The impact of single substitutions on multiple sequence alignments

We introduce another view of sequence evolution. Contrary to other approaches, we model the substitution process in two steps. First we assume (arbitrary) scaled branch lengths on a given phylogenetic tree. Second we allocate a Poisson distributed number of substitutions on the branches. The probability to place a mutation on a branch is proportional to its relative branch length. More importantly, the action of a single mutation on an alignment column is described by a doubly stochastic matrix, the so-called one-step mutation matrix. This matrix leads to analytical formulae for the posterior probability distribution of the number of substitutions for an alignment column.     

Expedited approaches to whole cell electron tomography and organelle mark-up in situ in high-pressure frozen pancreatic islets

We have developed a simplified, efficient approach for the 3D reconstruction and analysis of mammalian cells in toto by electron microscope tomography (ET), to provide quantitative information regarding ‘global’ cellular organization at 15–20 nm resolution. Two insulin-secreting beta cells—deemed ‘functionally equivalent’ by virtue of their location at the periphery of the same pancreatic islet—were reconstructed in their entirety in 3D after fast-freezing/freeze-substitution/plastic embedment in situ within a glucose-stimulated islet of Langerhans isolated intact from mouse pancreata. These cellular reconstructions have afforded several unique insights into fundamental structure–function relationships among key organelles involved in the biosynthesis and release of the crucial metabolic hormone, insulin, that could not be provided by other methods. The Golgi ribbon, mitochondria and insulin secretory granules in each cell were segmented for comparative analysis. We propose that relative differences between the two cells in terms of the number, dimensions and spatial distribution (and for mitochondria, also the extent of branching) of these organelles per cubic micron of cellular volume reflects differences in the two cells’ individual capacity (and/or readiness) to respond to secretagogue stimulation, reflected by an apparent inverse relationship between the number/size of insulin secretory granules versus the number/size of mitochondria and the Golgi ribbon. We discuss the advantages of this approach for quantitative cellular ET of mammalian cells, briefly discuss its application relevant to other complementary techniques, and summarize future strategies for overcoming some of its current limitations.     

A near atomic resolution structure of a Melanocarpus albomyces laccase

It is becoming routine for cryoEM single particle reconstructions to result in 3D electron density maps with resolutions of 10 Å, but maps with resolutions of 5 Å or better are still celebrated events. The electron microscope has a resolving power to better than 2 Å, and thus should not be a limiting factor; instead the practical limitations in resolution most likely arise from a combination of specimen preparation methods, data collection parameters, and data analysis procedures. With the aid of a highly automated system for acquiring images, coupled to a relational database to keep track of all processing parameters, we have taken a systematic approach to optimizing parameters affecting the resolution of single particle reconstructions. Using GroEL as a test-bed, we performed a series of 3D reconstructions where we systematically varied the number of particles used in computing the map, the accelerating voltage of the microscope, and the electron dose used to acquire the images. We also investigated methods for excluding unacceptable or “bad” particles from contributing to the final 3D map. Using relatively standard instrumentation (Tecnai F20, 4K × 4K CCD, side entry cold stage) and a completely automated approach, these approaches resulted in a map with a nominal resolution of 5.4 Å (FSC_0.5) in which secondary structure is clearly discernable and the handedness of some of the α-helices in the GroEL structure can be determined.     

Conditional chromosome splitting in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> using the homing endonuclease PI-SceI

A novel chromosome engineering technology is described which enables conditional splitting of natural chromosomes in haploid cells of the yeast Saccharomyces cerevisiae. The technology consists of introduction of a recognition sequence for the homing endonuclease PI-SceI into the S. cerevisiae genome and conditional expression of the gene encoding the PI-SceI enzyme under the control of the MET3 promoter. To test the technology, we split chromosome V upstream of GLC7 by use of the autonomously replicating sequence (ARS)-added polymerase-chain-reaction-mediated chromosome-splitting (ARS-PCS) method that we recently developed. A recognition sequence for PI-SceI was subsequently introduced downstream of the GLC7 locus. Splitting was analyzed following induction of the PI-SceI-encoding gene. Approximately 50% of the clones tested had the expected minichromosome harboring only the GLC7 gene, suggesting that any desired chromosomal region may be converted into a new chromosome by use of this method. Because this technology allows initial construction of a strain harboring multiple constructs prior to subsequent induction of random chromosome loss events under specific selective conditions, we propose that this technology may be applicable to reconstructing the S. cerevisiae genome by means of combinatorial loss of minichromosomes.     

