Using subsite coupling to predict signal peptides.
(49/692)
Given a nascent protein sequence, how can one predict its signal peptide or "Zipcode" sequence? This is a first important problem for scientists to use signal peptides as a vehicle to find new drugs or to reprogram cells for gene therapy. Based on a model that takes into account the coupling effect among some key subsites, the so-called [-3, -1, +1] coupling model, a new prediction algorithm is developed. The overall rate of correct prediction for 1939 secretory proteins and 1440 non-secretary proteins was over 92%. It has not escaped our attention that the new method may also serve as a useful tool for helping investigate further many unclear details regarding the molecular mechanism of the ZIP code protein-sorting system in cells. (+info)
Relationship of 16S rRNA sequence similarity to DNA hybridization in prokaryotes.
(50/692)
The relationship between 16S rRNA sequence similarity (S) and the extent of DNA hybridization (D) was well described by the equation In(-InD) = 0.53 [In(-InS)]+2.201 when D was determined by either the S1 nuclease or membrane filter methods. When the presence of nonultrametric rRNA sequences and differences between genera or families were controlled, this relationship accounted for 78% of the variability of D given S, and it was possible to estimate the distribution of D from S with a known precision. Thus, D<0.70 was expected to occur 50, 95 and 99% of the time when S was 0.998, 0.992 and 0.986, respectively. The relationship between D and S varied between prokaryotic taxa even within the same subphylum, and more precise estimates of D could be made when the relationship for a particular taxon was known. The relationship between D and S was not significantly different between the prokaryotic domains, and S appeared to be a quasi-molecular clock of approximately constant rate when averaging effects and stochastic factors were taken into account. The relationship between logD and logS was nonlinear, and D provided a very poor measure of relatedness for distantly related organisms. For instance, within the range 1.0 >S>> 0.95, D decreased from 1.0 to 0.15; and within the range 0.95 >S> 0.90, D decreased from 0.15 to 0.06. Lastly, at least some of the rRNA sequences from about one-third of the taxa examined had nonultrametric properties where S was much lower than expected from the value of D. For these taxa, S was a poor indicator of relatedness for closely related strains. Thus, the ultrametric properties of rRNA sequences should be tested before making taxonomic or phylogenetic conclusions based upon S. (+info)
Structural adaptation of enzymes to low temperatures.
(51/692)
A systematic comparative analysis of 21 psychrophilic enzymes belonging to different structural families from prokaryotic and eukaryotic organisms is reported. The sequences of these enzymes were multiply aligned to 427 homologous proteins from mesophiles and thermophiles. The net flux of amino acid exchanges from meso/thermophilic to psychrophilic enzymes was measured. To assign the observed preferred exchanges to different structural environments, such as secondary structure, solvent accessibility and subunit interfaces, homology modeling was utilized to predict the secondary structure and accessibility of amino acid residues for the psychrophilic enzymes for which no experimental three-dimensional structure is available. Our results show a clear tendency for the charged residues Arg and Glu to be replaced at exposed sites on alpha-helices by Lys and Ala, respectively, in the direction from 'hot' to 'cold' enzymes. Val is replaced by Ala at buried regions in alpha-helices. Compositional analysis of psychrophilic enzymes shows a significant increase in Ala and Asn and a decrease in Arg at exposed sites. Buried sites in beta-strands tend to be depleted of VAL: Possible implications of the observed structural variations for protein stability and engineering are discussed. (+info)
Cytokinesis in prokaryotes and eukaryotes: common principles and different solutions.
(52/692)
Cytokinesis requires duplication of cellular structures followed by bipolarization of the predivisional cell. As a common principle, this applies to prokaryotes as well as eukaryotes. With respect to eukaryotes, the discussion has focused mainly on Saccharomyces cerevisiae and on Schizosaccharomyces pombe. Escherichia coli and to a lesser extent Bacillus subtilis have been used as prokaryotic examples. To establish a bipolar cell, duplication of a eukaryotic origin of DNA replication as well as its genome is not sufficient. Duplication of the microtubule-organizing center is required as a prelude to mitosis, and it is here that the dynamic cytoskeleton with all its associated proteins comes to the fore. In prokaryotes, a cytoskeleton that pervades the cytoplasm appears to be absent. DNA replication and the concomitant DNA segregation seem to occur without help from extensive cytosolic supramacromolecular assemblies but with help from the elongating cellular envelope. Prokaryotic cytokinesis proceeds through a contracting ring, which has a roughly 100-fold-smaller circumference than its eukaryotic counterpart. Although the ring contains proteins that can be considered as predecessors of actin, tubulin, and microtubule-associated proteins, its macromolecular composition is essentially different. (+info)
A single vector containing modified cre recombinase and LOX recombination sequences for inducible tissue-specific amplification of gene expression.
