Nucleotide composition and CpG and CpNpG content of ITS1, ITS2, and the 5.8S rRNA in representatives of the phylogenetic branches melanthiales-liliales and melanthiales-asparagales (Angiospermae,Monocotyledones) reflect the specifics of their evolution

The nucleotide composition and the contents of CpG and CpNpG in internal transcribed spacers 1 and 2 (ITS1 and ITS2) and the 5.8S rRNA gene of the nuclear genome were studied in two phylogenetic lineages of monocotyledonous angiosperms. The evolutionary advance of taxa by morphological characters proved to positively correlate with an increase in the contents of C, CpG, and CpNpG, contrasting the views that genome evolution in vertebrate and higher plants tends to decrease or, at least, preserve the amount of CpG and CpNpG, potentially subject to methylation, in nuclear DNA. Cryptaffinity taxa, which are intermediates between morphologically distinct taxonomic groups, displayed higher contents of CpG and CpNpG as compared with neighboring taxa. Changes in the contents of these elements in the regions of cryptaffinity taxa are intricate, suggesting a reciprocating character for their accumulation. Cryptaffinity taxa and their close phylogenetic relatives from the ancestral and descendant groups were assumed to reflect the key macroevolutionary changes and to correspond to saltatory periods separating the periods of gradual evolution.     

Computational method for predicting protein functional sites with the use of specificity determinants

The currently available body of decoded amino acid sequences of various proteins exceeds manifold the experimental capabilities of their functional annotation. Therefore, in silico annotation using bioinformatics methods becomes increasingly important. Such annotation is actually a prediction; however, this can be an important starting point for further laboratory research. This work describes a new method for predicting functionally important protein sites, SDPsite, on the basis of identification of specificity determinants. The algorithm proposed utilizes a protein family aglinment and a phylogenetic tree to predict the conserved positions and specificity determinants, map them onto the protein structure, and search for clusters of the predicted positions. Comparison of the resulting predictions with experimental data and published predictions of functional sites by other methods demonstrates that the results of SDPsite agree well with experimental data and exceed the results obtained with the majority of previous methods. SDPsite is publicly available at http://bioinf.fbb.msu.ru/SDPsite.     

THE SHAPE OF THE PHANEROZOIC MARINE PALAEODIVERSITY CURVE: HOW MUCH CAN BE PREDICTED FROM THE SEDIMENTARY ROCK RECORD OF WESTERN EUROPE?

Abstract:  Palaeodiversity curves are constructed from counts of fossils collected at outcrop and thus potentially biased by variation in the rock record, specifically by the amount of sedimentary rock representative of different time intervals that has been preserved at outcrop. To investigate how much of a problem this poses we have compiled a high-resolution record of marine rock outcrop area in Western Europe for the Phanerozoic and use this to generate a model that predicts the sampled diversity curve. We find that we can predict with high accuracy the variance of the marine genus diversity curve (itself dominated by European taxa) from rock outcrop data and a three-step model of diversity that tracks supercontinent fragmentation, coalescence and fragmentation. The size and position of two of the five major mass extinction spikes are largely predicted by rock outcrop data. We conclude that the long-term trends in taxonomic diversity and the end-Cretaceous extinction are not the result of rock area bias, but cannot rule out that rock outcrop area bias explains many of the short-term rises and falls in sampled diversity that palaeontologists have previously sought to explain biologically.     

Stone artifacts and hominins in island Southeast Asia: new insights from Flores, eastern Indonesia

This study reexamines the current understanding of Pleistocene stone-artifact assemblages in island Southeast Asia. A differentiation has long been made between assemblages of large-sized "core tools" and assemblages of small-sized "flake tools." "Core tool" assemblages are often argued to be the handiwork of early hominin species such as Homo erectus, while small-sized "flake tool" assemblages have been attributed to Homo sapiens. We argue that this traditional Southeast Asian perspective on stone tools assumes that the artifacts recovered from a site reflect a complete technological sequence. Our analyses of Pleistocene-age artifact assemblages from Flores, Indonesia, demonstrate that large pebble-based cores and small flake-based cores are aspects of one reduction sequence. We propose that the Flores pattern applies across island Southeast Asia: large-sized "core tool" assemblages are in fact a missing element of the small-sized flake-based reduction sequences found in many Pleistocene caves and rock-shelters. We conclude by discussing the implications of this for associating stone-artifact assemblages with hominin species in island Southeast Asia.     

Pulling apart catalytically active Tn5 synaptic complexes using magnetic tweezers

The Tn5 transposase is an example of a class of proteins that move DNA sequences (transposons) via a process called transposition. DNA transposition is a widespread genetic mobility mechanism that has profoundly affected the genomes of nearly all organisms. We have used single-DNA micromanipulation experiments to study the process by which Tn5 DNA transposons are identified and processed by their transposase protein. We have determined that the energy barrier to disassemble catalytically active synaptic complexes is 16 kcal mol(-1). However, we have found that the looping organization of DNA segments by transposase is less sequence-driven than previously thought. Loops anchored at some non-transposon end sequences display a disassembly energy barrier of 14 kcal mol(-1), nearly as stable as the synapses formed at known transposon end sequences. However, these non-transposon end sequence independent complexes do not mediate DNA cleavage. Therefore, the sequence-sensitivity for DNA binding and looping by Tn5 transposase is significantly less than that required for DNA cleavage. These results have implications for the in vivo down regulation of transposition and the cis-transposition bias of transposase.     

