Genomic methods for measuring DNA replication dynamics.

Genomic DNA replicates according to a defined temporal program in which early-replicating loci are associated with open chromatin, higher gene density, and increased gene expression levels, while late-replicating loci tend to be heterochromatic and show higher rates of genomic instability. The ability to measure DNA replication dynamics at genome scale has proven crucial for understanding the mechanisms and cellular consequences of DNA replication timing. Several methods, such as quantification of nucleotide analog incorporation and DNA copy number analyses, can accurately reconstruct the genomic replication timing profiles of various species and cell types. More recent developments have expanded the DNA replication genomic toolkit to assays that directly measure the activity of replication origins, while single-cell replication timing assays are beginning to reveal a new level of replication timing regulation. The combination of these methods, applied on a genomic scale and in multiple biological systems, promises to resolve many open questions and lead to a holistic understanding of how eukaryotic cells replicate their genomes accurately and efficiently.

Phosphate: from stardust to eukaryotic cell cycle control

Phosphorus is a pivotal element in all biochemical systems: it serves to store metabolic energy as ATP, it forms the backbone of genetic material such as RNA and DNA, and it separates cells from the environment as phospholipids. In addition to this 'big hits', phosphorus has recently been shown to play an important role in other important processes such as cell cycle regulation. In the present review, we briefly summarize the biological processes in which phosphorus is involved in the yeast Saccharomyces cerevisiae before discussing our latest findings on the role of this element in the regulation of DNA replication in this eukaryotic model organism. We describe both the role of phosphorus in the regulation of G1 progression by means of the Cyclin Dependent Kinase (CDK) Pho85 and the stabilization of the cyclin Cln3, as well as the role of other molecule composed of phosphorus-the polyphosphate-in cell cycle progression, dNTP synthesis, and genome stability. Given the eminent role played by phosphorus in life, we outline the future of phosphorus in the context of one of the main challenges in human health: cancer treatment (AU)

No disponible

Investigation of Ribosomes Using Molecular Dynamics Simulation Methods.

The ribosome as a complex molecular machine undergoes significant conformational changes while synthesizing a protein molecule. Molecular dynamics simulations have been used as complementary approaches to X-ray crystallography and cryoelectron microscopy, as well as biochemical methods, to answer many questions that modern structural methods leave unsolved. In this review, we demonstrate that all-atom modeling of ribosome molecular dynamics is particularly useful in describing the process of tRNA translocation, atomic details of behavior of nascent peptides, antibiotics, and other small molecules in the ribosomal tunnel, and the putative mechanism of allosteric signal transmission to functional sites of the ribosome.

Modes of deformation of walled cells.

The bewildering morphological diversity found in cells is one of the starkest illustrations of life's ability to self-organize. Yet the morphogenetic mechanisms that produce the multifarious shapes of cells are still poorly understood. The shared similarities between the walled cells of prokaryotes, many protists, fungi, and plants make these groups particularly appealing to begin investigating how morphological diversity is generated at the cell level. In this review, I attempt a first classification of the different modes of surface deformation used by walled cells. Five modes of deformation were identified: inextensional bending, equi-area shear, elastic stretching, processive intussusception, and chemorheological growth. The two most restrictive modes-inextensional and equi-area deformations-are embodied in the exine of pollen grains and the wall-like pellicle of euglenoids, respectively. For these modes, it is possible to express the deformed geometry of the cell explicitly in terms of the undeformed geometry and other easily observable geometrical parameters. The greatest morphogenetic power is reached with the processive intussusception and chemorheological growth mechanisms that underlie the expansive growth of walled cells. A comparison of these two growth mechanisms suggests a possible way to tackle the complexity behind wall growth.

Imaging living cells surface and quantifying its properties at high resolution using AFM in QI™ mode.

Since the last 10 years, AFM has become a powerful tool to study biological samples. However, the classical modes offered (imaging or tapping mode) often damage sample that are too soft or loosely immobilized. If imaging and mechanical properties are required, it requests long recording time as two different experiments must be conducted independently. In this study we compare the new QI™ mode against contact imaging mode and force volume mode, and we point out its benefit in the new challenges in biology on six different models: Escherichia coli, Candida albicans, Aspergillus fumigatus, Chinese hamster ovary cells and their isolated nuclei, and human colorectal tumor cells.

