Using atomic force microscopy to study chromatin structure and nucleosome remodeling

Atomic force microscopy (AFM) is a technique that can directly image single molecules in solution and it therefore provides a powerful tool for obtaining unique insights into the basic properties of biological materials and the functional processes in which they are involved. We have used AFM to analyze basic features of nucleosomes in arrays, such as DNA-histone binding strength, cooperativity in template occupation, nucleosome stabilities, nucleosome locations and the effects of acetylation, to compare these features in different types of arrays and to track the response of array nucleosomes to the action of the human Swi-Snf ATP-dependent nucleosome remodeling complex. These experiments required several specific adaptations of basic AFM methods, such as repetitive imaging of the same fields of molecules in liquid, the ability to change the environmental conditions of the sample being imaged and detection of specific types of molecules within compositionally complex samples. Here, we describe the techniques that allowed such analyses to be carried out.     

Deer do not affect short‐term rates of vegetation recovery in overwash fans on Fire Island after Hurricane Sandy

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A Long-Acting PYY3–36 Analog Mediates Robust Anorectic Efficacy with Minimal Emesis in Nonhuman Primates

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Bacterial Nonhomologous End Joining Requires Teamwork

All living organisms must repair DNA double-stranded breaks (DSBs) in order to survive. Many bacteria rely on nonhomologous end joining (NHEJ) when only a single copy of the genome is available and maintain NHEJ pathways with a minimum of two proteins. In this issue, Bhattarai and colleagues identify additional factors that can work together to aid in survival of stationary-phase cells with chromosomal breaks.     

Age-Related Shifts in the Density and Distribution of Genetic Marker Water Quality Indicators in Cow and Calf Feces

Calves make up about 16% of the current bovine population in the United States and can excrete high levels of human pathogens in their feces. We describe the density and distribution of genetic markers from 9 PCR- and real-time quantitative PCR-based assays, including CF128, CF193, CowM2, CowM3, GenBac3, Entero1, EC23S857, CampF2, and ttr-6, commonly used to help assess ambient surface water quality. Each assay was tested against a collection of 381 individual bovine fecal samples representing 31 mother and calf pairings collected over a 10-month time period from time of birth through weaning. Genetic markers reported to be associated with ruminant and/or bovine fecal pollution were virtually undetected in calves for up to 115 days from birth, suggesting that physiological changes in calf ruminant function impact host-associated genetic marker shedding. In addition, general fecal indicator markers for Bacteroidales, Escherichia coli, and Enterococcus spp. exhibited three separate trends across time, indicating that these bacteria respond differently to age-related physiological and dietary changes during calf development. The results of this study suggest that currently available PCR-based water quality indicator technologies can under- or overestimate fecal pollution originating from calves and identify a need for novel calf-associated source identification methods.     

Exploring the genetic architecture of alcohol dependence in African-Americans via analysis of a genomewide set of common variants

Alcohol dependence (AD) is a complex psychiatric disorder that affects about 12.5 % of US adults. Genetic factors play a major role in the development of AD. We conducted a genomewide association study in 2,875 African-Americans including 1,719 AD cases and 1,156 controls. We used the Illumina Omni 1-Quad microarray, which yielded 769,498 single-nucleotide polymorphisms (SNPs) after quality control. To explore the genetic architecture of AD, we estimated the variance that could be explained by all SNPs and subsets of SNPs using two different approaches to genome partitioning. We found that 23.9 % (s.e. 9.3 %) of the phenotypic variance could be explained by using all of the common SNPs on the array. We also found a significant linear relationship between the proportion of the top SNPs used and the phenotypic variance explained by them. Based on genome partitioning of common variants, we also observed a significant linear relationship between the variance explained by a chromosome and its length. Chromosome 4, known to contain several AD risk genes, accounted for excess risk in proportion to its length. By functional partitioning, we found that the genetic variants within 20 kb of genes explained 17.5 % (s.e. 11.4 %) of the phenotypic variance. Our findings are consistent with the generally accepted view that AD is a highly polygenic trait, i.e., the genetic risk in AD appears to be conferred by multiple variants, each of which may have a small or moderate effect.     

Epizootic Pneumonia of Bighorn Sheep following Experimental Exposure to Mycoplasma ovipneumoniae

Background

Bronchopneumonia is a population limiting disease of bighorn sheep (Ovis canadensis). The cause of this disease has been a subject of debate. Leukotoxin expressing Mannheimia haemolytica and Bibersteinia trehalosi produce acute pneumonia after experimental challenge but are infrequently isolated from animals in natural outbreaks. Mycoplasma ovipneumoniae, epidemiologically implicated in naturally occurring outbreaks, has received little experimental evaluation as a primary agent of bighorn sheep pneumonia.

Methodology/Principal Findings

In two experiments, bighorn sheep housed in multiple pens 7.6 to 12 m apart were exposed to M. ovipneumoniae by introduction of a single infected or challenged animal to a single pen. Respiratory disease was monitored by observation of clinical signs and confirmed by necropsy. Bacterial involvement in the pneumonic lungs was evaluated by conventional aerobic bacteriology and by culture-independent methods. In both experiments the challenge strain of M. ovipneumoniae was transmitted to all animals both within and between pens and all infected bighorn sheep developed bronchopneumonia. In six bighorn sheep in which the disease was allowed to run its course, three died with bronchopneumonia 34, 65, and 109 days after M. ovipneumoniae introduction. Diverse bacterial populations, predominantly including multiple obligate anaerobic species, were present in pneumonic lung tissues at necropsy.

Conclusions/Significance

Exposure to a single M. ovipneumoniae infected animal resulted in transmission of infection to all bighorn sheep both within the pen and in adjacent pens, and all infected sheep developed bronchopneumonia. The epidemiologic, pathologic and microbiologic findings in these experimental animals resembled those seen in naturally occurring pneumonia outbreaks in free ranging bighorn sheep.     

The budding index of Saccharomyces cerevisiae deletion strains identifies genes important for cell cycle progression

Budding marks initiation of cell division in Saccharomyces cerevisiae. Consequently, cell cycle progression can be monitored by the fraction of budded cells (budding index) in a proliferating cell population. We determined the budding index of a large collection of deletion strains, to systematically identify genes involved in cell cycle progression.     

Phylogenetic analysis of cryptic speciation in the polychaete Pygospio elegans

Development in marine invertebrate species can take place through a variety of modes and larval forms, but within a species, developmental mode is typically uniform. Poecilogony refers to the presence of more than one mode of development within a single species. True poecilogony is rare, however, and in some cases, apparent poecilogony is actually the result of variation in development mode among recently diverged cryptic species. We used a phylogenetic approach to examine whether poecilogony in the marine polychaete worm, Pygospio elegans, is the result of cryptic speciation. Populations of worms identified as P. elegansooded, and intermediate larvae; these modes are found both within and among populations. We examined sequence variation among partial mitochondrial cytochrome c oxidase subunit I sequences obtained for 279 individual worms sampled across broad geographic and environmental scales. Despite a large number of unique haplotypes (121 haplotypes from 279 individuals), sequence divergence among European samples was low (1.7%) with most of the sequence variation observed within populations, relative to the variation among regions. More importantly, we observed common haplotypes that were widespread among the populations we sampled, and the two most common haplotypes were shared between populations differing in developmental mode. Thus, our results support an earlier conclusion of poecilogony in P elegans. In addition, predominantly planktonic populations had a larger number of population-specific low-frequency haplotypes. This finding is largely consistent with interspecies comparisons showing high diversity for species with planktonic developmental modes in contrast to low diversity in species with brooded developmental modes.