Allosteric communication in dihydrofolate reductase: signaling network and pathways for closed to occluded transition and back

Escherichia coli dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate to tetrahydrofolate. During the catalytic cycle, DHFR undergoes conformational transitions between the closed (CS) and occluded (OS) states that, respectively, describe whether the active site is closed or occluded by the Met20 loop. The CS→OS and the reverse transition may be viewed as allosteric transitions. Using a sequence-based approach, we identify a network of residues that represents the allostery wiring diagram. Many of the residues in the allostery wiring diagram, which are dispersed throughout the adenosine-binding domain as well as the loop domain, are not conserved. Several of the residues in the network have been previously shown by NMR experiments, mutational studies, and molecular dynamics simulations to be linked to equilibration conformational fluctuations of DHFR. To further probe the nature of events that occur during conformational fluctuations, we use a self-organized polymer model to monitor the kinetics of the CS→OS and the reverse transitions. During the CS→OS transition, coordinated changes in a number of residues in the loop domain enable the Met20 loop to slide along the α-helix in the adenosine-binding domain. Sliding is triggered by pulling of the Met20 loop by the βG-βH loop and the pushing action of the βG-βH loop. The residues that facilitate the Met20 loop motion are part of the network of residues that transmit allosteric signals during the CS→OS transition. Replacement of M16 and G121, whose C^α atoms are about 4.3 Å in the CS, by a disulfide cross-link impedes that CS→OS transition. The order of events in the OS→CS transition is not the reverse of the forward transition. The contact Glu18-Ser49 in the OS persists until the sliding of the Met20 loop is nearly complete. The ensemble of structures in the transition state in both the allosteric transitions is heterogeneous. The most probable transition-state structure resembles the OS (CS) in the CS→OS (OS→CS) transition, which is in accord with the Hammond postulate. Structures resembling the OS (CS) are present as minor (∼ 1-3%) components in equilibrated CS (OS) structures.     

NMR structure and functional characterization of a human cancer-related nucleoside triphosphatase

A screen of the human cancer genome anatomy project (CGAP) database was performed to search for new proteins involved in tumorigenesis. The resulting hits were further screened for recombinant expression, solubility and protein aggregation, which led to the identification of the previously unknown human cancer-related (HCR) protein encoded by the mRNA NM_032324 as a target for structure determination by NMR. The three-dimensional structure of the protein in its complex with ATPgammaS forms a three-layered alpha/beta sandwich, with a central nine-stranded beta-sheet surrounded by five alpha-helices. Sequence and three-dimensional structure comparisons with AAA+ ATPases revealed the presence of Walker A (GPPGVGKT) and Walker B (VCVIDEIG) motifs. Using 1D (31)P-NMR spectroscopy and a coupled enzymatic assay for the determination of inorganic phosphate, we showed that the purified recombinant protein is active as a non-specific nucleoside triphosphatase, with k(cat)=7.6x10(-3) s(-1). The structural basis for the enzymatic activity of HCR-NTPase was further characterized by site-directed mutagenesis of the Walker B motif, which further contributes to making the HCR-NTPase an attractive new target for further biochemical characterization in the context of its presumed role in human tumorigenesis.     

Defective signal joint recombination in fanconi anemia fibroblasts reveals a role for Rad50 in V(D)J recombination

V(D)J recombination of immunoglobulin loci is dependent on the immune cell-specific Rag1 and Rag2 proteins as well as a number of ubiquitously expressed cellular DNA repair proteins that catalyze non-homologous end-joining of DNA double-strand breaks. The evolutionarily conserved Rad50/Mre11/Nibrin protein complex has a role in DNA double-strand break-repair, suggesting that these proteins, too, may participate in V(D)J recombination. Recent findings demonstrating that Rad50 function is defective in cells from patients afflicted with Fanconi anemia provide a possible mechanistic explanation for previous findings that lymphoblasts derived from these patients exhibit subtle defects in V(D)J recombination of extrachromosomal plasmid molecules. Here, we describe a series of findings that provide convincing evidence for a role of the Rad50 protein complex in V(D)J recombination. We found that the fidelity of V(D)J signal joint recombination in fibroblasts from patients afflicted with Fanconi anemia was reduced by nearly tenfold, compared to that observed in fibroblasts from normal donors. Second, we observed that antibody-mediated inhibition of the Rad50, Mre11, or Nibrin proteins reduced the fidelity of signal joint recombination significantly in wild-type cells. The latter finding was somewhat unexpected, because signal joint rejoining in cells from patients with Nijmegen breakage syndrome, which results from mutations in the Nibrin gene, occurs with normal fidelity. However, introduction of anti-Nibrin antibodies into these cells reduced the fidelity of signal joint recombination dramatically. These data reveal for the first time a role for the Rad50 complex in V(D)J recombination, and demonstrate that the protein product of the disease-causing allele responsible for Nijmegen breakage syndrome encodes a protein with residual DNA double-strand break repair activity.