Population genetic variation, geographic structure, and multiple origins of autopolyploidy in Galax urceolata.

PREMISE OF THE STUDY: Whereas population genetic studies have examined allopolyploids, comparable studies of naturally occurring autopolyploids remain rare. To address fundamental questions regarding autopolyploidy, we undertook a detailed population genetic study of one of the classic examples of autopolyploidy, Galax urceolata (Diapensiaceae), which comprises diploid, triploid, and autotetraploid cytotypes. Galax is endemic to the Appalachian Mountains, the adjacent piedmont, sandhills, and coastal plain and represents perhaps the most widely known example of autopolyploidy in nature. METHODS: Flow cytometry was used to diagnose ploidal level of ∼1000 individuals across 71 populations. We used 10 microsatellite markers to examine genetic variation across the geographic range of Galax and assessed multiple origins though comparisons of diploid, triploid, and tetraploid accessions using multiple analytical approaches. KEY RESULTS: Tetraploids had higher levels of heterozygosity than diploids did. Genetic variation in diploid and tetraploid Galax is geographically structured among the ecoregions of the southeastern United States. Autotetraploidy in Galax urceolata has occurred independently at least 46 times, with triploidy having occurred a minimum of 31 times. CONCLUSIONS: Genetic differentiation among ecoregions suggests historical patterns of local adaptation. The numerous independent origins of tetraploid Galax reported here are among the highest frequencies of independent polyploidizations ever reported for any polyploid (auto- or allopolyploid).

Microsatellite marker development for Galax urceolata (Diapensiaceae).

PREMISE OF THE STUDY: Microsatellite markers were developed for Galax urceolata to investigate genetic diversity, population structure, and polyploid origins (auto- vs. allopolyploid), and to estimate the minimum number of independent cytotype origins. METHODS AND RESULTS: Ten primer sets have been developed, and preliminary study indicates that all loci are appropriate for population-level genetic investigations. All loci are polymorphic with 6 to 46 alleles per locus. Expected heterozygosity ranges from 0.1007 to 0.6085. CONCLUSIONS: The microsatellite markers presented will facilitate analyses of polyploid origins, genetic diversity, geographic structure, and gene flow.

Shift work in nurses: contribution of phenotypes and genotypes to adaptation.

BACKGROUND: Daily cycles of sleep/wake, hormones, and physiological processes are often misaligned with behavioral patterns during shift work, leading to an increased risk of developing cardiovascular/metabolic/gastrointestinal disorders, some types of cancer, and mental disorders including depression and anxiety. It is unclear how sleep timing, chronotype, and circadian clock gene variation contribute to adaptation to shift work. METHODS: Newly defined sleep strategies, chronotype, and genotype for polymorphisms in circadian clock genes were assessed in 388 hospital day- and night-shift nurses. RESULTS: Night-shift nurses who used sleep deprivation as a means to switch to and from diurnal sleep on work days (∼25%) were the most poorly adapted to their work schedule. Chronotype also influenced efficacy of adaptation. In addition, polymorphisms in CLOCK, NPAS2, PER2, and PER3 were significantly associated with outcomes such as alcohol/caffeine consumption and sleepiness, as well as sleep phase, inertia and duration in both single- and multi-locus models. Many of these results were specific to shift type suggesting an interaction between genotype and environment (in this case, shift work). CONCLUSIONS: Sleep strategy, chronotype, and genotype contribute to the adaptation of the circadian system to an environment that switches frequently and/or irregularly between different schedules of the light-dark cycle and social/workplace time. This study of shift work nurses illustrates how an environmental "stress" to the temporal organization of physiology and metabolism can have behavioral and health-related consequences. Because nurses are a key component of health care, these findings could have important implications for health-care policy.

Genetic differences in human circadian clock genes among worldwide populations.

