Małgorzata Dudkiewicz

High correlation between the turnover of nucleotides under mutational pressure and the DNA composition

BackgroundAny DNA sequence is a result of compromise between the selection and mutation pressures exerted on it during evolution. It is difficult to estimate the relative influence of each of these pressures on the rate of accumulation of substitutions. However, it is important to discriminate between the effect of mutations, and the effect of selection, when studying the phylogenic relations between taxa.ResultsWe have tested in computer simulations, and analytically, the available substitution matrices for many genomes, and we have found that DNA strands in equilibrium under mutational pressure have unique feature: the fraction of each type of nucleotide is linearly dependent on the time needed for substitution of half of nucleotides of a given type, with a correlation coefficient close to 1. Substitution matrices found for sequences under selection pressure do not have this property. A substitution matrix for the leading strand of the Borrelia burgdorferi genome, having reached equilibrium in computer simulation, gives a DNA sequence with nucleotide composition and asymmetry corresponding precisely to the third positions in codons of protein coding genes located on the leading strand.ConclusionsParameters of mutational pressure allow us to count DNA composition in equilibrium with this mutational pressure. Comparing any real DNA sequence with the sequence in equilibrium it is possible to estimate the distance between these sequences, which could be used as a measure of the selection pressure. Furthermore, the parameters of the mutational pressure enable direct estimation of the relative mutation rates in any DNA sequence in the studied genome.

A novel protein kinase-like domain in a selenoprotein, widespread in the tree of life.

Selenoproteins serve important functions in many organisms, usually providing essential oxidoreductase enzymatic activity, often for defense against toxic xenobiotic substances. Most eukaryotic genomes possess a small number of these proteins, usually not more than 20. Selenoproteins belong to various structural classes, often related to oxidoreductase function, yet a few of them are completely uncharacterised. Here, the structural and functional prediction for the uncharacterised selenoprotein O (SELO) is presented. Using bioinformatics tools, we predict that SELO protein adopts a three-dimensional fold similar to protein kinases. Furthermore, we argue that despite the lack of conservation of the “classic” catalytic aspartate residue of the archetypical His-Arg-Asp motif, SELO kinases might have retained catalytic phosphotransferase activity, albeit with an atypical active site. Lastly, the role of the selenocysteine residue is considered and the possibility of an oxidoreductase-regulated kinase function for SELO is discussed. The novel kinase prediction is discussed in the context of functional data on SELO orthologues in model organisms, FMP40 a.k.a.YPL222W (yeast), and ydiU (bacteria). Expression data from bacteria and yeast suggest a role in oxidative stress response. Analysis of genomic neighbourhoods of SELO homologues in the three domains of life points toward a role in regulation of ABC transport, in oxidative stress response, or in basic metabolism regulation. Among bacteria possessing SELO homologues, there is a significant over-representation of aquatic organisms, also of aerobic ones. The selenocysteine residue in SELO proteins occurs only in few members of this protein family, including proteins from Metazoa, and few small eukaryotes (Ostreococcus, stramenopiles). It is also demonstrated that enterobacterial mchC proteins involved in maturation of bactericidal antibiotics, microcins, form a distant subfamily of the SELO proteins. The new protein structural domain, with a putative kinase function assigned, expands the known kinome and deserves experimental determination of its biological role within the cell-signaling network.

How many protein-coding genes are there in the Saccharomyces cerevisiae genome?

We have compared the results of estimations of the total number of protein‐coding genes in the Saccharomyces cerevisiae genome, which have been obtained by many laboratories since the yeast genome sequence was published in 1996. We propose that there are 5300–5400 genes in the genome. This makes the first estimation of the number of intronless ORFs longer than 100 codons, based on the features of the set of genes with phenotypes known in 1997 to be correct. This estimation assumed that the set of the first 2300 genes with known phenotypes was representative for the whole set of protein‐coding genes in the genome. The same method used in this paper for the approximation of the total number of protein‐coding sequences among more than 40 000 ORFs longer than 20 codons gives a result that is only slightly higher. This suggests that there are still some non‐coding ORFs in the databases and a few dozen small ORFs, not yet annotated, which probably code for proteins. Copyright

A novel predicted calcium-regulated kinase family implicated in neurological disorders.

