SNP analysis to dissect human traits

The analysis of complex human diseases has been spurred by the number of published genomic sequence variants - many identified in the course of sequencing the human genome. But, to be useful for genetic analysis, variants have to be mapped accurately, their frequencies in various populations determined, and automated high-throughput assay techniques developed. Recently proposed methods address these issues: the use of 'reduced representation shotgun' methods for more efficient detection of single nucleotide polymorphisms (SNPs), the employment of high-throughput genotyping techniques, the development of SNP maps that incorporate information about linkage disequilibrium, and the use of SNPs in identifying susceptibility genes for common illnesses.     

How to dissect the Node of Ranvier

Voltage-gated sodium channels are unique in that they combine action potential conduction with cell adhesion. Mammalian sodium channels are heterotrimers, composed of a central, pore-forming α subunit and two auxiliary β subunits. The α subunits are members of a large gene family containing the voltage-gated sodium, potassium, and calcium channels. Sodium channel α subunits form a gene subfamily with at least 11 members. Mutations in sodium channel α subunit genes have been linked to paroxysmal disorders such as epilepsy, long QT syndrome (LQT), and hyperkalemic periodic paralysis in humans, and motor endplate disease and cerebellar ataxia in mice. Three genes encode the sodium channel β subunits with at least one alternative splice product. Unlike the pore-forming α subunits, the sodium channel β subunits are not structurally related to β subunits of calcium and potassium channels. Sodium channel β subunits are multifunctional. They modulate channel gating and regulate the level of channel expression at the plasma membrane. We have shown that β subunits also function as cell adhesion molecules (CAMs) in terms of interaction with extracellular matrix molecules, regulation of cell migration, cellular aggregation, and interaction with the cytoskeleton. A mutation in SCN1B has been shown to cause GEFS + 1 epilepsy in human families. We propose that the sodium channel signalling complex at nodes of Ranvier involves β subunits as channel modulators as well as CAMs, other CAMs such as neurofascin and contactin, RPTPβ, and extracellular matrix molecules such as tenascin.     

Mining the HapMap to dissect complex traits

A report on the Keystone Symposium 'Genome Sequence Variation and the Inherited Basis of Common Disease and Complex Traits', Big Sky, USA, 8-13 January 2006.     

Genetic approaches to dissect plant nonhost resistance mechanisms

Nonhost resistance (NHR) refers to the immunity of most tested genotypes of a plant species to most tested variants of a pathogen species. Thus, NHR is broad spectrum and durable in nature and constitutes a major safety barrier against invasion of a myriad of potentially pathogenic microbes in any plants including domesticated crops. Genetic study of NHR is generally more difficult compared to host resistance mainly because NHR is genetically more complicated and often lacks intraspecific polymorphisms. Nevertheless, substantial progress has been made towards the understanding of the molecular basis of NHR in the past two decades using various approaches. Not surprisingly, molecular mechanisms of NHR revealed so far encompasses pathogen-associated molecular pattern-triggered immunity and effector-triggered immunity. In this review, we briefly discuss the inherent difficulty in genetic studies of NHR and summarize the main approaches that have been taken to identify genes contributing to NHR. We also discuss new enabling strategies for dissecting multilayered NHR in model plants with a focus on NHR against filamentous pathogens, especially biotrophic pathogens such as powdery mildew and rust fungi.     

A genetic approach to dissect sexually dimorphic behaviors

It has been known since antiquity that gender-specific behaviors are regulated by the gonads. We now know that testosterone is required for the appropriate display of male patterns of behavior. Estrogen and progesterone, on the other hand, are essential for female typical responses. Research from several groups also indicates that estrogen signaling is required for male typical behaviors. This finding raises the issue of the relative contribution of these two hormonal systems in the control of male typical behavioral displays. In this review we discuss the findings that led to these conclusions and suggest various genetic strategies that may be required to understand the relative roles of testosterone and estrogen signaling in the control of gender-specific behavior.     

