Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

A nucleoside kinase as a dual selector for genetic switches and circuits.

ABSTRACT: The development of genetic switches and their integrated forms (genetic circuits) with desired specifications/functions is key for success in synthetic biology. Due to the difficulty in rational design, genetic switches and circuits with desirable specifications are mostly obtained by directed evolution. Based on a virus-derived nucleotide kinase as a single-gene dual selector, we constructed a robust, efficient and stringent selection system for genetic switches. This method exhibited unprecedented enrichment efficacy (>30,000-fold) of functional switches from non-functional ones in a single selection cycle. In addition, negative (OFF) selection was exceptionally stringent, allowing the rapid and efficient selection of non-leaky from leaky circuits.

SUBMITTER: Tashiro Y

PROVIDER: S-EPMC3035434 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

A nucleoside kinase as a dual selector for genetic switches and circuits.

Tashiro Yohei Y Fukutomi Hiroki H Terakubo Kei K Saito Kyoichi K Umeno Daisuke D

Nucleic acids research 20101109 3

The development of genetic switches and their integrated forms (genetic circuits) with desired specifications/functions is key for success in synthetic biology. Due to the difficulty in rational design, genetic switches and circuits with desirable specifications are mostly obtained by directed evolution. Based on a virus-derived nucleotide kinase as a single-gene dual selector, we constructed a robust, efficient and stringent selection system for genetic switches. This method exhibited unprecede ...[more]

PMID: 21062820

Similar Datasets

Rapid and liquid-based selection of genetic switches using nucleoside kinase fused with aminoglycoside phosphotransferase.

Project description:The evolutionary design of genetic switches and circuits requires iterative rounds of positive (ON-) and negative (OFF-) selection. We previously reported a rapid OFF selection system based on the kinase activity of herpes simplex virus thymidine kinase (hsvTK) on the artificial mutator nucleoside dP. By fusing hsvTK with the kanamycin resistance marker aminoglycoside-(3')-phosphotransferase (APH), we established a novel selector system for genetic switches. Due to the bactericidal nature of kanamycin and nucleoside-based lethal mutagenesis, both positive and negative selection could be completed within several hours. Using this new selector system, we isolated a series of homoserine lactone-inducible genetic switches with different expression efficiencies from libraries of the Vibrio fischeri lux promoter in two days, using only liquid handling.

| S-EPMC4366196 | biostudies-literature

Dual protein kinase and nucleoside kinase modulators for rationally designed polypharmacology.

Project description:Masitinib, a highly selective protein kinase inhibitor, can sensitise gemcitabine-refractory cancer cell lines when used in combination with gemcitabine. Here we report a reverse proteomic approach that identifies the target responsible for this sensitisation: the deoxycytidine kinase (dCK). Masitinib, as well as other protein kinase inhibitors, such as imatinib, interact with dCK and provoke an unforeseen conformational-dependent activation of this nucleoside kinase, modulating phosphorylation of nucleoside analogue drugs. This phenomenon leads to an increase of prodrug phosphorylation of most of the chemotherapeutic drugs activated by this nucleoside kinase. The unforeseen dual activity of protein kinase inhibition/nucleoside kinase activation could be of great therapeutic benefit, through either reducing toxicity of therapeutic agents by maintaining effectiveness at lower doses or by counteracting drug resistance initiated via down modulation of dCK target.

| S-EPMC5681654 | biostudies-literature

Dual protein kinase/ nucleoside kinase modulat ors for rationally designed polypharmacology

Project description:Masitinib, a highly selective protein kinase inhibitor, can sensitise gemcitabine-refractory cancer cell lines when used in combination with gemcitabine. A reverse proteomic approach has identified the target responsible for this sensitisation: the deoxycytidine kinase (dCK). Masitinib, as well as other protein kinase inhibitors such as imatinib, interact with dCK and provoke an unforeseen conformational-dependent activation of this nucleoside kinase, modulating phosphorylation of nucleoside analogue drugs. This phenomenon leads to an increase of prodrug phosphorylation of most of the chemotherapeutic drugs activated by this nucleoside kinase. The unforeseen dual activity of protein kinase inhibition/nucleoside kinase activation could be of great therapeutic benefit, through either reducing toxicity of therapeutic agents by maintaining effectiveness at lower doses or by counteracting drug resistance initiated via down modulation of dCK target.

2017-11-13 | PXD007765 | Pride

Dynamic rewiring of the Drosophila retinal determination network switches its function from selector to differentiation.