The catalogues of protein kinases, the essential effectors of cellular signaling, have been charted in Metazoan genomes for a decade now. Yet, surprisingly, using bioinformatics tools, we predicted protein kinase structure for proteins coded by five related human genes and their Metazoan homologues, the FAM69 family. Analysis of three-dimensional structure models and conservation of the classic catalytic motifs of protein kinases present in four out of five human FAM69 proteins suggests they might have retained catalytic phosphotransferase activity. An EF-hand Ca2+-binding domain in FAM69A and FAM69B proteins, inserted within the structure of the kinase domain, suggests they may function as Ca2+-dependent kinases. The FAM69 genes, FAM69A, FAM69B, FAM69C, C3ORF58 (DIA1) and CXORF36 (DIA1R), are by large uncharacterised molecularly, yet linked to several neurological disorders in genetics studies. The C3ORF58 gene is found deleted in autism, and resides in the Golgi. Unusually high cysteine content and presence of signal peptides in some of the family members suggest that FAM69 proteins may be involved in phosphorylation of proteins in the secretory pathway and/or of extracellular proteins.

The Differential Killing of Genes by Inversions in Prokaryotic Genomes

We have elaborated a method which has allowed us to estimate the direction of translocation of orthologs which have changed, during the phylogeny, their positions on chromosome in respect to the leading or lagging role of DNA strands. We have shown that the relative number of translocations which have switched positions of genes from the leading to the lagging DNA strand is lower than the number of translocations which have transferred genes from the lagging strand to the leading strand of prokaryotic genomes. This paradox could be explained by assuming that the stronger mutation pressure and selection after inversion preferentially eliminate genes transferred from the leading to the lagging DNA strand.

Replication associated mutational pressure generating long-range correlation in DNA

There are many biological mechanisms which introduce long-range correlations into the DNA molecule. One of the most important is replication of chromosomes, its mechanisms and topology. Replication associated mutational pressure, defined as specific preferences in nucleotide substitutions during replication, generates asymmetry in the genome. On the other hand, substitution rates, which determine the evolutionary turnover time of a nucleotide, are highly correlated with the fraction of that nucleotide in the genome. Assuming the Azbel hypothesis that the number of mutations per genome per generation is invariant and universal, a general rule for mutational pressure can be formulated: the half-time of a nucleotide turnover in the genome is linearly dependent on the number of this nucleotide in the genome.

Present data on organ donation and transplantation in Poland.

At present, organ transplantation activity in Poland is located in the middle among European Union countries. There are appropriate law regulations, well-organized legal structures, well-educated transplant teams, good transplantation results, and case registries. There are 24 organ transplant teams in 20 centers, including 46 programs. Since 1966, over 18,000 organs have been transplanted 14,300 kidneys; 1800 livers; 1700 hearts; 250 kidney/pancreases and 30 lungs. Every year almost 1500 organs are procured from about 500 cadaveric heart-beating donors (9-14 per million people [pmp]) with 50 from living donors: 800 to 1000 kidneys (21-28 pmp); over 200 livers (5-6 pmp); and 60 to 100 hearts (1.6-2.7 pmp). National transplant registries are maintained in Poltransplant including a central registry of refusals (the policy of presumed consent with registered objection on donation after death is implemented), waiting lists, a cadaveric and living donor registry, and a transplant registry. There are still some actions that should be undertaken to strengthen the transplantation system in Poland, to increase organ availability, to enhance the efficiency and accessibility of transplantation systems, and to improve quality and safety: namely, increasing deceased donations to their full potential, appointing transplant coordinators in every hospital where there is the potential for organ donation, promoting quality improvement programs, and promoting living donation.

