Heterologous Stop Codon Readthrough of Metazoan Readthrough Candidates in Yeast

Recent analysis of genomic signatures in mammals, flies, and worms indicates that functional translational stop codon readthrough is considerably more abundant in metazoa than previously recognized, but this analysis provides only limited clues about the function or mechanism of readthrough. If an mRNA known to be read through in one species is also read through in another, perhaps these questions can be studied in a simpler setting. With this end in mind, we have investigated whether some of the readthrough genes in human, fly, and worm also exhibit readthrough when expressed in S. cerevisiae. We found that readthrough was highest in a gene with a post-stop hexamer known to trigger readthrough, while other metazoan readthrough genes exhibit borderline readthrough in S. cerevisiae.National Institutes of Health (U.S.) (5U54HG004555-03

CodonLogo: a sequence logo-based viewer for codon patterns

Motivation: Conserved patterns across a multiple sequence alignment can be visualized by generating sequence logos. Sequence logos show each column in the alignment as stacks of symbol(s) where the height of a stack is proportional to its informational content, whereas the height of each symbol within the stack is proportional to its frequency in the column. Sequence logos use symbols of either nucleotide or amino acid alphabets. However, certain regulatory signals in messenger RNA (mRNA) act as combinations of codons. Yet no tool is available for visualization of conserved codon patterns

A Mathematical Model of Liver Cell Aggregation In Vitro

The behavior of mammalian cells within three-dimensional structures is an area of intense biological research and underpins the efforts of tissue engineers to regenerate human tissues for clinical applications. In the particular case of hepatocytes (liver cells), the formation of spheroidal multicellular aggregates has been shown to improve cell viability and functionality compared to traditional monolayer culture techniques. We propose a simple mathematical model for the early stages of this aggregation process, when cell clusters form on the surface of the extracellular matrix (ECM) layer on which they are seeded. We focus on interactions between the cells and the viscoelastic ECM substrate. Governing equations for the cells, culture medium, and ECM are derived using the principles of mass and momentum balance. The model is then reduced to a system of four partial differential equations, which are investigated analytically and numerically. The model predicts that provided cells are seeded at a suitable density, aggregates with clearly defined boundaries and a spatially uniform cell density on the interior will form. While the mechanical properties of the ECM do not appear to have a significant effect, strong cell-ECM interactions can inhibit, or possibly prevent, the formation of aggregates. The paper concludes with a discussion of our key findings and suggestions for future work

A Viable Hypomorphic Allele of the Essential IMP3 Gene Reveals Novel Protein Functions in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the essential IMP3 gene encodes a component of the SSU processome, a large ribonucleoprotein complex required for processing of small ribosomal subunit RNA precursors. Mutation of the IMP3 termination codon to a sense codon resulted in a viable mutant allele producing a C-terminal elongated form of the Imp3 protein. A strain expressing the mutant allele displayed ribosome biogenesis defects equivalent to IMP3 depletion. This hypomorphic allele represented a unique opportunity to investigate and better understand the Imp3p functions. We demonstrated that the +1 frameshifting was increased in the mutant strain. Further characterizations revealed involvement of the Imp3 protein in DNA repair and telomere length control, pointing to a functional relationship between both pathways and ribosome biogenesis

Increased incidence of rare codon clusters at 5' and 3' gene termini:implications for function

<p>Abstract</p> <p>Background</p> <p>The process of translation can be affected by the use of rare versus common codons within the mRNA transcript.</p> <p>Results</p> <p>Here, we show that rare codons are enriched at the 5' and 3' termini of genes from <it>E. coli </it>and other prokaryotes. Genes predicted to be secreted show significant enrichment in 5' rare codon clusters, but not 3' rare codon clusters. Surprisingly, no correlation between 5' mRNA structure and rare codon usage was observed.</p> <p>Conclusions</p> <p>Potential functional roles for the enrichment of rare codons at terminal positions are explored.</p

Contact-inhibited chemotaxis in de novo and sprouting blood-vessel growth

Blood vessels form either when dispersed endothelial cells (the cells lining the inner walls of fully-formed blood vessels) organize into a vessel network (vasculogenesis), or by sprouting or splitting of existing blood vessels (angiogenesis). Although they are closely related biologically, no current model explains both phenomena with a single biophysical mechanism. Most computational models describe sprouting at the level of the blood vessel, ignoring how cell behavior drives branch splitting during sprouting. We present a cell-based, Glazier-Graner-Hogeweg-model simulation of the initial patterning before the vascular cords form lumens, based on plausible behaviors of endothelial cells. The endothelial cells secrete a chemoattractant, which attracts other endothelial cells. As in the classic Keller-Segel model, chemotaxis by itself causes cells to aggregate into isolated clusters. However, including experimentally-observed adhesion-driven contact inhibition of chemotaxis in the simulation causes randomly-distributed cells to organize into networks and cell aggregates to sprout, reproducing aspects of both de novo and sprouting blood-vessel growth. We discuss two branching instabilities responsible for our results. Cells at the surfaces of cell clusters attempting to migrate to the centers of the clusters produce a buckling instability. In a model variant that eliminates the surface-normal force, a dissipative mechanism drives sprouting, with the secreted chemical acting both as a chemoattractant and as an inhibitor of pseudopod extension. The branching instabilities responsible for our results, which result from contact inhibition of chemotaxis, are both generic developmental mechanisms and interesting examples of unusual patterning instabilities.Comment: Thoroughly revised version, now in press in PLoS Computational Biology. 53 pages, 13 figures, 2 supporting figures, 56 supporting movies, source code and parameters files for computer simulations provided. Supporting information: http://www.psb.ugent.be/~romer/ploscompbiol/ Source code: http://sourceforge.net/projects/tst