Microparticles in physiological and in pathological conditions.

Chronic diseases pose a severe burden to modern National Health Systems. Individuals nowadays have a far more extended lifespan than in the past, but healthy living was only scantily extended. As much as longer life is desirable, it is saddened by chronic diseases and organ malfunctions. One contributor to these problems was recognized to be represented by microparticles (MPs). Our purpose is to better understand MPs, to contrast their ominous threat and possible clinical importance. For this intent we correlated MPs with thrombotic pathologies, hemophilia, malaria, diabetes, cardiovascular diseases, endothelial dysfunctions, pulmonary hypertension, ischemic stroke, pre-eclampsia, rheumatologic diseases-rheumatoid arthritis, polymyositis-dermatomyositis, angiogenesis and tumor progression-cancer; we listed the possibilities of using them to improve transfusion methods, as a marker for acute allograft rejection, in stem cell transplantation, as neuronal biomarkers, to understand gender-specific susceptibility for diseases and to improve vaccination methods and we presented some methods for the detection of MPs.

Nutrient control of eukaryote cell growth: a systems biology study in yeast.

BACKGROUND: To elucidate the biological processes affected by changes in growth rate and nutrient availability, we have performed a comprehensive analysis of the transcriptome, proteome and metabolome responses of chemostat cultures of the yeast, Saccharomyces cerevisiae, growing at a range of growth rates and in four different nutrient-limiting conditions. RESULTS: We find significant changes in expression for many genes in each of the four nutrient-limited conditions tested. We also observe several processes that respond differently to changes in growth rate and are specific to each nutrient-limiting condition. These include carbohydrate storage, mitochondrial function, ribosome synthesis, and phosphate transport. Integrating transcriptome data with proteome measurements allows us to identify previously unrecognized examples of post-transcriptional regulation in response to both nutrient and growth-rate signals. CONCLUSIONS: Our results emphasize the unique properties of carbon metabolism and the carbon substrate, the limitation of which induces significant changes in gene regulation at the transcriptional and post-transcriptional level, as well as altering how many genes respond to growth rate. By comparison, the responses to growth limitation by other nutrients involve a smaller set of genes that participate in specific pathways. See associated commentary http://www.biomedcentral.com/1741-7007/8/62.

Trends in selenium utilization in marine microbial world revealed through the analysis of the global ocean sampling (GOS) project.

Selenium is an important trace element that occurs in proteins in the form of selenocysteine (Sec) and in tRNAs in the form of selenouridine. Recent large-scale metagenomics projects provide an opportunity for understanding global trends in trace element utilization. Herein, we characterized the selenoproteome of the microbial marine community derived from the Global Ocean Sampling (GOS) expedition. More than 3,600 selenoprotein gene sequences belonging to 58 protein families were detected, including sequences representing 7 newly identified selenoprotein families, such as homologs of ferredoxin-thioredoxin reductase and serine protease. In addition, a new eukaryotic selenoprotein family, thiol reductase GILT, was identified. Most GOS selenoprotein families originated from Cys-containing thiol oxidoreductases. In both Pacific and Atlantic microbial communities, SelW-like and SelD were the most widespread selenoproteins. Geographic location had little influence on Sec utilization as measured by selenoprotein variety and the number of selenoprotein genes detected; however, both higher temperature and marine (as opposed to freshwater and other aquatic) environment were associated with increased use of this amino acid. Selenoproteins were also detected with preference for either environment. We identified novel fusion forms of several selenoproteins that highlight redox activities of these proteins. Almost half of Cys-containing SelDs were fused with NADH dehydrogenase, whereas such SelD forms were rare in terrestrial organisms. The selenouridine utilization trait was also analyzed and showed an independent evolutionary relationship with Sec utilization. Overall, our study provides insights into global trends in microbial selenium utilization in marine environments.

[Construction expression and biologic activity of recombinant human sCR1 eucaryotic cell].

