Detailed protein sequence alignment based on Spectral Similarity Score (SSS)

Background  

The chemical property and biological function of a protein is a direct consequence of its primary structure. Several algorithms have been developed which determine alignment and similarity of primary protein sequences. However, character based similarity cannot provide insight into the structural aspects of a protein. We present a method based on spectral similarity to compare subsequences of amino acids that behave similarly but are not aligned well by considering amino acids as mere characters. This approach finds a similarity score between sequences based on any given attribute, like hydrophobicity of amino acids, on the basis of spectral information after partial conversion to the frequency domain.     

Staurosporine-induced apoptotic water loss is cell- and attachment-specific

Apoptotic volume decrease (AVD) is a characteristic cell shrinkage observed during apoptosis. There are at least two known processes that may result in the AVD: exit of intracellular water and splitting of cells into smaller fragments. Although AVD has traditionally been attributed to water loss, direct evidence for that is often lacking. In this study, we quantified intracellular water in staurosporine-treated cells using a previously described optical microscopic technique that combines volume measurements with quantitative phase analysis. Water loss was observed in detached HeLa and in adherent MDCK but not in adherent HeLa cells. At the same time, adherent HeLa and adherent MDCK cells exhibited visually similar apoptotic morphology, including fragmentation and activation of caspase-3. Morphological changes and caspase activation were prevented by chloride channel blockers DIDS and NPPB in both adherent and suspended HeLa cells, while potassium channel blocker TEA was ineffective. We conclude that staurosporine-induced dehydration is not a universal cell response but depends on the cell type and substrate attachment and can only be judged by direct water measurements. The effects of potassium or chloride channel blockers do not always correlate with the AVD.     

Do metacommunity mass effects predict changes in species incidence and abundance?

Metacommunity matrices contain data on species incidence or abundance across sites, compactly portraying community composition and how it varies over sites. We constructed models based on an initial metacommunity matrix of either species incidence or abundance to test whether such data suffice to predict subsequent changes in incidence or abundance at each site. The models reflect both species and site mass effects in using products of the row and column totals to predict the incidence or abundance of each species expected at each site. We tested these models against empirical data on vascular plant incidence and abundance collected from two sets of forested sites in both the 1950s and 2000s. Predictions from these models parallel observed changes in species incidence and abundance in these distinctly different metacommunities and differ greatly from null model predictions. The abundance model shows greater power than the incidence model reflecting its higher information content. Predictions were more accurate for the more diverse forests of southern Wisconsin that are changing quickly in response to succession and fragmentation. Simulations show that these results are robust but sensitive to sampling intensity. Because these models require no data on site conditions or species’ characteristics, they provide a useful baseline to assess more complex models based on species’ functional traits, local site conditions, or landscape context. They may also prove useful to conservation biologists seeking to predict local population declines and extinction risks.     

Estimating synonymous and nonsynonymous substitution rates

Partitioning the total substitution rate into synnonymous and nonsynonymous components is a key aspect of many analyses in molecular evolution. Numerous methods exist for estimating these rates. However, until recently none of the estimation procedures were based on a sound statistical footing. In this paper, the evolutionary model of Muse and Gaut (1994) is used as the basis for two sets of parameters quantifying silent and replacement substitution rates. The parameters are shown to be equal when the four nucleotides are equally frequent and unequal otherwise. Maximum-likelihood estimation of these parameters is described, and the performance of these estimates is compared to that of existing estimation procedures. It is shown that the estimates of Nei and Gojobori (1986) are not unbiased for either set of parameters, although they provide very good estimates for one set as long as sequence divergence is not too high. However, some disturbing properties are found for the Nei and Gojobori estimates. In particular, it is shown that the expected value of the Nei and Gojobori estimate of silent substitution rate is a function of both the silent and replacement substitution rates. The maximum-likelihood estimates have no such problems.      

