Closing gaps in the human genome using sequencing by synthesis

The most recent release of the finished human genome contains 260 euchromatic gaps (excluding chromosome Y). Recent work has helped explain a large number of these unresolved regions as 'structural' in nature. Another class of gaps is likely to be refractory to clone-based approaches, and cannot be approached in ways previously described. We present an approach for closing these gaps using 454 sequencing. As a proof of principle, we closed all three remaining non-structural gaps in chromosome 15.     

Rational and computational design of stabilized variants of cyanovirin-N that retain affinity and specificity for glycan ligands

Cyanovirin-N (CVN) is an 11 kDa pseudosymmetric cyanobacterial lectin that has been shown to inhibit infection by the human immunodeficiency virus by binding to high-mannose oligosaccharides on the surface of the viral envelope glycoprotein gp120. In this work, we describe rationally designed CVN variants that stabilize the protein fold while maintaining high affinity and selectivity for their glycan targets. Poisson-Boltzmann calculations and protein repacking algorithms were used to select stabilizing mutations in the protein core. By substituting the buried polar side chains of Ser11, Ser20, and Thr61 with aliphatic groups, we stabilized CVN by nearly 12 °C against thermal denaturation, and by 1 M GuaHCl against chemical denaturation, relative to a previously characterized stabilized mutant. Glycan microarray binding experiments confirmed that the specificity profile of carbohydrate binding is unperturbed by the mutations and is identical for all variants. In particular, the variants selectively bound glycans containing the Manα(1→2)Man linkage, which is the known minimal binding unit of CVN. We also report the slow denaturation kinetics of CVN and show that they can complicate thermodynamic analysis; in particular, the unfolding of CVN cannot be described as a fixed two-state transition. Accurate thermodynamic parameters are needed to describe the complicated free energy landscape of CVN, and we provide updated values for CVN unfolding.     

Effect of vascular cannulation on serum concentrations of LH, FSH, prolactin and cortisol in prepubertal gilts

Two experiments were conducted to determine whether cannulation of the jugular vein in gilts alters serum concentrations of LH, FSH, prolactin (PRL) or cortisol (C). In Experiment 1, 12 crossbred prepubertal gilts weighing 95 +/- 1.3 kg were immobilized by snaring, and tygon tubing was threaded into the anterior vena cava through a 12-gauge needle inserted into the jugular vein. Five hours later, blood samples were drawn at 20-min intervals for 4 h (Day 0). Samples were also drawn at 20-min intervals for 4-h periods 24 h (Day 1) and 48 h (Day 2) after cannulation. Serum concentrations of LH were similar (P=0.26) among Day 0 (0.40 ng/ml), Day 1 (0.39 ng/ml) and Day 2 (0.34 ng/ml). Serum PRL was similar (P=0.07) among Day 0 (4.10 ng/ml), Day 1 (3.87 ng/ml) and Day 2 (3.43 ng/ml). Serum concentrations of C were greater (P < 0.001) on Day 0 (8.32 ng/ml) than Day 1 (4.48 ng/ml) or Day 2 (3.54 ng/ml). In Experiment 2, cannulas were placed in 29 prepubertal gilts. Two days after initial cannulation, six blood samples were drawn at 20-min intervals. Gilts were then immobilized by snaring, and a second cannulae was inserted into the contralateral vein. Five blood samples were taken at 2-min intervals during the second cannulation and then six samples were drawn at 20-min intervals. Serum LH and FSH were not altered by cannulation or elevated during the subsequent 2-h sampling period (P>0.05). In contrast, serum concentrations of PRL rose slowly (P<0.05) during cannulation and remained elevated for 60 min before returning to baseline. Serum concentrations of C rose within 6 min of cannulation, remained elevated for 30 min, and then declined over the next 90 min. From these two experiments, it appears that secretory patterns of LH and FSH can be accurately assessed immediately after cannulation in prepubertal gilts. Measurements of serum PRL and C that reflect nonstressed conditions, however, cannot be obtained until at least 2 h or 1 d after cannulation, respectively.     

Demographic mechanism of a historical bird population collapse reconstructed using museum specimens

Long-term studies of demographic rates provide clues about the external causes of animal population declines, but systematic monitoring is rarely in place until after the decline has occurred. This study evaluates alternative hypotheses about the demographic mechanisms underlying the historical collapse of corncrake (Crexcrex) populations in Britain and Ireland in the late nineteenth and early twentieth centuries using characteristics of museum specimens. The proportion of adult corncrakes that are 1-year old was estimated from feather characteristics of birds collected before, during and after the population decline and showed a marked transitory reduction during the decline. This pattern would be expected if the decline was caused by a large reduction in the recruitment of young birds to the breeding population and is the opposite of what would be expected if a change in adult survival had caused the decline. These results are consistent with previous suggestions that the corncrake population decline was caused by adverse effects on breeding productivity caused by the mechanization of the harvesting of hay crops.     

Effects of seed ingestion and herbivory by waterfowl on seedling establishment: a field experiment with wigeongrass Ruppia maritima in Doñana,south-west Spain

The ingestion of seeds by vertebrates usually affects the viability and/or germination rate of seeds. Increases in germination rate following passage through the vertebrate gut have often been assumed to be favourable for seedling survival and plant fitness, but this assumption has never been tested experimentally. Given that numbers of herbivorous waterfowl are higher in winter in Mediterranean wetlands, herbivory pressure there will be higher for early growing plants. In a factorial experiment we investigated the effects of seed ingestion by ducks (shoveler, Anas clypeata) on the survival of wigeongrass Ruppia maritima seedlings in the field in Doñana (south-west Spain), under differing exposures to herbivory by waterfowl and fish. We planted ingested and non-ingested seeds in December, using exclosures to protect half of them from herbivores. When they were protected inside exclosures, there was no difference between ingested and non-ingested seeds in the number of plants that survived until June-July. However, fewer plants survived from ingested seeds when exposed to natural levels of herbivory because they were exposed for longer than plants germinating from non-ingested seeds. In conclusion, increases in germination rate after ingestion are not necessarily beneficial for the plant, and the final outcome depends on complex interactions with other factors such as herbivore abundance.     

Use of tritiated 3-O-methyl-d-glucose for studies of membrane transport caveat

Tritiated 3-O-methyl-d-glucose has many useful attributes as a model substance for studies of the transport of glucose across cell membranes. However, preparations of high specific radioactivity can decompose within a few months, producing radioactive impurities that can cause a several-fold increase in the apparent rate of sugar transport. In our investigation radioactive contaminants entered frog skeletal muscle cells by free diffusion rather than by facilitated transport. Much of the contaminating radioactive material could be removed by evaporating the solvent and redissolving the sugar. Tritiated sugar samples that had a specific activity below 0.1 Ci/mmol remained stable and suitable for transport measurements after several years of storage at -20°C. In order to evaluate the suitability of a given tritiated preparation of sugar for transport measurements, it is recommended that its behavior be compared with that of a stable reference standard of low specific activity.