Phosphoprotein analysis: from proteins to proteomes

Characterization of protein modification by phosphorylation is one of the major tasks that have to be accomplished in the post-genomic era. Phosphorylation is a key reversible modification occurring mainly on serine, threonine and tyrosine residues that can regulate enzymatic activity, subcellular localization, complex formation and degradation of proteins. The understanding of the regulatory role played by phosphorylation begins with the discovery and identification of phosphoproteins and then by determining how, where and when these phosphorylation events take place. Because phosphorylation is a dynamic process difficult to quantify, we must at first acquire an inventory of phosphoproteins and characterize their phosphorylation sites. Several experimental strategies can be used to explore the phosphorylation status of proteins from individual moieties to phosphoproteomes. In this review, we will examine and catalogue how proteomics techniques can be used to answer specific questions related to protein phosphorylation. Hence, we will discuss the different methods for enrichment of phospho-proteins and -peptides, and then the various technologies for their identification, quantitation and validation.     

Use of minigene systems to dissect alternative splicing elements

Pre-mRNA splicing is an essential step for gene expression in higher eukaryotes. The splicing efficiency of individual exons is determined by multiple features involving gene architecture, a variety of cis-acting elements within the exons and flanking introns, and interactions with components of the basal splicing machinery (called the spliceosome) and auxiliary regulatory factors which transiently co-assemble with the spliceosome. Both alternative and constitutive exons are recognized by multiple weak protein:RNA interactions and different exons differ in the interactions which are determinative for exon usage. Alternative exons are often regulated according to cell-specific patterns and regulation is mediated by specific sets of cis-acting elements and trans-acting factors. Transient expression of minigenes is a commonly used in vivo assay to identify the intrinsic features of a gene that control exon usage, identify specific cis-acting elements that control usage of constitutive and alternative exons, identify cis-acting elements that control cell-specific usage of alternative exons, and once regulatory elements have been identified, to identify the trans-acting factors that bind to these elements and modulate splicing. This chapter describes approaches and strategies for using minigenes to define the cis-acting elements that determine splice site usage and to identify and characterize the trans-acting factors that bind to these elements and regulate alternative splicing.     

Proteomic analysis of zygote and ookinete stages of the avian malaria parasite Plasmodium gallinaceum delineates the homologous proteomes of the lethal human malaria parasite Plasmodium falciparum

Delineation of the complement of proteins comprising the zygote and ookinete, the early developmental stages of Plasmodium within the mosquito midgut, is fundamental to understand initial molecular parasite-vector interactions. The published proteome of Plasmodium falciparum does not include analysis of the zygote/ookinete stages, nor does that of P. berghei include the zygote stage or secreted proteins. P. gallinaceum zygote, ookinete, and ookinete-secreted/released protein samples were prepared and subjected to Multidimensional protein identification technology (MudPIT). Peptides of P. gallinaceum zygote, ookinete, and ookinete-secreted proteins were identified by MS/MS, mapped to ORFs (> 50 amino acids) in the extent P. gallinaceum whole genome sequence, and then matched to homologous ORFs in P. falciparum. A total of 966 P. falciparum ORFs encoding orthologous proteins were identified; just over 40% of these predicted proteins were found to be hypothetical. A majority of putative proteins with predicted secretory signal peptides or transmembrane domains were hypothetical proteins. This analysis provides a more comprehensive view of the hitherto unknown proteome of the early mosquito midgut stages of P. falciparum. The results underpin more robust study of Plasmodium-mosquito midgut interactions, fundamental to the development of novel strategies of blocking malaria transmission.     

Chlamydia persistence -- a tool to dissect chlamydia--host interactions

Under stress, chlamydiae can enter a non-infectious but viable state termed persistence. In the absence of a tractable genetic system, persistence induction provides an important experimental tool with which to study these fascinating organisms. This review will discuss examples of: i) persistence studies that have illuminated critical chlamydiae/host interactions; and ii) novel persistence models that will do so in the future.     