NEMix: Single-cell Nested Effects Models for Probabilistic Pathway Stimulation

Nested effects models have been used successfully for learning subcellular networks from high-dimensional perturbation effects that result from RNA interference (RNAi) experiments. Here, we further develop the basic nested effects model using high-content single-cell imaging data from RNAi screens of cultured cells infected with human rhinovirus. RNAi screens with single-cell readouts are becoming increasingly common, and they often reveal high cell-to-cell variation. As a consequence of this cellular heterogeneity, knock-downs result in variable effects among cells and lead to weak average phenotypes on the cell population level. To address this confounding factor in network inference, we explicitly model the stimulation status of a signaling pathway in individual cells. We extend the framework of nested effects models to probabilistic combinatorial knock-downs and propose NEMix, a nested effects mixture model that accounts for unobserved pathway activation. We analyzed the identifiability of NEMix and developed a parameter inference scheme based on the Expectation Maximization algorithm. In an extensive simulation study, we show that NEMix improves learning of pathway structures over classical NEMs significantly in the presence of hidden pathway stimulation. We applied our model to single-cell imaging data from RNAi screens monitoring human rhinovirus infection, where limited infection efficiency of the assay results in uncertain pathway stimulation. Using a subset of genes with known interactions, we show that the inferred NEMix network has high accuracy and outperforms the classical nested effects model without hidden pathway activity. NEMix is implemented as part of the R/Bioconductor package ‘nem’ and available at www.cbg.ethz.ch/software/NEMix.     

Frameshift mutagenesis: the roles of primer-template misalignment and the nonhomologous end-joining pathway in Saccharomyces cerevisiae

Small insertions or deletions that alter the reading frame of a gene typically occur in simple repeats such as mononucleotide runs and are thought to reflect spontaneous primer-template misalignment during DNA replication. The resulting extrahelical repeat is efficiently recognized by the mismatch repair machinery, which specifically replaces the newly replicated strand to restore the original sequence. Frameshift mutagenesis is most easily studied using reversion assays, and previous studies in Saccharomyces cerevisiae suggested that the length threshold for polymerase slippage in mononucleotide runs is 4N. Because the probability of slippage is strongly correlated with run length, however, it was not clear whether shorter runs were unable to support slippage or whether the resulting frameshifts were obscured by the presence of longer runs. To address this issue, we removed all mononucleotide runs >3N from the yeast lys2ΔBgl and lys2ΔA746 frameshift reversion assays, which detect net 1-bp deletions and insertions, respectively. Analyses demonstrate that 2N and 3N runs can support primer-template misalignment, but there is striking run-specific variation in the frequency of slippage, in the accumulation of +1 vs. -1 frameshifts and in the apparent efficiency of mismatch repair. We suggest that some of this variation reflects the role of flanking sequence in initiating primer-template misalignment and that some reflects replication-independent frameshifts generated by the nonhomologous end-joining pathway. Finally, we demonstrate that nonhomologous end joining is uniquely required for the de novo creation of tandem duplications from noniterated sequence.     

Length variation and secondary structure of introns in the Mlc1 gene in six species of Drosophila

A nearly universal feature of intron sequences is that even closely related species exhibit a large number of insertion/deletion differences. The goal of the analysis described here is to test whether the observed pattern of insertion/deletion events in the genealogy of the myosin alkali light chain (Mlc1) gene is consistent with neutrality, and if not, to determine the underlying forces of evolutionary change. Mlc1 pre-mRNA is alternatively spliced, and one constraint is that signals necessary for tissue-specificity of directed splicing must be conserved. If the total length of an intron is functionally constrained, then the distribution of indels on branches of the gene genealogy should reflect a departure from randomness. Here we perform a phylogenetic analysis, inferring ancestral states wherever possible on a phylogeny of 29 alleles of Mlc1 from six species of Drosophila. Observed patterns of indels on the genealogy were compared to those from simulated data, with the result that we cannot reject the null hypothesis of neutrality. A clear departure from a neutral prediction was seen in the excess folding free energy predicted for the introns flanking the alternatively spliced exon. Relative rate tests also suggest a retardation in the rate of Mlc1 sequence evolution in the simulans clade.      

Polyomavirus large and small T antigens cooperate in induction of the S phase in serum-starved 3T3 mouse fibroblasts.

