Mapping of the influenza A hemagglutinin serotypes evolution by the ISSCOR method.

Analyses and visualizations by the ISSCOR method of influenza virus hemagglutinin genes of different A-subtypes revealed some rather striking temporal relationships between groups of individual gene subsets. Based on these findings we consider application of the ISSCOR-PCA method for analyses of large sets of homologous genes to be a worthwhile addition to a toolbox of genomics--allowing for a rapid diagnostics of trends, and ultimately even aiding an early warning of newly emerging epidemiological threats.

Alignment free characterization of the influenza-A hemagglutinin genes by the ISSCOR method.

Analyses and visualizations by the ISSCOR method of the influenza virus hemagglutinin genes of three different A-subtypes revealed some rather striking temporal (for A/H3N3), and spatial relationships (for A/H5N1) between groups of individual gene subsets. The application to the A/H1N1 set revealed also relationships between the seasonal H1, and the swine-like novel 2009 H1v variants in a quick and unambiguous manner. Based on these examples we consider the application of the ISSCOR method for analysis of large sets of homologous genes as a worthwhile addition to a toolbox of genomics-it allows a rapid diagnostics of trends, and possibly can even aid an early warning of newly emerging epidemiological threats.

ISSCOR: Intragenic, Stochastic Synonymous Codon Occurrence Replacement--a new method for an alignment-free genome sequence analysis.

Synonymous codons do not occur at equal frequencies. Codon usage and codon bias have been extensively studied. However, the sequential order in which synonymous codons appear within a gene has not been studied until now. Here we describe an in silico method, which is the first attempt to tackle this problem: to what extent this sequential order is unique, and to what extent the succession of synonymous codons is important. This method, which we called Intragenic, Stochastic Synonymous Codon Occurrence Replacement (ISSCOR), generates, by a Monte Carlo approach, a set of genes which code for the same amino acid sequence, and display the same codon usage, but have random permutations of the synonymous codons, and therefore different sequential codon orders from the original gene. We analyze the complete genome of the bacterium Helicobacter pylori (containing 1574 protein coding genes), and show by various, alignment-free computational methods (e.g., frequency distribution of codon-pairs, as well as that of nucleotide bigrams in codon-pairs), that: (i) not only the succession of adjacent synonymous codons is far from random, but also, which is totally unexpected, the occurrences of non-adjacent synonymous codon-pairs are highly constrained, at strikingly long distances of dozens of nucleotides; (ii) the statistical deviations from the random synonymous codon order are overwhelming; and (iii) the pattern of nucleotide bigrams in codon-pairs can be used in a novel way for characterizing and comparing genes and genomes. Our results demonstrate that the sequential order of synonymous codons within a gene must be under a strong selective pressure, which is superimposed on the classical codon usage. This new dimension can be measured by the ISSCOR method, which is simple, robust, and should be useful for comparative and functional genomics.

Periodic oscillations of the genomic nucleotide sequences disclose major differences in the way of constructing homologous proteins from different procaryotic species.

In this study, a set of 80 completely sequenced procaryotic genomes has been analysed by an alignment-free method, namely the expectancy-rectified frequency of bigrams or 2-tuples, representing the 16 combinations of A, T, G, C. It demonstrates that all genomes exhibit periodic oscillations of their nucleotide sequence, with a period close to 11 phosphodiester bonds, and resembling in shape an exponentially dampened sinusoid at the distance from 5 to 49 bonds. Interestingly, the amplitude of nucleotide oscillation (but not the period) can differ drastically from one species to another. I show that these differences are due neither to the (G + C) content, nor to the size of the genome. They are not directly related to phylogeny, since specific genomes from Archaea and Bacteria can display large as well as small amplitudes. I have compared also a set of genes coding for proteins rich in alpha helical structures (as determined by X-ray diffraction) with a set of genes coding for proteins devoid of alpha helices. The first set has periodic oscillations of large amplitude, with an 11-bond period, while the second has none. Furthermore, I analysed a large number of sets of homologous genes from several different species. They exhibit very different amplitudes of oscillations. Altogether, the data with their statistical analyses strongly suggest that the nucleotide oscillations are due to the 'genomic style of proteins', which means that homologous proteins, having the same biochemical function in different organisms, may have different secondary structures or may use different ways to be constructed. I realize that this idea is a heterodox one, but I believe that it can shed a new light both on phylogenies and on constraints between proteins and their coding sequences.

Primary sequences of proteins from complete genomes display a singular periodicity: Alignment-free N-gram analysis.

A method is proposed to represent and to analyze complete genome sequences (52 species from procaryotes and eukaryotes), based upon n-gram sequence's frequencies of amino acid pairs (bigrams), separated by a given number of other residues. For each of the species analyzed, it allows us to construct over-abundant and over-deficient occurrence profiles, summarizing amino acid bigram frequencies over the entire genome. The method deals efficiently with a sparseness of statistical representations of individual sequences, and describes every gene sequence in the same way, independently of its length and of the genome sizes. The frequency of over-abundant and over-deficient occurrences of bigrams presents a singular periodicity around 3.5 peptide bonds, suggesting a relation with the alpha helical secondary structure.

Yeast cells lacking the mitochondrial gene encoding the ATP synthase subunit 6 exhibit a selective loss of complex IV and unusual mitochondrial morphology.

