Non-homogeneous models of sequence evolution in the Bio++ suite of libraries and programs

Background  

Accurately modeling the sequence substitution process is required for the correct estimation of evolutionary parameters, be they phylogenetic relationships, substitution rates or ancestral states; it is also crucial to simulate realistic data sets. Such simulation procedures are needed to estimate the null-distribution of complex statistics, an approach referred to as parametric bootstrapping, and are also used to test the quality of phylogenetic reconstruction programs. It has often been observed that homologous sequences can vary widely in their nucleotide or amino-acid compositions, revealing that sequence evolution has changed importantly among lineages, and may therefore be most appropriately approached through non-homogeneous models. Several programs implementing such models have been developed, but they are limited in their possibilities: only a few particular models are available for likelihood optimization, and data sets cannot be easily generated using the resulting estimated parameters.     

Mutation detection using ENDO1: Application to disease diagnostics in humans and TILLING and Eco-TILLING in plants

Background  

Most enzymatic mutation detection methods are based on the cleavage of heteroduplex DNA by a mismatch-specific endonuclease at mismatch sites and the analysis of the digestion product on a DNA sequencer. Important limitations of these methods are the availability of a mismatch-specific endonuclease, their sensitivity in detecting one allele in pool of DNA, the cost of the analysis and the ease by which the technique could be implemented in a standard molecular biology laboratory.     

piggyBac-based mosaic screen identifies a postmitotic function for cohesin in regulating developmental axon pruning

Developmental axon pruning is widely used to refine neural circuits. We performed a mosaic screen to identify mutations affecting axon pruning of Drosophila mushroom body gamma neurons. We constructed a modified piggyBac vector with improved mutagenicity and generated insertions in >2000 genes. We identified two cohesin subunits (SMC1 and SA) as being essential for axon pruning. The cohesin complex maintains sister-chromatid cohesion during cell division in eukaryotes. However, we show that the pruning phenotype in SMC1(-/-) clones is rescued by expressing SMC1 in neurons, revealing a postmitotic function. SMC1(-/-) clones exhibit reduced levels of the ecdysone receptor EcR-B1, a key regulator of axon pruning. The pruning phenotype is significantly suppressed by overexpressing EcR-B1 and is enhanced by a reduced dose of EcR, supporting a causal relationship. We also demonstrate a postmitotic role for SMC1 in dendrite targeting of olfactory projection neurons. We suggest that cohesin regulates diverse aspects of neuronal morphogenesis.     

A generic approach for the purification of signaling complexes that specifically interact with the carboxyl-terminal domain of G protein-coupled receptors

G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors and are major drug targets. Recent progress has shown that GPCRs are part of large protein complexes that regulate their activity. We present here a generic approach for identification of these complexes that is based on the use of receptor subdomains and that overcomes the limitations of currently used genetics and proteomics approaches. Our approach consists of a carefully balanced combination of chemically synthesized His6-tagged baits, immobilized metal affinity chromatography, one- and two-dimensional gel electrophoresis separation and mass spectrometric identification. The carboxyl-terminal tails (C-tails) of the human MT1 and MT2 melatonin receptors, two class A GPCRs, were used as models to purify protein complexes from mouse brain lysates. We identified 32 proteins that interacted with the C-tail of MT1, 14 proteins that interacted with the C-tail of MT2, and eight proteins that interacted with both C-tails. Several randomly selected proteins were validated by Western blotting, and the functional relevance of our data was further confirmed by showing the interaction between the full-length MT1 and the regulator of G protein signaling Z1 in transfected HEK 293 cells and native tissue. Taken together, we have established an integrated and generic purification strategy for the identification of high quality and functionally relevant GPCR-associated protein complexes that significantly widens the repertoire of available techniques.     

Molecular signals in the trafficking of Toxoplasma gondii protein MIC3 to the micronemes

The protozoan parasite Toxoplasma gondii is equipped with a sophisticated secretory apparatus, including three distinct exocytic organelles, named micronemes, rhoptries, and dense granules. We have dissected the requirements for targeting the microneme protein MIC3, a key component of T. gondii infection. We have shown that MIC3 is processed in a post-Golgi compartment and that the MIC3 propeptide and epidermal growth factor (EGF) modules contain microneme-targeting information. The minimal requirement for microneme delivery is defined by the propeptide plus any one of the three EGF domains. We have demonstrated that the cleavage of the propeptide, the dimerization of MIC3, and the chitin binding-like sequence, which are crucial for host cell binding and virulence, are dispensable for proper targeting. Finally, we have shown that part of MIC3 is withheld in the secretory pathway in a cell cycle-dependent manner.     

A forward chemical genetic screen reveals an inhibitor of the Mre11-Rad50-Nbs1 complex

The MRN (Mre11-Rad50-Nbs1)-ATM (ataxia-telangiectasia mutated) pathway is essential for sensing and signaling from DNA double-strand breaks. The MRN complex acts as a DNA damage sensor, maintains genome stability during DNA replication, promotes homology-dependent DNA repair and activates ATM. MRN is essential for cell viability, which has limited functional studies of the complex. Small-molecule inhibitors of MRN could circumvent this experimental limitation and could also be used as cellular radio- and chemosensitization compounds. Using cell-free systems that recapitulate faithfully the MRN-ATM signaling pathway, we designed a forward chemical genetic screen to identify inhibitors of the pathway, and we isolated 6-(4-hydroxyphenyl)-2-thioxo-2,3-dihydro-4(1H)-pyrimidinone (mirin, 1) as an inhibitor of MRN. Mirin prevents MRN-dependent activation of ATM without affecting ATM protein kinase activity, and it inhibits Mre11-associated exonuclease activity. Consistent with its ability to target the MRN complex, mirin abolishes the G2/M checkpoint and homology-dependent repair in mammalian cells.     

