84 research outputs found

    Transforming a Pair of Orthogonal tRNA-aminoacyl-tRNA Synthetase from Archaea to Function in Mammalian Cells

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    A previously engineered Methanocaldococcus jannaschii –tyrosyl-tRNA synthetase pair orthogonal to Escherichia coli was modified to become orthogonal in mammalian cells. The resulting -tyrosyl-tRNA synthetase pair was able to suppress an amber codon in the green fluorescent protein, GFP, and in a foldon protein in mammalian cells. The methodology reported here will allow rapid transformation of the much larger collection of existing tyrosyl-tRNA synthetases that were already evolved for the incorporation of an array of over 50 unnatural amino acids into proteins in Escherichia coli into proteins in mammalian cells. Thus we will be able to introduce a large array of possibilities for protein modifications in mammalian cells

    Genetic Code Mutations: The Breaking of a Three Billion Year Invariance

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    The genetic code has been unchanging for some three billion years in its canonical ensemble of encoded amino acids, as indicated by the universal adoption of this ensemble by all known organisms. Code mutations beginning with the encoding of 4-fluoro-Trp by Bacillus subtilis, initially replacing and eventually displacing Trp from the ensemble, first revealed the intrinsic mutability of the code. This has since been confirmed by a spectrum of other experimental code alterations in both prokaryotes and eukaryotes. To shed light on the experimental conversion of a rigidly invariant code to a mutating code, the present study examined code mutations determining the propagation of Bacillus subtilis on Trp and 4-, 5- and 6-fluoro-tryptophans. The results obtained with the mutants with respect to cross-inhibitions between the different indole amino acids, and the growth effects of individual nutrient withdrawals rendering essential their biosynthetic pathways, suggested that oligogenic barriers comprising sensitive proteins which malfunction with amino acid analogues provide effective mechanisms for preserving the invariance of the code through immemorial time, and mutations of these barriers open up the code to continuous change

    Covert Genetic Selections to Optimize Phenotypes

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    In many high complexity systems (cells, organisms, institutions, societies, economies, etc.), it is unclear which components should be regulated to affect overall performance. To identify and prioritize molecular targets which impact cellular phenotypes, we have developed a selection procedure (β€œSPI”–single promoting/inhibiting target identification) which monitors the abundance of ectopic cDNAs. We have used this approach to identify growth regulators. For this purpose, complex pools of S. cerevisiae cDNA transformants were established and we quantitated the evolution of the spectrum of cDNAs which was initially present. These data emphasized the importance of translation initiation and ER-Golgi traffic for growth. SPI provides functional insight into the stability of cellular phenotypes under circumstances in which established genetic approaches cannot be implemented. It provides a functional β€œsynthetic genetic signature” for each state of the cell (i.e. genotype and environment) by surveying complex genetic libraries, and does not require specialized arrays of cDNAs/shRNAs, deletion strains, direct assessment of clonal growth or even a conditional phenotype. Moreover, it establishes a hierarchy of importance of those targets which can contribute, either positively or negatively, to modify the prevailing phenotype. Extensions of these proof-of-principle experiments to other cell types should provide a novel and powerful approach to analyze multiple aspects of the basic biology of yeast and animal cells as well as clinically-relevant issues

    A Screen for RNA-Binding Proteins in Yeast Indicates Dual Functions for Many Enzymes

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    Hundreds of RNA-binding proteins (RBPs) control diverse aspects of post-transcriptional gene regulation. To identify novel and unconventional RBPs, we probed high-density protein microarrays with fluorescently labeled RNA and selected 200 proteins that reproducibly interacted with different types of RNA from budding yeast Saccharomyces cerevisiae. Surprisingly, more than half of these proteins represent previously known enzymes, many of them acting in metabolism, providing opportunities to directly connect intermediary metabolism with posttranscriptional gene regulation. We mapped the RNA targets for 13 proteins identified in this screen and found that they were associated with distinct groups of mRNAs, some of them coding for functionally related proteins. We also found that overexpression of the enzyme Map1 negatively affects the expression of experimentally defined mRNA targets. Our results suggest that many proteins may associate with mRNAs and possibly control their fates, providing dense connections between different layers of cellular regulation

