complement, and other general immune and defense responses. By 144 hours, we observed gene expression changes indicating host-mediated cell death, inflammation, and cytotoxicity. Conclusion Although A. mexicanum appears to mount a robust innate immune response, we did not observe gene expression changes indicative of lymphocyte proliferation in the spleen, which is associated with clearance of Frog 3 iridovirus in adult Xenopus. We speculate that ATV may be especially lethal to A. mexicanum and related tiger salamanders because they lack proliferative lymphocyte responses that are needed to clear highly virulent iridoviruses. Genes identified from this study provide important new resources to investigate ATV disease pathology and host-pathogen dynamics in natural populations

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

Using a comparative approach to investigate the relationship between landscape and genetic connectivity among woodland salamander population

For many amphibian species, reduced landscape connectivity results in reduced genetic connectivity among populations. However, large efective population sizes (Ne) slow the rate of genetic drift, causing subdivided populations to remain genetically similar despite little gene fow among them. Therefore, it is important to address the combined efects of Ne and matrix permeability to quantify the relative importance of gene fow and genetic drift on isolated amphibian populations. We applied a landscape genetic approach to investigate how patterns of gene fow (m), Ne (inferred via θ) and genetic difer- entiation difer among Eastern Red-backed Salamander (Plethodon cinereus) populations in a fragmented landscape (n=4) compared to a continuous forest (n=4). We assayed a panel of 10 microsatellite markers for population genetic analyses. Additionally, we constructed and validated a distribution model to generate resistance surfaces for examining the relation- ship between landscape connectivity, m, θ, and genetic diferentiation (FST) using maximum-likelihood population-efects models (MLPE). Populations in continuous habitat were undiferentiated, whereas fragmented populations exhibited genetic structure driven by a single population. Results of the MLPE models in the fragmented landscape revealed spatial variation in θ as the best predictor of pairwise FST, followed by estimates of m, suggesting migration-drift interactions have a stronger infuence on genetic diferentiation than matrix permeability. Moreover, model coefcients for landscape resistance were comparable between landscapes. Overall, our results provide insight as to how the interaction of gene fow and genetic drift shapes population structure for a dispersal-limited species within a predominately anthropogenic landscape

Effect of thyroid hormone concentration on the transcriptional response underlying induced metamorphosis in the Mexican axolotl (\u3ci\u3eAmbystoma\u3c/i\u3e)

Background Thyroid hormones (TH) induce gene expression programs that orchestrate amphibian metamorphosis. In contrast to anurans, many salamanders do not undergo metamorphosis in nature. However, they can be induced to undergo metamorphosis via exposure to thyroxine (T4). We induced metamorphosis in juvenile Mexican axolotls (Ambystoma mexicanum) using 5 and 50 nM T4, collected epidermal tissue from the head at four time points (Days 0, 2, 12, 28), and used microarray analysis to quantify mRNA abundances. Results Individuals reared in the higher T4 concentration initiated morphological and transcriptional changes earlier and completed metamorphosis by Day 28. In contrast, initiation of metamorphosis was delayed in the lower T4 concentration and none of the individuals completed metamorphosis by Day 28. We identified 402 genes that were statistically differentially expressed by ≥ two-fold between T4 treatments at one or more non-Day 0 sampling times. To complement this analysis, we used linear and quadratic regression to identify 542 and 709 genes that were differentially expressed by ≥ two-fold in the 5 and 50 nM T4 treatments, respectively. Conclusion We found that T4 concentration affected the timing of gene expression and the shape of temporal gene expression profiles. However, essentially all of the identified genes were similarly affected by 5 and 50 nM T4. We discuss genes and biological processes that appear to be common to salamander and anuran metamorphosis, and also highlight clear transcriptional differences. Our results show that gene expression in axolotls is diverse and precise, and that axolotls provide new insights about amphibian metamorphosis