Antibodies targeting the PfRH1 binding domain inhibit invasion of Plasmodium falciparum merozoites

Invasion by the malaria merozoite depends on recognition of specific erythrocyte surface receptors by parasite ligands. Plasmodium falciparum uses multiple ligands, including at least two gene families, reticulocyte binding protein homologues (RBLs) and erythrocyte binding proteins/ligands (EBLs). The combination of different RBLs and EBLs expressed in a merozoite defines the invasion pathway utilized and could also play a role in parasite virulence. The binding regions of EBLs lie in a conserved cysteine-rich domain while the binding domain of RBL is still not well characterized. Here, we identify the erythrocyte binding region of the P. falciparum reticulocyte binding protein homologue 1 (PfRH1) and show that antibodies raised against the functional binding region efficiently inhibit invasion. In addition, we directly demonstrate that changes in the expression of RBLs can constitute an immune evasion mechanism of the malaria merozoite.     

Genetic variation in an individual human exome

There is much interest in characterizing the variation in a human individual, because this may elucidate what contributes significantly to a person's phenotype, thereby enabling personalized genomics. We focus here on the variants in a person's 'exome,' which is the set of exons in a genome, because the exome is believed to harbor much of the functional variation. We provide an analysis of the approximately 12,500 variants that affect the protein coding portion of an individual's genome. We identified approximately 10,400 nonsynonymous single nucleotide polymorphisms (nsSNPs) in this individual, of which approximately 15-20% are rare in the human population. We predict approximately 1,500 nsSNPs affect protein function and these tend be heterozygous, rare, or novel. Of the approximately 700 coding indels, approximately half tend to have lengths that are a multiple of three, which causes insertions/deletions of amino acids in the corresponding protein, rather than introducing frameshifts. Coding indels also occur frequently at the termini of genes, so even if an indel causes a frameshift, an alternative start or stop site in the gene can still be used to make a functional protein. In summary, we reduced the set of approximately 12,500 nonsilent coding variants by approximately 8-fold to a set of variants that are most likely to have major effects on their proteins' functions. This is our first glimpse of an individual's exome and a snapshot of the current state of personalized genomics. The majority of coding variants in this individual are common and appear to be functionally neutral. Our results also indicate that some variants can be used to improve the current NCBI human reference genome. As more genomes are sequenced, many rare variants and non-SNP variants will be discovered. We present an approach to analyze the coding variation in humans by proposing multiple bioinformatic methods to hone in on possible functional variation.     

Live analysis of endodermal layer formation identifies random walk as a novel gastrulation movement

During gastrulation, dramatic movements rearrange cells into three germ layers expanded over the entire embryo [1-3]. In fish, both endoderm and mesoderm are specified as a belt at the embryo margin. Mesodermal layer expansion is achieved through the combination of two directed migrations. The outer ring of precursors moves toward the vegetal pole and continuously seeds mesodermal cells inside the embryo, which then reverse their movement in the direction of the animal pole [3-6]. Unlike mesoderm, endodermal cells internalize at once and must therefore adopt a different strategy to expand over the embryo [7, 8]. With live imaging of YFP-expressing zebrafish endodermal cells, we demonstrate that in contrast to mesoderm, internalized endodermal cells display a nonoriented/noncoordinated movement fit by a random walk that rapidly disperses them over the yolk surface. Transplantation experiments reveal that this behaviour is largely cell autonomous, induced by TGF-beta/Nodal, and dependent on the downstream effector Casanova. At midgastrulation, endodermal cells switch to a convergence movement. We demonstrate that this switch is triggered by environmental cues. These results uncover random walk as a novel Nodal-induced gastrulation movement and as an efficient strategy to transform a localized cell group into a layer expanded over the embryo.     

Correlation between 13Calpha chemical shifts and helix content of peptide ensembles

Replica exchange molecular dynamics simulations are used to generate three ensembles of an S-peptide analog (AETAAAKFLREHMDS). Percent helicity of the peptide ensembles calculated using STRIDE is compared to percent helicity calculated from (13)C(alpha) chemical shift deviations (CSD) from random coil in order to test the assumption that CSD can be correlated to percent helicity. The two estimates of helicity, one based on structure and the other on CSD, are in close to quantitative agreement, except at the edges of helical stretches where disagreements of as much as 50% can be found. These disagreements can occur by CSDs both as an under- and an overestimate of peptide helicity. We show that underestimation arises due to ensemble averaging of positive CSDs from conformers with torsion angles in the helical region of Ramachandran space with negative CSDs corresponding to conformers of the peptide in the extended region. In contrast, overestimation comes about due to the fact that a large number of conformations with torsion angles in the helical region are not counted as helical by STRIDE due to a lack of correlated helical torsion angles in neighboring residues.