AIM: To express human soluble complement receptor type 1(sCR1)protein using ferment cell secreting type carrier and study the extraorgan biologic activity of recombinant human sCR1 fusion protein. METHODS: Total human RNA was extracted from peripheral blood. The full length cDNA of human sCR1 gene was obtained by RT-PCR and them, cloned into Pichia pastoris eukaryotic expression vector pPIC9k to construct the recombinant plasmid pPIC9k-sCR1 containing human sCR1.After identified by DNA sequencing, the recombinant plasmid pPIC9k-sCR1 was transformed into Pichia pastoris SMD1168. The ferment cell line of the recombinant sCR1 which was chosen by G418 resistance was identified by PCR, After methanol induction, the expressed protein products were verified by SDS-PAGE and Western blot, purified by Ni(2+)-NTA agarose affinity chromatography, and its biologic activity was identified. RESULTS: The obtained Pichia pastoris secretion type yeast carrier pPIC9k-sCR1 was chosen by G418 and identified by PCR to get a highly copied and integral recombinant ferment cell line. The recombinant human sCR1 fusion protein was expressed by yeast cells containing pPIC9k-sCR1 induced by methanol. It was a protein band about M(r) 31 000 in gel, which could be identified by CD35 of anti-sCR1 protein monoclonal antibody with Western blotting technique. The highly purified sCR1 fusion protein and its biologic activity were detected obtained by Ni(2+)-NTA agarose affinity chromatography. CONCLUSION: The recombinant human sCR1 fusion protein can be highly expressed in the Pichia pastoris expression system, which resembles the human natural protein's antigenicity and biologic activity.

Lipid accumulation in Schizochytrium G13/2S produced in continuous culture.

Lipid and docosahexaenoic acid (DHA) accumulation into Schizochytrium G13/2S was studied under batch and continuous culture. Different glucose and glutamate concentrations were supplemented in a defined medium. During batch cultivation, lipid accumulation, 35% total fatty acids (TFA) occurred at the arithmetic growth phase but ceased when cell growth stopped. When continuous culture was performed under different glutamate concentrations, nitrogen-growth-limiting conditions induced the accumulation of 30-28% TFA in Schizochytrium. As the dilution rate decreased from 0.08 to 0.02 h(-1), both cell dry weight and TFA content of the cell increased. Under a constant dilution rate of 0.04 h(-1), carbon-limiting conditions decreased the TFA to 22%. Fatty acid profile was not affected by the different nutrient concentrations provided during continuous culture. Consequently, lipid accumulation can be induced through the carbon and nitrogen source concentration in the medium to maximise the TFA and subsequently DHA productivity by this microorganism.

Diffusion-limited phase separation in eukaryotic chemotaxis.

The ability of cells to sense spatial gradients of chemoattractant factors governs the development of complex eukaryotic organisms. Cells exposed to shallow chemoattractant gradients respond with strong accumulation of the enzyme phosphatidylinositol 3-kinase (PI3K) and its D3-phosphoinositide product (PIP(3)) on the plasma membrane side exposed to the highest chemoattractant concentration, whereas PIP(3)-degrading enzyme PTEN and its product PIP(2) localize in a complementary pattern. Such an early symmetry-breaking event is a mandatory step for directed cell movement elicited by chemoattractants, but its physical origin is still mysterious. Here, we propose that directional sensing is the consequence of a phase-ordering process mediated by phosphoinositide diffusion and driven by the distribution of chemotactic signal. By studying a realistic reaction-diffusion lattice model that describes PI3K and PTEN enzymatic activity, recruitment to the plasma membrane, and diffusion of their phosphoinositide products, we show that the effective enzyme-enzyme interaction induced by catalysis and diffusion introduces an instability of the system toward phase separation for realistic values of physical parameters. In this framework, large reversible amplification of shallow chemotactic gradients, selective localization of chemical factors, macroscopic response timescales, and spontaneous polarization arise naturally. The model is robust with respect to order-of-magnitude variations of the parameters.

A novel eukaryotic selenoprotein in the haptophyte alga Emiliania huxleyi.

