Computer Modeling of the Solution Conformation of Cyclic Enkephalins

Summary The probable conformations of two cyclic enkephalin analogs, DNS-cyclo[d-Dab-Gly-Trp-Leu] (I) and DNS-cyclo[d-Dab-Gly-Trp-d-Leu] (II) (DNS=dansyl), were determined by combining the results of NOE, vicinal coupling constant and fluorescence energy transfer measurements with theoretical calculations. The common feature of the conformations for both peptides is the presence of a β-turn at residues 2 and 3.     

A universal TagModule collection for parallel genetic analysis of microorganisms

Systems-level analyses of non-model microorganisms are limited by the existence of numerous uncharacterized genes and a corresponding over-reliance on automated computational annotations. One solution to this challenge is to disrupt gene function using DNA tag technology, which has been highly successful in parallelizing reverse genetics in Saccharomyces cerevisiae and has led to discoveries in gene function, genetic interactions and drug mechanism of action. To extend the yeast DNA tag methodology to a wide variety of microorganisms and applications, we have created a universal, sequence-verified TagModule collection. A hallmark of the 4280 TagModules is that they are cloned into a Gateway entry vector, thus facilitating rapid transfer to any compatible genetic system. Here, we describe the application of the TagModules to rapidly generate tagged mutants by transposon mutagenesis in the metal-reducing bacterium Shewanella oneidensis MR-1 and the pathogenic yeast Candida albicans. Our results demonstrate the optimal hybridization properties of the TagModule collection, the flexibility in applying the strategy to diverse microorganisms and the biological insights that can be gained from fitness profiling tagged mutant collections. The publicly available TagModule collection is a platform-independent resource for the functional genomics of a wide range of microbial systems in the post-genome era.     

A Simple Genetic Architecture Underlies Morphological Variation in Dogs

Domestic dogs exhibit tremendous phenotypic diversity, including a greater variation in body size than any other terrestrial mammal. Here, we generate a high density map of canine genetic variation by genotyping 915 dogs from 80 domestic dog breeds, 83 wild canids, and 10 outbred African shelter dogs across 60,968 single-nucleotide polymorphisms (SNPs). Coupling this genomic resource with external measurements from breed standards and individuals as well as skeletal measurements from museum specimens, we identify 51 regions of the dog genome associated with phenotypic variation among breeds in 57 traits. The complex traits include average breed body size and external body dimensions and cranial, dental, and long bone shape and size with and without allometric scaling. In contrast to the results from association mapping of quantitative traits in humans and domesticated plants, we find that across dog breeds, a small number of quantitative trait loci (≤3) explain the majority of phenotypic variation for most of the traits we studied. In addition, many genomic regions show signatures of recent selection, with most of the highly differentiated regions being associated with breed-defining traits such as body size, coat characteristics, and ear floppiness. Our results demonstrate the efficacy of mapping multiple traits in the domestic dog using a database of genotyped individuals and highlight the important role human-directed selection has played in altering the genetic architecture of key traits in this important species.     

Development of a Novel Aluminum Tolerance Phenotyping Platform Used for Comparisons of Cereal Aluminum Tolerance and Investigations into Rice Aluminum Tolerance Mechanisms