Project description:Organ development is directed by selector gene networks. Eye development in the fruit fly Drosophila melanogaster is driven by the highly conserved selector gene network referred to as the "retinal determination gene network," composed of approximately 20 factors, whose core comprises twin of eyeless (toy), eyeless (ey), sine oculis (so), dachshund (dac), and eyes absent (eya). These genes encode transcriptional regulators that are each necessary for normal eye development, and sufficient to direct ectopic eye development when misexpressed. While it is well documented that the downstream genes so, eya, and dac are necessary not only during early growth and determination stages but also during the differentiation phase of retinal development, it remains unknown how the retinal determination gene network terminates its functions in determination and begins to promote differentiation. Here, we identify a switch in the regulation of ey by the downstream retinal determination genes, which is essential for the transition from determination to differentiation. We found that central to the transition is a switch from positive regulation of ey transcription to negative regulation and that both types of regulation require so. Our results suggest a model in which the retinal determination gene network is rewired to end the growth and determination stage of eye development and trigger terminal differentiation. We conclude that changes in the regulatory relationships among members of the retinal determination gene network are a driving force for key transitions in retinal development.

| S-EPMC3757064 | biostudies-literature

Dual Functions of V/SiOx/AlOy/p++Si Device as Selector and Memory.

Project description:This letter presents dual functions including selector and memory switching in a V/SiOx/AlOy/p++Si resistive memory device by simply controlling compliance current limit (CCL). Unidirectional threshold switching is observed after a positive forming with low CCL of 1 ?A. The shifts to the V-electrode side of the oxygen form the VOx layer, where the threshold switching can be explained by the metal-insulation-transition phenomenon. For higher CCL (30 ?A) applied to the device, a bipolar memory switching is obtained, which is attributed to formation and rupture of the conducting filament in SiOy layer. 1.5-nm-thick AlOy layer with high thermal conductivity plays an important role in lowering the off-current for memory and threshold switching. Through the temperature dependence, high-energy barrier (0.463 eV) in the LRS is confirmed, which can cause nonlinearity in a low-resistance state. The smaller the CCL, the higher the nonlinearity, which provides a larger array size in the cross-point array. The coexistence of memory and threshold switching in accordance with the CCL provides the flexibility to control the device for its intended use.

| S-EPMC6107449 | biostudies-literature

Synthetic dual-input mammalian genetic circuits enable tunable and stringent transcription control by chemical and light.

Project description:Programmable transcription factors can enable precise control of gene expression triggered by a chemical inducer or light. To obtain versatile transgene system with combined benefits of a chemical inducer and light inducer, we created various chimeric promoters through the assembly of different copies of the tet operator and Gal4 operator module, which simultaneously responded to a tetracycline-responsive transcription factor and a light-switchable transactivator. The activities of these chimeric promoters can be regulated by tetracycline and blue light synergistically or antagonistically. Further studies of the antagonistic genetic circuit exhibited high spatiotemporal resolution and extremely low leaky expression, which therefore could be used to spatially and stringently control the expression of highly toxic protein Diphtheria toxin A for light regulated gene therapy. When transferring plasmids engineered for the gene switch-driven expression of a firefly luciferase (Fluc) into mice, the Fluc expression levels of the treated animals directly correlated with the tetracycline and light input program. We suggest that dual-input genetic circuits using TET and light that serve as triggers to achieve expression profiles may enable the design of robust therapeutic gene circuits for gene- and cell-based therapies.

| S-EPMC4824083 | biostudies-other

Long-range transcriptional interference in E. coli used to construct a dual positive selection system for genetic switches.

Project description:We have investigated transcriptional interference between convergent genes in E. coli and demonstrate substantial interference for inter-promoter distances of as far as 3 kb. Interference can be elicited by both strong σ(70) dependent and T7 promoters. In the presented design, a strong promoter driving gene expression of a 'forward' gene interferes with the expression of a 'reverse' gene by a weak promoter. This arrangement allows inversely correlated gene expression without requiring further regulatory components. Thus, modulation of the activity of the strong promoter alters expression of both the forward and the reverse gene. We used this design to develop a dual selection system for conditional operator site binding, allowing positive selection both for binding and for non-binding to DNA. This study demonstrates the utility of this novel system using the Lac repressor as a model protein for conditional DNA binding, and spectinomycin and chloramphenicol resistance genes as positive selection markers in liquid culture. Randomized LacI libraries were created and subjected to subsequent dual selection, but mispairing IPTG and selection cues in respect to the wild-type LacI response, allowing the isolation of a LacI variant with a reversed IPTG response within three rounds of library generation and dual selection.

| S-EPMC4889929 | biostudies-literature

How Hsp90 and Cdc37 Lubricate Kinase Molecular Switches.