The diversity of selenoproteins raises the question of why many life forms require selenium. Especially in photosynthetic organisms, the biochemical basis for the requirement for selenium is unclear because there is little information on selenoproteins. We found six selenium-containing proteins in a haptophyte alga, Emiliania huxleyi, which requires selenium for growth. The 27-kDa protein EhSEP2 was isolated, and its cDNA was cloned. The deduced amino acid sequence revealed that EhSEP2 is homologous to protein disulfide isomerase (PDI) and contains a highly conserved thioredoxin domain. The nucleotide sequence contains an in-frame TGA codon encoding selenocysteine at the position corresponding to the cysteine residue in the reaction center of known PDIs. However, no typical selenocysteine insertion sequence was found in the EhSEP2 cDNA. The EhSEP2 mRNA level was related to the abundance of selenium. E. huxleyi possesses a novel PDI-like selenoprotein and may have a novel type of selenocysteine insertion machinery.

Chemotaxis assays for eukaryotic cells.

Chemotaxis is a complex response of a cell to an external stimulus. It involves detecting and measuring the concentration of the chemoattractant, biochemical transmission of the information, and the motility and adhesive changes associated with the response. This unit describes a number of chemotaxis assays that can be used to identify chemoattractants individually and in large-scale screenings, to distinguish chemotaxis from chemokinesis, and to analyze cellular behavioral and biochemical responses. Some of these assays such as the filter, under agarose, and small population assays, can be used to monitor the behavior of large groups of cells; the bridge, pipet, and upshift assays can be used to analyze the responses of single cells.

MAGPIE/EGRET annotation of the 2.9-Mb Drosophila melanogaster Adh region.

Our challenge in annotating the 2.91-Mb Adh region of the Drosophila melanogaster genome was to identify genetic and genomic features automatically, completely, and precisely within a 6-week period. To do so, we augmented the MAGPIE microbial genome annotation system to handle eukaryotic genomic sequence data. The new configuration required the integration of eukaryotic gene-finding tools and DNA repeat tools into the automatic data collection module. It also required us to define in MAGPIE new strategies to combine data about eukaryotic exon predictions with functional data to refine the exon predictions. At the heart of the resulting new eukaryotic genome annotation system is a reverse comparison of public protein and complementary DNA sequences against the input genome to identify missing exons and to refine exon boundaries. The software modules that add eukaryotic genome annotation capability to MAGPIE are available as EGRET (Eukaryotic Genome Rapid Evaluation Tool).

[Role of hypothalamic nonapeptides in interaction of prokaryotic and eukaryotic cells]. / Rol' gipotalamicheskikh nonapeptidov vo vzaimodeistvii pro- i éukarioticheskikh kletok.

The role of hypothalamic nonapeptides in the interaction of prokaryotic and eukaryotic cells was studied in the experimental setting. Nonapeptides were found to stimulate the adaptive and regenerative properties of eukaryotic cells and they are likely to have an antimicrobic effect on prokaryotic ones. The paper discusses the modulating role of nonapeptides in the establishment of symbiotic relations in the bacterial agent-host system.

A complementary circular code in the protein coding genes.