Atp6p is an essential subunit of the ATP synthase proton translocating domain, which is encoded by the mitochondrial DNA (mtDNA) in yeast. We have replaced the coding sequence of Atp6p gene with the non-respiratory genetic marker ARG8m. Due to the presence of ARG8m, accumulation of rho-/rho0 petites issued from large deletions in mtDNA could be restricted to 20-30% by growing the atp6 mutant in media lacking arginine. This moderate mtDNA instability created favorable conditions to investigate the consequences of a specific lack in Atp6p. Interestingly, in addition to the expected loss of ATP synthase activity, the cytochrome c oxidase respiratory enzyme steady-state level was found to be extremely low (<5%) in the atp6 mutant. We show that the cytochrome c oxidase-poor accumulation was caused by a failure in the synthesis of one of its mtDNA-encoded subunits, Cox1p, indicating that, in yeast mitochondria, Cox1p synthesis is a key target for cytochrome c oxidase abundance regulation in relation to the ATP synthase activity. We provide direct evidence showing that in the absence of Atp6p the remaining subunits of the ATP synthase can still assemble. Mitochondrial cristae were detected in the atp6 mutant, showing that neither Atp6p nor the ATP synthase activity is critical for their formation. However, the atp6 mutant exhibited unusual mitochondrial structure and distribution anomalies, presumably caused by a strong delay in inner membrane fusion.

Increasing mitochondrial substrate-level phosphorylation can rescue respiratory growth of an ATP synthase-deficient yeast.

In a previous study we have identified Fmc1p, a mitochondrial protein involved in the assembly/stability of the yeast F0F1-ATP synthase at elevated temperatures. The deltafmc1 mutant was shown to exhibit a severe phenotype of very slow growth on respiratory substrates at 37 degrees C. We have isolated ODC1 as a multicopy suppressor of the fmc1 deletion restoring a good respiratory growth. Odc1p expression level was estimated to be at least 10 times higher in mitochondria isolated from the deltafmc1/ODC1 transformant as compared with wild type mitochondria. Interestingly, ODC1 encodes an oxodicarboxylate carrier, which transports alpha-ketoglutarate and alpha-ketoadipate or any other transported tricarboxylic acid cycle intermediate in a counter-exchange through the inner mitochondrial membrane. We show that the suppression of the respiratory-growth-deficient fmc1 by the overexpressed Odc1p was not due to a restored stable ATP synthase. Instead, the rescuing mechanism involves an increase in the flux of tricarboxylic acid cycle intermediate from the cytosol into the mitochondria, leading to an increase in the alpha-ketoglutarate oxidative decarboxylation, resulting in an increase in mitochondrial substrate-level-dependent ATP synthesis. This mechanism of metabolic bypass of a defective ATP synthase unravels the physiological importance of intramitochondrial substrate-level phosphorylations. This unexpected result might be of interest for the development of therapeutic solutions in pathologies associated with defects in the oxidative phosphorylation system.

Hierarchical clustering based upon contextual alignment of proteins: a different way to approach phylogeny.

We perform a computational study using a new approach to the analysis of protein sequences. The contextual alignment model, proposed recently by Gambin et al. (2002), is based on the assumption that, while constructing an alignment, the score of a substitution of one residue by another depends on the surrounding residues. The contextual alignment scores calculated in this model were used to hierarchical clustering of several protein families from the database of Clusters of Orthologous Groups (COG). The clustering has been also constructed based on the standard approach. The comparative analysis shows that the contextual model results in more consistent clustering trees. The difference, although small, is with no exception in favour of the contextual model. The consistency of the family of trees is measured by several consensus and agreement methods, as well as by the inter-tree distance approach.

The bromodomain-containing protein Bdf1p acts as a phenotypic and transcriptional multicopy suppressor of YAF9 deletion in yeast.

It was observed previously that the deletion of the open reading frame YNL107w (YAF9) was highly pleiotropic in yeast and caused defective growth phenotypes in the presence of several unrelated inhibitors, including caesium chloride. We have selected multicopy extragenic suppressor genes, revealing that this phenotype can be suppressed by overdosing the transcription factors BDF1 and GAT1 in the yaf9Delta strain. We focused our analysis on suppression by BDF1 and performed a genome-wide transcript analysis on a yaf9Delta strain, compared with the wild-type and BDF1-suppressed strains. YAF9 deletion has a clear effect on transcription and leads to modulation of the level of expression of several genes. Transcription of a considerable portion of the underexpressed genes is restored to wild-type levels in the BDF1-suppressed strain. We show by chromatin immunoprecipitation that both Yaf9p and Bdf1p bind to promoters of some of these genes and that the level of H3 and H4 acetylation at one of these promoters is significantly lowered in the yaf9 deleted strain, compared with the wild-type and the BDF1-suppressed strains.

F1-catalysed ATP hydrolysis is required for mitochondrial biogenesis in Saccharomyces cerevisiae growing under conditions where it cannot respire.

Mutant strains of yeast Saccharomyces cerevisiae lacking a functional F1-ATPase were found to grow very poorly under anaerobic conditions. A single amino acid replacement (K222 > E222) that locally disrupts the adenine nucleotide catalytic site in the beta-F1 subunit was sufficient to compromise anaerobic growth. This mutation also affected growth in aerated conditions when ethidium bromide (an intercalating agent impairing mtDNA propagation) or antimycin (an inhibitor of respiration) was included in the medium. F1-deficient cells forced to grow in oxygen-limited conditions were shown to lose their mtDNA completely and to accumulate Hsp60p mainly under its precursor form. Fluorescence microscopy analyses with a modified GFP containing a mitochondrial targeting presequence revealed that aerobically growing F1-deficient cells stopped importing the GFP when antimycin was added to the medium. Finally, after total inactivation of the catalytic alpha3beta3 subcomplex of F1, mitochondria could no longer be energized by externally added ATP because of either a block in assembly or local disruption of the adenine nucleotide processing site. Altogether these data strengthen the notion that in the absence of respiration, and whether the proton translocating domain (F0) of complex V is present or not, F1-catalysed hydrolysis of ATP is essential for the occurrence of vital cellular processes depending on the maintenance of an electrochemical potential across the mitochondrial inner membrane.