Statistical Analysis of Readthrough Levels for Nonsense Mutations in Mammalian Cells Reveals a Major Determinant of Response to Gentamicin

The efficiency of translation termination depends on the nature of the stop codon and the surrounding nucleotides. Some molecules, such as aminoglycoside antibiotics (gentamicin), decrease termination efficiency and are currently being evaluated for diseases caused by premature termination codons. However, the readthrough response to treatment is highly variable and little is known about the rules governing readthrough level and response to aminoglycosides. In this study, we carried out in-depth statistical analysis on a very large set of nonsense mutations to decipher the elements of nucleotide context responsible for modulating readthrough levels and gentamicin response. We quantified readthrough for 66 sequences containing a stop codon, in the presence and absence of gentamicin, in cultured mammalian cells. We demonstrated that the efficiency of readthrough after treatment is determined by the complex interplay between the stop codon and a larger sequence context. There was a strong positive correlation between basal and induced readthrough levels, and a weak negative correlation between basal readthrough level and gentamicin response (i.e. the factor of increase from basal to induced readthrough levels). The identity of the stop codon did not affect the response to gentamicin treatment. In agreement with a previous report, we confirm that the presence of a cytosine in +4 position promotes higher basal and gentamicin-induced readthrough than other nucleotides. We highlight for the first time that the presence of a uracil residue immediately upstream from the stop codon is a major determinant of the response to gentamicin. Moreover, this effect was mediated by the nucleotide itself, rather than by the amino-acid or tRNA corresponding to the −1 codon. Finally, we point out that a uracil at this position associated with a cytosine at +4 results in an optimal gentamicin-induced readthrough, which is the therapeutically relevant variable

Stereochemical Basis for Engineered Pyrrolysyl-tRNA Synthetase and the Efficient in Vivo Incorporation of Structurally Divergent Non-native Amino Acids

bS Supporting Information Incorporation of Uaas into proteins using a host’s endogenoustranslation machinery opens the door to addressing questions with chemical precision that is unattainable using naturally occurring amino acids. This expanded toolset allows one to pose and answer more in-depth molecular questions without the limitations imposed by the 20 natural amino acids used in traditional mutagenic analyses.1,2 Aminoacyl-tRNA synthetases (RSs) obtained by structure-based engineering and directed evolution efficiently recognize and activate Uaas through ATP-dependent adenylation and subsequently catalyze transfer to their cognate tRNA. To date, more than 70 Uaas are now amenable to translational insertion into proteins in bacteria, yeast, or mammalian cells using these artificially evolved tRNA/ RS pairs.3 By choosing particular matching sets of tRNA/RSs from diverse organisms, the pairs can function in vivo in a

A bacterial genome in transition - an exceptional enrichment of IS elements but lack of evidence for recent transposition in the symbiont Amoebophilus asiaticus

<p>Abstract</p> <p>Background</p> <p>Insertion sequence (IS) elements are important mediators of genome plasticity and are widespread among bacterial and archaeal genomes. The 1.88 Mbp genome of the obligate intracellular amoeba symbiont <it>Amoebophilus asiaticus </it>contains an unusually large number of transposase genes (n = 354; 23% of all genes).</p> <p>Results</p> <p>The transposase genes in the <it>A. asiaticus </it>genome can be assigned to 16 different IS elements termed ISCaa1 to ISCaa16, which are represented by 2 to 24 full-length copies, respectively. Despite this high IS element load, the <it>A. asiaticus </it>genome displays a GC skew pattern typical for most bacterial genomes, indicating that no major rearrangements have occurred recently. Additionally, the high sequence divergence of some IS elements, the high number of truncated IS element copies (n = 143), as well as the absence of direct repeats in most IS elements suggest that the IS elements of <it>A. asiaticus </it>are transpositionally inactive. Although we could show transcription of 13 IS elements, we did not find experimental evidence for transpositional activity, corroborating our results from sequence analyses. However, we detected contiguous transcripts between IS elements and their downstream genes at nine loci in the <it>A. asiaticus </it>genome, indicating that some IS elements influence the transcription of downstream genes, some of which might be important for host cell interaction.</p> <p>Conclusions</p> <p>Taken together, the IS elements in the <it>A. asiaticus </it>genome are currently in the process of degradation and largely represent reflections of the evolutionary past of <it>A. asiaticus </it>in which its genome was shaped by their activity.</p