Using mouse models to dissect the genetics of obesity

Mice have proved to be powerful models for understanding obesity in humans and farm animals. Single-gene mutants and genetically modified mice have been used successfully to discover genes and pathways that can regulate body weight. For polygenic obesity, the most common pattern of inheritance, many quantitative trait loci (QTLs) have been mapped in crosses between selected and inbred mouse lines. Most QTL effects are additive, and diet, age and gender modify the genetic effects. Congenic, recombinant inbred, advanced intercross, and chromosome substitution strains are needed to map QTLs finely, to identify the genes underlying the traits, and to examine interactions between them.     

Combining mechanical and optical approaches to dissect cellular mechanobiology

Mechanical force modulates a wide array of cell physiological processes. Cells sense and respond to mechanical stimuli using a hierarchy of structural complexes spanning multiple length scales, including force-sensitive molecules and cytoskeletal networks. Understanding mechanotransduction, i.e., the process by which cells convert mechanical inputs into biochemical signals, has required the development of novel biophysical tools that allow for probing of cellular and subcellular components at requisite time, length, and force scales and technologies that track the spatio-temporal dynamics of relevant biomolecules. In this review, we begin by discussing the underlying principles and recent applications of atomic force microscopy, magnetic twisting cytometry, and traction force microscopy, three tools that have been widely used for measuring the mechanical properties of cells and for probing the molecular basis of cellular mechanotransduction. We then discuss how such tools can be combined with advanced fluorescence methods for imaging biochemical processes in living cells in the context of three specific problem spaces. We first focus on fluorescence resonance energy transfer, which has enabled imaging of intra- and inter-molecular interactions and enzymatic activity in real time based on conformational changes in sensor molecules. Next, we examine the use of fluorescence methods to probe force-dependent dynamics of focal adhesion proteins. Finally, we discuss the use of calcium ratiometric signaling to track fast mechanotransductive signaling dynamics. Together, these studies demonstrate how single-cell biomechanical tools can be effectively combined with molecular imaging technologies for elucidating mechanotransduction processes and identifying mechanosensitive proteins.     

Using quantitative redox proteomics to dissect the yeast redoxome

To understand and eventually predict the effects of changing redox conditions and oxidant levels on the physiology of an organism, it is essential to gain knowledge about its redoxome: the proteins whose activities are controlled by the oxidation status of their cysteine thiols. Here, we applied the quantitative redox proteomic method OxICAT to Saccharomyces cerevisiae and determined the in vivo thiol oxidation status of almost 300 different yeast proteins distributed among various cellular compartments. We found that a substantial number of cytosolic and mitochondrial proteins are partially oxidized during exponential growth. Our results suggest that prevailing redox conditions constantly control central cellular pathways by fine-tuning oxidation status and hence activity of these proteins. Treatment with sublethal H(2)O(2) concentrations caused a subset of 41 proteins to undergo substantial thiol modifications, thereby affecting a variety of different cellular pathways, many of which are directly or indirectly involved in increasing oxidative stress resistance. Classification of the identified protein thiols according to their steady-state oxidation levels and sensitivity to peroxide treatment revealed that redox sensitivity of protein thiols does not predict peroxide sensitivity. Our studies provide experimental evidence that the ability of protein thiols to react to changing peroxide levels is likely governed by both thermodynamic and kinetic parameters, making predicting thiol modifications challenging and de novo identification of peroxide sensitive protein thiols indispensable.     

Meet the neighbours: tools to dissect nuclear structure and function

The eukaryotic cell nucleus displays a high degree of spatial organization, with discrete functional subcompartments that provide microenvironments where specialized processes take place. Concordantly, the genome also adopts defined conformations that, in part, enable specific genomic regions to interface with these functional centers. Yet the roles of many subcompartments and the genomic regions that contact them have not been explored fully. More fundamentally, it is not entirely clear how genome organization impacts function, and vice versa. The past decade has witnessed the development of a new breed of methods that are capable of assessing the spatial organization of the genome. These stand to further our understanding of the relationship between genome structure and function, and potentially assign function to various nuclear subcompartments. Here, we review the principal techniques used for analyzing genomic interactions, the functional insights they have afforded and discuss the outlook for future advances in nuclear structure and function dynamics.