The induction of an S phase in the host cell is a prerequisite for the lytic replication cycle of polyomavirus. This function was attributed to proteins coded for by the early region of the viral DNA, the T antigens. A consideration of the role of the T antigens in the initiation of a mitogenic response of the host cell has to take into account the recent discovery that virus adsorption is sufficient to induce the synthesis of proteins which are known to appear early after quiescent cells are stimulated by the addition of serum, namely fos, jun, and myc (J. Zullo, C.D. Stiles, and R.L. Garcea, Proc. Natl. Acad. Sci. USA 84:1210-1214, 1987; G. M. Glenn and W. Eckhart, J. Virol. 64:2193-2201, 1990). This induction is followed by an initiation of DNA synthesis. It is therefore important to dissociate the effects of the T antigens on the host cell from those of virus adsorption. To do so, we used dexamethasone-regulated versions of the large and small T antigens of polyomavirus stably integrated into the genome of Swiss 3T3 cells to study their function in S-phase induction. When the production of the large or small T antigen in serum-starved 3T3 mouse fibroblasts was activated, only a small fraction of cells was able to leave G0/G1 despite the synthesis of considerable amounts of the respective T antigen. Activation of both T antigens within the same cell, on the other hand, resulted in S-phase induction in a notable percentage of cells, suggesting that the two proteins cooperate in this activity. Polyomavirus T antigens appear to bypass the pathway of growth regulation involving the activation of c-fos. These results are discussed in relation to other known functions of the two virally coded proteins.     

A likelihood approach for comparing synonymous and nonsynonymous nucleotide substitution rates, with application to the chloroplast genome

A model of DNA sequence evolution applicable to coding regions is presented. This represents the first evolutionary model that accounts for dependencies among nucleotides within a codon. The model uses the codon, as opposed to the nucleotide, as the unit of evolution, and is parameterized in terms of synonymous and nonsynonymous nucleotide substitution rates. One of the model's advantages over those used in methods for estimating synonymous and nonsynonymous substitution rates is that it completely corrects for multiple hits at a codon, rather than taking a parsimony approach and considering only pathways of minimum change between homologous codons. Likelihood-ratio versions of the relative-rate test are constructed and applied to data from the complete chloroplast DNA sequences of Oryza sativa, Nicotiana tabacum, and Marchantia polymorpha. Results of these tests confirm previous findings that substitution rates in the chloroplast genome are subject to both lineage-specific and locus-specific effects. Additionally, the new tests suggest tha the rate heterogeneity is due primarily to differences in nonsynonymous substitution rates. Simulations help confirm previous suggestions that silent sites are saturated, leaving no evidence of heterogeneity in synonymous substitution rates.      

Adaptive change in protective coloration in adult striated shieldbugs Graphosoma lineatum (Heteroptera: Pentatomidae): test of detectability of two colour forms by avian predators

1. Protective coloration in insects may be aposematic or cryptic, and some species change defensive strategy between instars. In Sweden, the adult striated shieldbugs Graphosoma lineatum (Heteroptera: Pentatomidae) undergo a seasonal colour change from pale brown and black striation in the pre‐hibernating adults, to red and black striation in the same post‐hibernating individuals. To the human eye the pre‐hibernating adults appear cryptic against the withered late summer vegetation, whereas the red and black post‐hibernating adults appear aposematic. This suggests a possibility of a functional colour change. However, what is cryptic to the human eye is not necessarily cryptic to a potential predator. 2. Therefore we tested the effect of coloration in adult G. lineatum on their detectability for avian predators. Great tits (Parus major) were trained to eat sunflower seeds hidden inside the emptied exoskeletons of pale or red G. lineatum. Then the detection time for both colour forms was measured in a dry vegetation environment. 3. The birds required a longer time to find the pale form of G. lineatum than the red one. The pale form appears more cryptic on withered late summer vegetation than the red form, not only to the human eye but also to avian predators. The result supports the idea that the adult individuals of G. lineatum undergo a functional change from a cryptic protective coloration to an aposematic one.