The genetic and physiological mechanisms of aluminum (Al) tolerance have been well studied in certain cereal crops, and Al tolerance genes have been identified in sorghum (Sorghum bicolor) and wheat (Triticum aestivum). Rice (Oryza sativa) has been reported to be highly Al tolerant; however, a direct comparison of rice and other cereals has not been reported, and the mechanisms of rice Al tolerance are poorly understood. To facilitate Al tolerance phenotyping in rice, a high-throughput imaging system and root quantification computer program was developed, permitting quantification of the entire root system, rather than just the longest root. Additionally, a novel hydroponic solution was developed and optimized for Al tolerance screening in rice and compared with the Yoshida''s rice solution commonly used for rice Al tolerance studies. To gain a better understanding of Al tolerance in cereals, comparisons of Al tolerance across cereal species were conducted at four Al concentrations using seven to nine genetically diverse genotypes of wheat, maize (Zea mays), sorghum, and rice. Rice was significantly more tolerant than maize, wheat, and sorghum at all Al concentrations, with the mean Al tolerance level for rice found to be 2- to 6-fold greater than that in maize, wheat, and sorghum. Physiological experiments were conducted on a genetically diverse panel of more than 20 rice genotypes spanning the range of rice Al tolerance and compared with two maize genotypes to determine if rice utilizes the well-described Al tolerance mechanism of root tip Al exclusion mediated by organic acid exudation. These results clearly demonstrate that the extremely high levels of rice Al tolerance are mediated by a novel mechanism, which is independent of root tip Al exclusion.Aluminum (Al) is the most abundant metal in the earth''s crust, constituting approximately 7% of the soil (Wolt, 1994). Al is predominately found as a key component of soil clays; however, under highly acidic soil conditions (pH < 5.0), Al³⁺ is solubilized into the soil solution and is highly phytotoxic. Al³⁺ causes a rapid inhibition of root growth that leads to a reduced and stunted root system, thus having a direct effect on the ability of a plant to acquire both water and nutrients. Approximately 30% of the world''s total land area and over 50% of potentially arable lands are acidic, with the majority (60%) found in the tropics and subtropics (von Uexkull and Mutert, 1995). Thus, acidic soils are a major limitation to crop production, particularly in the developing world.As a whole, cereal crops (Poaceae) provide an excellent model for studying Al tolerance because of their abundant genetic resources, large, active research communities, and importance to agriculture. In addition, work in one cereal species can rapidly translate into impact throughout the family. Previous research has focused on understanding the genetic and physiological mechanisms of Al tolerance in maize (Zea mays), sorghum (Sorghum bicolor), and wheat (Triticum aestivum). The most recognized physiological mechanism conferring Al tolerance in plants involves exclusion of Al from the root tip (Miyasaka et al., 1991; Delhaize and Ryan, 1995; Kochian, 1995; Kochian et al., 2004a, 2004b). The exclusion mechanism is primarily mediated by Al-activated exudation of organic acids such as malate, citrate, or oxalate from the root apex, the site of Al toxicity (Ryan et al., 1993, 2001; Ma et al., 2001). These organic acids chelate Al in the rhizosphere, reducing the concentration and toxicity of Al at the growing root tip (Ma et al., 2001). Phosphate has also been identified as a class of root exudates involved in cation chelation and therefore can be considered a potential exudate involved in Al exclusion from the root tip (Pellet et al., 1996).Al-activated malate and citrate anion efflux transporters have been cloned from wheat (ALMT1; Sasaki et al., 2004) and sorghum (SbMATE; Magalhaes et al., 2007), and root citrate efflux transporters have been implicated in Al tolerance in maize (Piñeros and Kochian, 2001; Zhang et al., 2001). Recently, a maize homolog of sorghum SbMATE was shown to be the root citrate efflux transporter that plays a role in maize Al tolerance (Maron et al., 2010). Although organic acids have been shown to play a major role in Al tolerance in these species, another exclusion mechanism has been identified in an Arabidopsis (Arabidopsis thaliana) mutant, where a root-mediated increase in rhizosphere pH lowers the Al³⁺ activity and thus participates in Al exclusion from the root apex (Degenhardt et al., 1998). Furthermore, there is clear evidence that tolerance in maize cannot be fully explained by organic acid release (Piñeros et al., 2005). These types of findings strongly suggest that multiple Al tolerance mechanisms exist in plants.Rice (Oryza sativa) has been reported to be the most Al-tolerant cereal crop under field conditions, capable of withstanding significantly higher concentrations of Al than other major cereals (Foy, 1988). Despite this fact, very little is known about the physiological mechanisms of Al tolerance in rice. Two independent studies have identified increased Al accumulation in the root apex in susceptible compared with Al-tolerant rice varieties, but no differences were observed in organic acid exudation or rhizosphere pH (Ma et al., 2002; Yang et al., 2008). These studies suggest that rice may contain novel physiological