    Host Cell Invasion and Virulence Mediated by Candida albicans Ssa1

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    Candida albicans Ssa1 and Ssa2 are members of the HSP70 family of heat shock proteins that are expressed on the cell surface and function as receptors for antimicrobial peptides such as histatins. We investigated the role of Ssa1 and Ssa2 in mediating pathogenic host cell interactions and virulence. A C. albicans ssa1Ξ”/Ξ” mutant had attenuated virulence in murine models of disseminated and oropharyngeal candidiasis, whereas an ssa2Ξ”/Ξ” mutant did not. In vitro studies revealed that the ssa1Ξ”/Ξ” mutant caused markedly less damage to endothelial cells and oral epithelial cell lines. Also, the ssa1Ξ”/Ξ” mutant had defective binding to endothelial cell N-cadherin and epithelial cell E-cadherin, receptors that mediate host cell endocytosis of C. albicans. As a result, this mutant had impaired capacity to induce its own endocytosis by endothelial cells and oral epithelial cells. Latex beads coated with recombinant Ssa1 were avidly endocytosed by both endothelial cells and oral epithelial cells, demonstrating that Ssa1 is sufficient to induce host cell endocytosis. These results indicate that Ssa1 is a novel invasin that binds to host cell cadherins, induces host cell endocytosis, and is critical for C. albicans to cause maximal damage to host cells and induce disseminated and oropharyngeal disease

    Cell populations can use aneuploidy to survive telomerase insufficiency

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    Telomerase maintains ends of eukaryotic chromosomes, telomeres. Telomerase loss results in replicative senescence and a switch to recombination-dependent telomere maintenance. Telomerase insufficiency in humans leads to telomere syndromes associated with premature ageing and cancer predisposition. Here we use yeast to show that the survival of telomerase insufficiency differs from the survival of telomerase loss and occurs through aneuploidy. In yeast grown at elevated temperatures, telomerase activity becomes limiting: haploid cell populations senesce and generate aneuploid survivorsβ€”near diploids monosomic for chromosome VIII. This aneuploidy results in increased levels of the telomerase components TLC1, Est1 and Est3, and is accompanied by decreased abundance of ribosomal proteins. We propose that aneuploidy suppresses telomerase insufficiency through redistribution of cellular resources away from ribosome synthesis towards production of telomerase components and other non-ribosomal proteins. The aneuploidy-induced re-balance of the proteome via modulation of ribosome biogenesis may be a general adaptive response to overcome functional insufficiencies

    Large-scale profiling of noncoding RNA function in yeast

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    Noncoding RNAs (ncRNAs) are emerging as key regulators of cellular function. We have exploited the recently developed barcoded ncRNA gene deletion strain collections in the yeast Saccharomyces cerevisiae to investigate the numerous ncRNAs in yeast with no known function. The ncRNA deletion collection contains deletions of tRNAs, snoRNAs, snRNAs, stable unannotated transcripts (SUTs), cryptic unstable transcripts (CUTs) and other annotated ncRNAs encompassing 532 different individual ncRNA deletions. We have profiled the fitness of the diploid heterozygous ncRNA deletion strain collection in six conditions using batch and continuous liquid culture, as well as the haploid ncRNA deletion strain collections arrayed individually onto solid rich media. These analyses revealed many novel environmental-specific haplo-insufficient and haplo-proficient phenotypes providing key information on the importance of each specific ncRNA in every condition. Co-fitness analysis using fitness data from the heterozygous ncRNA deletion strain collection identified two ncRNA groups required for growth during heat stress and nutrient deprivation. The extensive fitness data for each ncRNA deletion strain has been compiled into an easy to navigate database called Yeast ncRNA Analysis (YNCA). By expanding the original ncRNA deletion strain collection we identified four novel essential ncRNAs; SUT527, SUT075, SUT367 and SUT259/691. We defined the effects of each new essential ncRNA on adjacent gene expression in the heterozygote background identifying both repression and induction of nearby genes. Additionally, we discovered a function for SUT527 in the expression, 3’ end formation and localization of SEC4, an essential protein coding mRNA. Finally, using plasmid complementation we rescued the SUT075 lethal phenotype revealing that this ncRNA acts in trans. Overall, our findings provide important new insights into the function of ncRNAs
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