Rates and patterns in the evolution of snake-like body form in squamate reptiles: evidence for repeated re-evolution of lost digits and long-term persistence of intermediate body forms

An important challenge in evolutionary biology is to understand how major changes in body form arise. The dramatic transition from a lizard-like to snake-like body form in squamate reptiles offers an exciting system for such research because this change is replicated dozens of times. Here, we use morphometric data for 258 species and a time-calibrated phylogeny to explore rates and patterns of body-form evolution across squamates. We also demonstrate how time-calibrated phylogenies may be used to make inferences about the time frame over which major morphological transitions occur. Using the morphometric data, we find that the transition from lizard-like to snake-like body form involves concerted evolution of limb reduction, digit loss, and body elongation. These correlations are similar across squamate clades, despite very different ecologies and >180 million years (My) of divergence. Using the time-calibrated phylogeny and ancestral reconstructions, we find that the dramatic transition between these body forms can occur in 20 My or less, but that seemingly intermediate morphologies can also persist for tens of millions of years. Finally, although loss of digits is common, we find statistically significant support for at least six examples of the re-evolution of lost digits in the forelimb and hind limb.     

Combining Harvest and Genetics to Estimate Reproduction in Wolves

Parameters of reproductive success are important to the management of wildlife populations. Genetic monitoring can be an effective approach for acquiring this important demographic information when traditional methods are unsuccessful, inefficient, or too expensive. This study demonstrates a novel application of genetic data opportunistically collected from harvested game to estimate a minimum annual count of breeding packs of gray wolves (Canis lupus) and to provide a coarse index of harvest vulnerability of young of the year (YOY) across packs. We used 18 microsatellite loci to genotype 98 gray wolf YOY from 2014 and 105 from 2015 harvested in Idaho, USA. Using this genotype data, we reconstructed sibling groups for each cohort using the program COLONY and treated full-sibling litters as proxies for unique packs. In addition to evaluating our marker panel using simulations, we assessed the accuracy of empirical relationship assignments by adding YOY of known relationship from long-term study packs to the dataset (27 individuals from 2014 and 61 from 2015) and tracking correctly reconstructed relationships. We varied COLONY input parameters to evaluate the power of relationship assignments under conditions that may be encountered when working with empirical data. We also compared COLONY's estimates of effective number of breeders based on sibship frequency to estimates based on a commonly used linkage-disequilibrium method. All COLONY runs for both cohorts correctly identified the known sibling relationships. Among the other individuals, changes in the geographic clustering of putative siblings, probabilities of inclusion and exclusion for reconstructed sibling groups, and consistency of relationship assignments across COLONY runs suggested that marker number had a larger effect on accuracy than access to population-level genetic data. Our estimates of breeding packs subjected to harvest within the state (52 for 2014 and 63 for 2015) differed from estimates reported by Idaho Department of Fish and Game by ≤6 for both years. Among packs that had pups harvested, most packs had 1–2 YOY harvested, whereas other packs had as many as 5 YOY harvested. All estimates of the number of effective breeders had overlapping confidence intervals regardless of method, though sibship frequency-based estimates had larger confidence intervals than estimates using the linkage disequilibrium method. Our study shows that sibling relationships can be accurately and reliably reconstructed from harvested gray wolves, and demonstrates a valuable new use of samples collected through harvest. © 2020 The Wildlife Society.     

Hydro-ecological analysis of the fen system Lieper Posse,eastern Germany

The Lieper Posse is a calcareous, ground water-fed fen system, situated in the end-moraine area of northeastern Germany. Nowadays the fen is partly drained and the vegetation adversely affected; hence regeneration measures are considered. Here we report a pilot survey of the actual vegetation cover in relation to the hydrological conditions of the system. The vegetation in the central part of the fen system can be assigned to the Caricetum lasiocarpae, with small areas of Eleocharitetum quinqueflorae. The southern part of the system includes a Ledo-Pinetum sylvestris bog. Along the edges eutrophic forest types are present. Peat analysis revealed that the system started as an open lake and subsequently changed into a rheophilous mire, ‘percolating mire’. The southern bog has only recently come into being. Hydrological investigations showed that the original mineral-rich ground water had to a large extent been replaced by acid rainwater; this is likely due to the construction of a ditch before 1850. The effects of this drainage on hydrological conditions and some aspects of soil fertility are discussed. Possible consequences for the restoration of calciphilous vegetation types are being considered. It is concluded that the vegetation development is predictable only to a limited extent, which is mainly due to a lack of knowledge on hysteresis effects in both vegetation and soil.     

CT reconstruction of the Glabellar bone

During systematic archaelogical excavations of the early medieval kingdom of Serbia, and adult male skull belonging to the skeleton of a Roman individual was unearthed at Babrež site in the Ras region. A supernumerary bone was found interposed between the frontal bones of the skull and the facial skeleton. Paleopathological investigation and CT reconstruction proved that this skull had been deformed during foetal development due to a disorder or disease affecting the mother. This probably led to the formation of the supernumerary Glabellar bone in the frontal region. This is the first case of Os Glabellae recorded so far.