and/or genetic mechanisms that confer significantly higher levels of Al tolerance than those found in other cereals. A more thorough analysis is required to clarify the mechanism of Al tolerance in rice.Cultivated rice is characterized by deep genetic divergence between the two major varietal groups: Indica and Japonica (Dally and Second, 1990; Garris et al., 2005; Hu et al., 2006; Londo et al., 2006). Extensive selection pressure over the last 10,000 years has resulted in the formation of five genetically distinct subpopulations: indica and aus within the Indica varietal group, and temperate japonica, tropical japonica, and aromatic/groupV within the Japonica varietal group (Garris et al., 2005; Caicedo et al., 2007; K. Zhao and S. McCouch, personal communication). (Note: When referring to varietal groups, the first letter will be capitalized, while lowercase letters will be used to refer to the subpopulation groups.) Subpopulation differences in trait performance are often significant, particularly with respect to biotic and abiotic stress (Champoux et al., 1995; Lilley et al., 1996; Garris et al. 2003; Xu et al., 2009). This can lead to confusion because trait or performance differences may be confounded with subpopulation structure, leading to false positives (type 1 error; Devlin and Roeder, 1999; Pritchard and Donnelly, 2001; Yu et al., 2006; Zhao et al., 2007). Therefore, it is important to consider the subpopulation origin of genotypes being compared when studying the genetics and physiology of Al tolerance in rice.Al tolerance screening is typically conducted by comparing root growth of seedlings grown in hydroponic solutions, with and without Al (Piñeros and Kochian, 2001; Magalhaes et al., 2004; Sasaki et al., 2004). Sorghum and maize are often screened for Al tolerance in Magnavaca''s nutrient solution (Piñeros and Kochian, 2001; Magalhaes et al., 2004; Piñeros et al., 2005), while rice seedlings are typically grown in Yoshida''s solution (Yoshida et al., 1976). Furthermore, Al concentrations used to screen for Al tolerance in maize (222 μm), sorghum (148 μm), and wheat (100 μm) are significantly lower than those used for screening Al tolerance in rice (1,112–1,482 μm; Wu et al., 2000; Nguyen et al., 2001, 2002, 2003). These differences in chemical composition of the nutrient solutions make it difficult to directly compare plant response to Al across these cereals. In rice, the high Al concentrations required to observe significant differences in root growth between susceptible and resistant varieties also complicate Al tolerance screening due to the precipitation of Al along with other elements. The result is that control (−Al) and treatment (+Al) solutions may differ with regard to essential mineral nutrients that react with Al, leading to differences in growth not directly attributable to Al. Additionally, because the active form of Al that is toxic to root growth is Al³⁺, any Al that precipitates out of solution has no effect on root growth (Kochian et al., 2004a). In a hydroponic solution, Al may be found in one of four forms: (1) as free Al³⁺, where it actively inhibits root growth; (2) precipitated with other elements and essentially unavailable to inhibit plant growth; (3) different hydroxyl monomers of Al, which are not believed to be toxic to roots (Parker et al., 1988); or (4) complexed with other elements in an equilibrium between its active and inactive states. The degree to which Al inhibits root growth is primarily dependent upon the activity of free Al³⁺ in solution (Kochian et al., 2004a).The objectives of this study were to (1) develop and optimize a suitable nutrient solution and high-throughput Al tolerance screening method for rice; (2) quantify and compare differences in Al tolerance between maize, sorghum, wheat, and rice; and (3) use the developed screening methods to determine if rice utilizes the organic acid-mediated Al exclusion mechanism that is observed in maize, sorghum, and wheat.     

Stimulation of mitochondrial reactive oxygen species production by unesterified,unsaturated fatty acids in defective human spermatozoa

Male infertility is a relatively common condition affecting 1 in 20 men of reproductive age. The etiology of this condition is thought to involve the excessive generation of reactive oxygen species by human spermatozoa; however, the cause of this aberrant activity is unknown. In this study we demonstrate that defective human sperm populations are characterized by high cellular contents of both esterified and unesterified fatty acids and a decrease in the proportion of the total fatty acid pool made up by docosahexaenoic acid. The free unsaturated fatty acid content of these cells was positively correlated with the induction of mitochondrial superoxide generation (P < 0.001). This relationship was causal and mediated by the range of unesterified, unsaturated fatty acids that are present in human spermatozoa. Thus direct exposure of these cells to free unsaturated fatty acids stimulated mitochondrial superoxide generation and precipitated a loss of motility and an increase in oxidative DNA damage, two key attributes of male infertility. We conclude that defective human spermatozoa are characterized by an abnormally high content of fatty acids that, in their unesterified, unsaturated form, promote ROS generation by sperm mitochondria, creating a state of oxidative stress and a concomitant loss of functional competence.