The daily biological clock regulates the timing of sleep and physiological processes that are of fundamental importance to human health, performance, and well-being. Environmental parameters of relevance to biological clocks include (1) daily fluctuations in light intensity and temperature, and (2) seasonal changes in photoperiod (day length) and temperature; these parameters vary dramatically as a function of latitude and locale. In wide-ranging species other than humans, natural selection has genetically optimized adaptiveness along latitudinal clines. Is there evidence for selection of clock gene alleles along latitudinal/photoperiod clines in humans? A number of polymorphisms in the human clock genes Per2, Per3, Clock, and AANAT have been reported as alleles that could be subject to selection. In addition, this investigation discovered several novel polymorphisms in the human Arntl and Arntl2 genes that may have functional impact upon the expression of these clock transcriptional factors. The frequency distribution of these clock gene polymorphisms is reported for diverse populations of African Americans, European Americans, Ghanaians, Han Chinese, and Papua New Guineans (including 5 subpopulations within Papua New Guinea). There are significant differences in the frequency distribution of clock gene alleles among these populations. Population genetic analyses indicate that these differences are likely to arise from genetic drift rather than from natural selection.

Imaging protein interactions with bioluminescence resonance energy transfer (BRET) in plant and mammalian cells and tissues.

FRET is a well established method for cellular and subcellular imaging of protein interactions. However, FRET obligatorily necessitates fluorescence excitation with its concomitant problems of photobleaching, autofluorescence, phototoxicity, and undesirable stimulation of photobiological processes. A sister technique, bioluminescence resonance energy transfer (BRET), avoids these problems because it uses enzyme-catalyzed luminescence; however, BRET signals usually have been too dim to image effectively in the past. Using a new generation electron bombardment-charge-coupled device camera coupled to an image splitter, we demonstrate that BRET can be used to image protein interactions in plant and animal cells and in tissues; even subcellular imaging is possible. We have applied this technology to image two different protein interactions: (i) dimerization of the developmental regulator, COP1, in plant seedlings; and (ii) CCAAT/enhancer binding protein alpha (C/EBPalpha) in the mammalian nucleus. This advance heralds a host of applications for imaging without fluorescent excitation and its consequent limitations.

Elimination of antigen-presenting cells and autoreactive T cells by Fas contributes to prevention of autoimmunity.

Fas (also known as Apo-1 and CD95) receptor has been suggested to control T cell expansion by triggering T cell-autonomous apoptosis. This paradigm is based on the extensive lymphoproliferation and systemic autoimmunity in mice and humans lacking Fas or its ligand. However, with systemic loss of Fas, it is unclear whether T cell-extrinsic mechanisms contribute to autoimmunity. We found that tissue-specific deletion of Fas in mouse antigen-presenting cells (APCs) was sufficient to cause systemic autoimmunity, implying that normally APCs are destroyed during immune responses via a Fas-mediated mechanism. Fas expression by APCs was increased by exposure to microbial stimuli. Analysis of mice with Fas loss restricted to T cells revealed that Fas indeed controls autoimmune T cells, but not T cells responding to strong antigenic stimulation. Thus, Fas-dependent elimination of APCs is a major regulatory mechanism curbing autoimmune responses and acts in concert with Fas-mediated regulation of chronically activated autoimmune T cells.

A suite of tools and application notes for in vivo protein interaction assays using bioluminescence resonance energy transfer (BRET).

Bioluminescence resonance energy transfer (BRET) is a natural biophysical phenomenon that underlies an emerging technique to monitor protein-protein interactions in living cells in real time. Here, we present a series of technical advances to enhance the utility of the BRET assay in plants. A series of recombination cloning vectors was generated to accelerate the expression of proteins tagged with Renilla luciferase or yellow fluorescent protein under transient assay conditions and in stable transgenic plants. Working in stably transformed Arabidopsis or tobacco, we then detected BRET between three pairs of candidate interaction partners: dimerization of the E3 ubiquitin ligase COP1, interaction between COP1 and the B-box protein STH, and interaction between the light regulatory bZip transcription factors HY5 and HYH. A codon-optimized version of the Renilla luciferase gene resulted in improved expression in Arabidopsis. Renilla luciferase was active in a variety of subcellular organelles, including plastids, mitochondria, peroxisomes and Golgi stacks. In a survey of the Arabidopsis light signaling machinery as a model system, we estimated the likelihood that a known protein-protein interaction can be documented using BRET. Finally, we show that Renilla luciferase may serve as a reporter of protein stability in a cycloheximide chase assay.