Project description:The Hsp90/Cdc37 chaperone system interacts with and supports 60% of the human kinome. Not only are Hsp90 and Cdc37 generally required for initial folding, but many kinases rely on the Hsp90/Cdc37 throughout their lifetimes. A large fraction of these 'client' kinases are key oncoproteins, and their interactions with the Hsp90/Cdc37 machinery are crucial for both their normal and malignant activity. Recently, advances in single-particle cryo-electron microscopy (cryoEM) and biochemical strategies have provided the first key molecular insights into kinase-chaperone interactions. The surprising results suggest a re-evaluation of the role of chaperones in the kinase lifecycle, and suggest that such interactions potentially allow kinases to more rapidly respond to key signals while simultaneously protecting unstable kinases from degradation and suppressing unwanted basal activity.

| S-EPMC5621984 | biostudies-literature

A Humanized Yeast Phenomic Model of Deoxycytidine Kinase to Predict Genetic Buffering of Nucleoside Analog Cytotoxicity.

Project description:Knowledge about synthetic lethality can be applied to enhance the efficacy of anticancer therapies in individual patients harboring genetic alterations in their cancer that specifically render it vulnerable. We investigated the potential for high-resolution phenomic analysis in yeast to predict such genetic vulnerabilities by systematic, comprehensive, and quantitative assessment of drug-gene interaction for gemcitabine and cytarabine, substrates of deoxycytidine kinase that have similar molecular structures yet distinct antitumor efficacy. Human deoxycytidine kinase (dCK) was conditionally expressed in the Saccharomyces cerevisiae genomic library of knockout and knockdown (YKO/KD) strains, to globally and quantitatively characterize differential drug-gene interaction for gemcitabine and cytarabine. Pathway enrichment analysis revealed that autophagy, histone modification, chromatin remodeling, and apoptosis-related processes influence gemcitabine specifically, while drug-gene interaction specific to cytarabine was less enriched in gene ontology. Processes having influence over both drugs were DNA repair and integrity checkpoints and vesicle transport and fusion. Non-gene ontology (GO)-enriched genes were also informative. Yeast phenomic and cancer cell line pharmacogenomics data were integrated to identify yeast-human homologs with correlated differential gene expression and drug efficacy, thus providing a unique resource to predict whether differential gene expression observed in cancer genetic profiles are causal in tumor-specific responses to cytotoxic agents.

| S-EPMC6826991 | biostudies-literature

Multiple genetic switches spontaneously modulating bacterial mutability.

Project description:BackgroundAll life forms need both high genetic stability to survive as species and a degree of mutability to evolve for adaptation, but little is known about how the organisms balance the two seemingly conflicting aspects of life: genetic stability and mutability. The DNA mismatch repair (MMR) system is essential for maintaining genetic stability and defects in MMR lead to high mutability. Evolution is driven by genetic novelty, such as point mutation and lateral gene transfer, both of which require genetic mutability. However, normally a functional MMR system would strongly inhibit such genomic changes. Our previous work indicated that MMR gene allele conversion between functional and non-functional states through copy number changes of small tandem repeats could occur spontaneously via slipped-strand mis-pairing during DNA replication and therefore may play a role of genetic switches to modulate the bacterial mutability at the population level. The open question was: when the conversion from functional to defective MMR is prohibited, will bacteria still be able to evolve by accepting laterally transferred DNA or accumulating mutations?ResultsTo prohibit allele conversion, we "locked" the MMR genes through nucleotide replacements. We then scored changes in bacterial mutability and found that Salmonella strains with MMR locked at the functional state had significantly decreased mutability. To determine the generalizability of this kind of mutability 'switching' among a wider range of bacteria, we examined the distribution of tandem repeats within MMR genes in over 100 bacterial species and found that multiple genetic switches might exist in these bacteria and may spontaneously modulate bacterial mutability during evolution.ConclusionsMMR allele conversion through repeats-mediated slipped-strand mis-pairing may function as a spontaneous mechanism to switch between high genetic stability and mutability during bacterial evolution.

| S-EPMC2955026 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data