The meaning of alignment: lessons from structural diversity

<p>Abstract</p> <p>Background</p> <p>Protein structural alignment provides a fundamental basis for deriving principles of functional and evolutionary relationships. It is routinely used for structural classification and functional characterization of proteins and for the construction of sequence alignment benchmarks. However, the available techniques do not fully consider the implications of protein structural diversity and typically generate a single alignment between sequences.</p> <p>Results</p> <p>We have taken alternative protein crystal structures and generated simulation snapshots to explicitly investigate the impact of structural changes on the alignments. We show that structural diversity has a significant effect on structural alignment. Moreover, we observe alignment inconsistencies even for modest spatial divergence, implying that the biological interpretation of alignments is less straightforward than commonly assumed. A salient example is the GroES 'mobile loop' where sub-Ångstrom variations give rise to contradictory sequence alignments.</p> <p>Conclusion</p> <p>A comprehensive treatment of ambiguous alignment regions is crucial for further development of structural alignment applications and for the representation of alignments in general. For this purpose we have developed an on-line database containing our data and new ways of visualizing alignment inconsistencies, which can be found at <url>http://www.ibi.vu.nl/databases/stralivari</url>.</p

CGHnormaliter: an iterative strategy to enhance normalization of array CGH data with imbalanced aberrations

Background: Array comparative genomic hybridization (aCGH) is a popular technique for detection of genomic copy number imbalances. These play a critical role in the onset of various types of cancer. In the analysis of aCGH data, normalization is deemed a critical pre-processing step. In general, aCGH normalization approaches are similar to those used for gene expression data, albeit both data-types differ inherently. A particular problem with aCGH data is that imbalanced copy numbers lead to improper normalization using conventional methods. Results: In this study we present a novel method, called CGHnormaliter, which addresses this issue by means of an iterative normalization procedure. First, provisory balanced copy numbers are identified and subsequently used for normalization. These two steps are then iterated to refine the normalization. We tested our method on three well-studied tumor-related aCGH datasets with experimentally confirmed copy numbers. Results were compared to a conventional normalization approach and two more recent state-of-the-art aCGH normalization strategies. Our findings show that, compared to these three methods, CGHnormaliter yields a higher specificity and precision in terms of identifying the 'true' copy numbers. Conclusion: We demonstrate that the normalization of aCGH data can be significantly enhanced using an iterative procedure that effectively eliminates the effect of imbalanced copy numbers. This also leads to a more reliable assessment of aberrations. An R-package containing the implementation of CGHnormaliter is available at http://www.ibi.vu.nl/programs/cghnormaliterwww. © 2009 van Houte et al; licensee BioMed Central Ltd

Comparative transcriptomics reveals commonalities and differences in the genetic underpinnings of a floral dimorphism

Distyly, a floral dimorphism associated with heteromorphic self-incompatibility and controlled by the S-locus supergene, evolved independently multiple times. Comparative analyses of the first transcriptome atlas for the main distyly model, Primula veris, with other distylous species produced the following findings. A set of 53 constitutively expressed genes in P. veris did not include any of the housekeeping genes commonly used to normalize gene expression in qPCR experiments. The S-locus gene CYP$^{T}$ acquired its role in controlling style elongation via a change in expression profile. Comparison of genes differentially expressed between floral morphs revealed that brassinosteroids and auxin are the main hormones controlling style elongation in P. veris and Fagopyrum esculentum, respectively. Furthermore, shared biochemical pathways might underlie the expression of distyly in the distantly related P. veris, F. esculentum and Turnera subulata, suggesting a degree of correspondence between evolutionary convergence at phenotypic and molecular levels. Finally, we provide the first evidence supporting the previously proposed hypothesis that distyly supergenes of distantly related species evolved via the recruitment of genes related to the phytochrome-interacting factor (PIF) signaling network. To conclude, this is the first study that discovered homologous genes involved in the control of distyly in distantly related taxa

Sequence harmony: detecting functional specificity from alignments

Multiple sequence alignments are often used for the identification of key specificity-determining residues within protein families. We present a web server implementation of the Sequence Harmony (SH) method previously introduced. SH accurately detects subfamily specific positions from a multiple alignment by scoring compositional differences between subfamilies, without imposing conservation. The SH web server allows a quick selection of subtype specific sites from a multiple alignment given a subfamily grouping. In addition, it allows the predicted sites to be directly mapped onto a protein structure and displayed. We demonstrate the use of the SH server using the family of plant mitochondrial alternative oxidases (AOX). In addition, we illustrate the usefulness of combining sequence and structural information by showing that the predicted sites are clustered into a few distinct regions in an AOX homology model. The SH web server can be accessed at www.ibi.vu.nl/programs/seqharmwww

Sequence harmony: detecting functional specificity from alignments

Multiple sequence alignments are often used for the identification of key specificity-determining residues within protein families. We present a web server implementation of the Sequence Harmony (SH) method previously introduced. SH accurately detects subfamily specific positions from a multiple alignment by scoring compositional differences between subfamilies, without imposing conservation. The SH web server allows a quick selection of subtype specific sites from a multiple alignment given a subfamily grouping. In addition, it allows the predicted sites to be directly mapped onto a protein structure and displayed. We demonstrate the use of the SH server using the family of plant mitochondrial alternative oxidases (AOX). In addition, we illustrate the usefulness of combining sequence and structural information by showing that the predicted sites are clustered into a few distinct regions in an AOX homology model. The SH web server can be accessed at www.ibi.vu.nl/programs/seqharmwww

Spread of carbapenem resistance by transposition and conjugation among Pseudomonas aeruginosa

The emergence of carbapenem-resistant Pseudomonas aeruginosa represents a worldwide problem. To understand the carbapenem-resistance mechanisms and their spreading among P. aeruginosa strains, whole genome sequences were determined of two extensively drug-resistant strains that are endemic in Dutch hospitals. Strain Carb01 63 is of O-antigen serotype O12 and of sequence type ST111, whilst S04 90 is a serotype O11 strain of ST446. Both strains carry a gene for metallo-β-lactamase VIM-2 flanked by two aacA29 genes encoding aminoglycoside acetyltransferases on a class 1 integron. The integron is located on the chromosome in strain Carb01 63 and on a plasmid in strain S04 90. The backbone of the 159-kb plasmid, designated pS04 90, is similar to a previously described plasmid, pND6-2, from Pseudomonas putida. Analysis of the context of the integron showed that it is present in both strains on a ~30-kb mosaic DNA segment composed of four different transposons that can presumably act together as a novel, active, composite transposon. Apart from the presence of a 1237-bp insertion sequence element in the composite transposon on pS04 90, these transposons show > 99% sequence identity indicating that transposition between plasmid and chromosome could have occurred only very recently. The pS04 90 plasmid could be transferred by conjugation to a susceptible P. aeruginosa strain. A second class 1 integron containing a gene for a CARB-2 β-lactamase flanked by an aacA4'-8 and an aadA2 gene, encoding an aminoglycoside acetyltransferase and adenylyltransferase, respectively, was present only in strain Carb01 63. This integron is located also on a composite transposon that is inserted in an integrative and conjugative element on the chromosome. Additionally, this strain contains a frameshift mutation in the oprD gene encoding a porin involved in the transport of carbapenems across the outer membrane. Together, the results demonstrate that integron-encoded carbapenem and carbapenicillin resistance can easily be disseminated by transposition and conjugation among Pseudomonas aeruginosa strains