Molecular cloning and phylogenetic analysis of cereal type II metacaspase cDNA from wheat

A new cereal type II metacaspase full-length cDNA from wheat (Triticum aestivum L.) leaves, TaeMCAII, was for the first time successfully amplified and sequenced. The full-length sequence of the TaeMCAII cDNA of 1 551 bp contains a 1 218 bp open reading frame. The deduced protein encoded by the TaeMCAII cDNA consists of 405 amino acids with a calculated molecular mass of 44 kDa and an isoelectric point of 5.29. In response to wounding or heat shock, a similar sequence of ultrastructural events including the tonoplast rupture, chromatin condensation, degradation of chloroplasts and disappearance of cytoplasm and organelles were observed using transmission electron microscopy. As the observed changes in TaeMCAII mRNA level did not occur to be statistically significant wounding-induced programmed cell death (PCD) seems to be metacaspase-independent pathway. Interestingly, in PCD caused by a heat-shock treatment, the level of TaeMCAII mRNA remained unaltered until 48 h after the stress what suggests that TaeMCAII participates in later stages of PCD triggered by heat-shock. Phylogenetic analysis enabled to classify TaeMCAII as a type II metacaspase. Finally, homology modelling of the putative three-dimensional structure of the TaeMCAII protein and a topology analysis of its probable active site were performed.

Phosphorylation of spore coat proteins by a family of atypical protein kinases

Significance The posttranslational modification of proteins with a molecule of phosphate, termed protein phosphorylation, is a mechanism used by cells to regulate cellular activities. Protein phosphorylation occurs in all life forms and is catalyzed by a superfamily of enzymes known as protein kinases. Using bioinformatics, we have identified a family of spore coat protein (Cot) kinases, which are related to the secreted kinase, family with sequence similarity 20C (Fam20C). The founding member of this family is CotH from the spore-forming bacterium Bacillus subtilis. We show that CotH-dependent phosphorylation of the spore proteins CotB and CotG is necessary for the proper germination of spores. Because several CotH-containing organisms are human pathogens, our work has important clinical implications to combat human diseases, such as anthrax. The modification of proteins by phosphorylation occurs in all life forms and is catalyzed by a large superfamily of enzymes known as protein kinases. We recently discovered a family of secretory pathway kinases that phosphorylate extracellular proteins. One member, family with sequence similarity 20C (Fam20C), is the physiological Golgi casein kinase. While examining distantly related protein sequences, we observed low levels of identity between the spore coat protein H (CotH), and the Fam20C-related secretory pathway kinases. CotH is a component of the spore in many bacterial and eukaryotic species, and is required for efficient germination of spores in Bacillus subtilis; however, the mechanism by which CotH affects germination is unclear. Here, we show that CotH is a protein kinase. The crystal structure of CotH reveals an atypical protein kinase-like fold with a unique mode of ATP binding. Examination of the genes neighboring cotH in B. subtilis led us to identify two spore coat proteins, CotB and CotG, as CotH substrates. Furthermore, we show that CotH-dependent phosphorylation of CotB and CotG is required for the efficient germination of B. subtilis spores. Collectively, our results define a family of atypical protein kinases and reveal an unexpected role for protein phosphorylation in spore biology.

Analysis of the expression, subcellular and tissue localisation of phosphoglucan, water dikinase (PWD/GWD3) in Solanum tuberosum L.: a bioinformatics approach for the comparative analysis of two α-glucan, water dikinases (GWDs) from Solanum tuberosum L.

There are several important factors affecting the rate of starch decomposition in plants, including the circadian clock, the regulation of gene expression, the regulation of enzyme activities and starch phosphorylation by glucan, water dikinase activities (GWDs). One isoform of glucan, water dikinase named GWD3 or PWD (EC 2.7.9.5) was isolated for the first time from Arabidopsis thaliana, and now we report its isolation and identification in Solanum tuberosum L. leaves and tubers. We compare StGWD3 sequence to the other GWDs sequences using bioinformatics tools and propose also structural models for the starch-binding domains in StGWD3 and StGWD1. The StGWD3 gene expression and protein were localised in different heterotrophic and autotrophic potato tissues and organs using in situ RT-PCR and immunolocalisation methods, respectively. Diurnal changes in the transcript abundance of StGWD3 in leaves were analysed using quantitative real-time PCR and they appeared to be typical for most genes involved in starch degradation in chloroplasts.