Recently, shifted periodicities 1 modulo 3 and 2 modulo 3 have been identified in protein (coding) genes of both prokaryotes and eukaryotes with autocorrelation functions analysing eight of 64 trinucleotides (Arquès et al., 1995). This observation suggests that the trinucleotides are associated with frames in protein genes. In order to verify this hypothesis, a distribution of the 64 trinucleotides AAA,..., TTT is studied in both gene populations by using a simple method based on the trinucleotide frequencies per frame. In protein genes, the trinucleotides can be read in three frames: the reading frame 0 established by the ATG start trinucleotide and frame 1 (resp. 2) which is the frame 0 shifted by 1 (resp. 2) nucleotide in the 5'-3' direction. Then, the occurrence frequencies of the 64 trinucleotides are computed in the three frames. By classifying each of the 64 trinucleotides in its preferential occurrence frame, i.e. the frame associated with its highest frequency, three subsets of trinucleotides can be identified in the three frames. This approach is applied in the two gene populations. Unexpectedly, the same three subsets of trinucleotides are identified in these two gene populations: Tzero = Xzero [symbol: see text] {AAA,TTT} with Xzero = {AAC,AAT,ACC,ATC,ATT, CAG,CTC,CTG,GAA,GAC,GAG, GAT,GCC,GGC,GGT,GTA,GTC,GTT,TAC,TTC} in frame 0, T1 = X1 [symbol: see text] {CCC} in frame 1 and T2 = X2 [symbol: see text] {GGG} in frame 2, each subset Xzero, X1 and X2 having 20 trinucleotides. Surprisingly, these three subsets have five important properties: (i) the property of maximal circular code for Xzero (resp. X1, X2) allowing the automatical retrieval of frame 0 (resp. 1, 2) in any region of a protein gene model (formed by a series of trinucleotides of Xzero) without using a start codon; (ii) the DNA complementarity property C (e.g. C(AAC) = GTT): C(T0) = T0, C(T1) = T2 and C(T2) = T1 allowing the two paired reading frames of a DNA double helix simultaneously to code for amino acids; (iii) the circular permutation property P (e.g. P(AAC) = ACA): P(Xzero) = X1 and P(X1) = X2 implying that the two subsets X1 and X2 can be deduced from Xzero; (iv) the rarity property with an occurrence probability of Xzero equal to 6 x 10(-8); and (v) the concatenation property with: a high frequency (27.5%) of misplaced trinucleotides in the shifted frames, a maximum (13 nucleotides) length of the minimal window to automatically retrieve the frame and an occurrence of the four types of nucleotides in the three trinucleotides sites, in favour of an evolutionary code. In the Discussion, the identified subsets Tzero, T1 and T2 replaced in the three two-letter genetic alphabets purine/pyrimidine, amino/ceto and strong/weak interaction, allow us to deduce that the RNY model (R = purine = A or G, Y = pyrimidine = C or T, N = R or Y) (Eigen & Schuster, 1978) is the closest two-letter codon model to the trinucleotides of Tzero. Then, these three subsets are related to the genetic code. The trinucleotides of Tzero code for 13 amino acids: Ala, Asn, Asp, Gln, Glu, Gly, Ile, Leu, Lys, Phe, Thr, Tyr and Val. Finally, a strong correlation between the usage of the trinucleotides of Tzero in protein genes and the amino acid frequencies in proteins is observed as six among seven amino acids not coded by Tzero, have as expected the lowest frequencies in proteins of both prokaryotes and eukaryotes.

Chronobiology at the cellular and molecular levels: models and mechanisms for circadian timekeeping.

This review considers cellular chronobiology and examines, at least in a superficial way, several classes of models and mechanisms that have been proposed for circadian rhythmicity and some of the experimental approaches that have appeared to be most productive. After a brief discussion of temporal organization and the metabolic, epigenetic, and circadian time domains, the general properties of circadian rhythms are enumerated. A survey of independent oscillations in isolated organs, tissues, and cells is followed by a review of selected circadian rhythms in eukaryotic microorganisms, with particular emphasis placed on the rhythm of cell division in the algal flagellate Euglena as a model system illustrating temporal differentiation. In the ensuing section, experimental approaches to circadian clock mechanisms are considered. The dissection of the clock by the use of chemical inhibitors is illustrated for the rhythm of bioluminescence in the marine dinoflagellate Gonyaulax and for the rhythm of photosynthetic capacity in the unicellular green alga Acetabularia. Alternatively, genetic analysis of circadian oscillators is considered in the green alga Chlamydomonas and in the bread mold Neurospora, both of which have yielded clock mutants and mutants having biochemical lesions that exhibit altered clock properties. On the basis of the evidence generated by these experimental approaches, several classes of biochemical and molecular models for circadian clocks have been proposed. These include strictly molecular models, feedback loop (network) models, transcriptional (tape-reading) models, and membrane models; some of their key elements and predictions are discussed. Finally, a number of general unsolved problems at the cellular level are briefly mentioned: cell cycle interfaces, the evolution of circadian rhythmicity, the possibility of multiple cellular oscillators, chronopharmacology and chronotherapy, and cell-cycle clocks in development and aging.