(53/692)
The selective alteration of the genome using Cre recombinase to target the rearrangement of genes flanked by LOX recognition sequences has required the use of two separate genetic constructs in trans, one containing cre and the other containing the gene of interest flanked by LOX sites. We have developed a strategy in which both the cre recombinase gene and LOX recombination sites may be cloned within a single vector in cis. This method uses a modified form of Cre (CREM) that contains alterations to the 5' region including the introduction of a Kozak consensus sequence and insertion of a functional intron. This system allows for the inducible, tissue-specific activation or inactivation of gene expression in a single vector and can be utilized for the 300-fold amplification of gene expression from a weak promoter. This approach can be applied to targeting strategies for generating genetically altered mice and gene therapy. (+info)
The ins(ide) and out(side) of dolichyl phosphate biosynthesis and recycling in the endoplasmic reticulum.
(54/692)
The precursor oligosaccharide donor for protein N-glycosylation in eukaryotes, Glc3Man9GlcNAc(2)-P-P-dolichol, is synthesized in two stages on both leaflets of the rough endoplasmic reticulum (ER). There is good evidence that the level of dolichyl monophosphate (Dol-P) is one rate-controlling factor in the first stage of the assembly process. In the current topological model it is proposed that ER proteins (flippases) then mediate the transbilayer movement of Man-P-Dol, Glc-P-Dol, and Man5GlcNAc(2)-P-P-Dol from the cytoplasmic leaflet to the lumenal leaflet. The rate of flipping of the three intermediates could plausibly influence the conversion of Man5GlcNAc(2)-P-P-Dol to Glc3Man(9)GlcNAc(2)-P-P-Dol in the second stage on the lumenal side of the rough ER. This article reviews the current understanding of the enzymes involved in the de novo biosynthesis of Dol-P and other polyisoprenoid glycosyl carrier lipids and speculates about the role of membrane proteins and enzymes that could be involved in the transbilayer movement of the lipid intermediates and the recycling of Dol-P and Dol-P-P discharged during glycosylphosphatidylinositol anchor biosynthesis, N-glycosylation, and O- and C-mannosylation reactions on the lumenal surface of the rough ER. (+info)
Intrinsic noise in gene regulatory networks.
(55/692)
Cells are intrinsically noisy biochemical reactors: low reactant numbers can lead to significant statistical fluctuations in molecule numbers and reaction rates. Here we use an analytic model to investigate the emergent noise properties of genetic systems. We find for a single gene that noise is essentially determined at the translational level, and that the mean and variance of protein concentration can be independently controlled. The noise strength immediately following single gene induction is almost twice the final steady-state value. We find that fluctuations in the concentrations of a regulatory protein can propagate through a genetic cascade; translational noise control could explain the inefficient translation rates observed for genes encoding such regulatory proteins. For an autoregulatory protein, we demonstrate that negative feedback efficiently decreases system noise. The model can be used to predict the noise characteristics of networks of arbitrary connectivity. The general procedure is further illustrated for an autocatalytic protein and a bistable genetic switch. The analysis of intrinsic noise reveals biological roles of gene network structures and can lead to a deeper understanding of their evolutionary origin. (+info)
Historical overview: searching for replication help in all of the rec places.
(56/692)
For several decades, research into the mechanisms of genetic recombination proceeded without a complete understanding of its cellular function or its place in DNA metabolism. Many lines of research recently have coalesced to reveal a thorough integration of most aspects of DNA metabolism, including recombination. In bacteria, the primary function of homologous genetic recombination is the repair of stalled or collapsed replication forks. Recombinational DNA repair of replication forks is a surprisingly common process, even under normal growth conditions. The new results feature multiple pathways for repair and the involvement of many enzymatic systems. The long-recognized integration of replication and recombination in the DNA metabolism of bacteriophage T4 has moved into the spotlight with its clear mechanistic precedents. In eukaryotes, a similar integration of replication and recombination is seen in meiotic recombination as well as in the repair of replication forks and double-strand breaks generated by environmental abuse. Basic mechanisms for replication fork repair can now inform continued research into other aspects of recombination. This overview attempts to trace the history of the search for recombination function in bacteria and their bacteriophages, as well as some of the parallel paths taken in eukaryotic recombination research. (+info)