scholarly journals An in vivo translation-reporter system for the study of protein synthesis in zebrafish embryos

Biology Open ◽  
2018 ◽  
Vol 7 (12) ◽  
pp. bio039362
Author(s):  
Inês Garcez Palha ◽  
Isabelle Anselme ◽  
Sylvie Schneider-Maunoury ◽  
François Giudicelli
Keyword(s):  
Protein Synthesis ◽  
Zebrafish Embryos ◽  
Reporter System

scholarly journals An in vivo translation-reporter system for the study of protein synthesis in zebrafish embryos

2018 ◽  
Author(s):  
Inês Garcez Palha ◽  
Isabelle Anselme ◽  
Sylvie Schneider-Maunoury ◽  
François Giudicelli
Keyword(s):  
Protein Synthesis ◽  
Protease Inhibitor ◽  
Fluorescent Protein ◽  
Mrna Translation ◽  
Transgenic Zebrafish ◽  
Zebrafish Embryos ◽  
Reporter System ◽  
Control Of Gene Expression ◽  
A Cell

ABSTRACTControl of gene expression at the translation level is increasingly regarded as a key feature in many biological processes. Simple, inexpensive, and reliable procedures to visualise sites of protein production are required to allow observation of the spatiotemporal patterns of mRNA translation at subcellular resolution. We present a method, named SPoT (for Subcellular Patterns of Translation), developed upon the original TimeStamp technique (Lin et al., 2008), consisting in the expression of a fluorescent protein fused to a tagged, self-cleavable protease domain. Addition of a cell-permeable protease inhibitor instantly stabilizes newly produced, tagged protein allowing to distinguish recently synthesized protein from more ancient one. After a brief protease inhibitor treatment, the ratio of tagged vs non-tagged forms is highest at sites where proteins are the most recent, i.e. sites of synthesis. Therefore, by comparing tagged and non-tagged protein it is possible to spotlight sites of translation. By specifically expressing the SPoT cassette in neurons of transgenic zebrafish embryos, we reveal sites of neuronal protein synthesis in diverse cellular compartments during early development.

scholarly journals Initiator-Elongator Discrimination in Vertebrate tRNAs for Protein Synthesis

1998 ◽  
Vol 18 (3) ◽  
pp. 1459-1466 ◽  
Author(s):  
Harold J. Drabkin ◽  
Melanie Estrella ◽  
Uttam L. Rajbhandary
Keyword(s):  
Protein Synthesis ◽  
Mammalian Cells ◽  
Elongation Factor ◽  
Reporter System ◽  
Base Pairs ◽  
Initiator Trna ◽  
Phosphate Backbone ◽  
Elongation Step

ABSTRACT Initiator tRNAs are used exclusively for initiation of protein synthesis and not for the elongation step. We show, in vivo and in vitro, that the primary sequence feature that prevents the human initiator tRNA from acting in the elongation step is the nature of base pairs 50:64 and 51:63 in the TΨC stem of the initiator tRNA. Various considerations suggest that this is due to sequence-dependent perturbation of the sugar phosphate backbone in the TΨC stem of initiator tRNA, which most likely blocks binding of the elongation factor to the tRNA. Because the sequences of all vertebrate initiator tRNAs are identical, our findings with the human initiator tRNA are likely to be valid for all vertebrate systems. We have developed reporter systems that can be used to monitor, in mammalian cells, the activity in elongation of mutant human initiator tRNAs carrying anticodon sequence mutations from CAU to CCU (the C35 mutant) or to CUA (the U35A36 mutant). Combination of the anticodon sequence mutation with mutations in base pairs 50:64 and 51:63 yielded tRNAs that act as elongators in mammalian cells. Further mutation of the A1:U72 base pair, which is conserved in virtually all eukaryotic initiator tRNAs, to G1:C72 in the C35 mutant background yielded tRNAs that were even more active in elongation. In addition, in a rabbit reticulocyte in vitro protein-synthesizing system, a tRNA carrying the TΨC stem and the A1:U72-to-G1:C72 mutations was almost as active in elongation as the elongator methionine tRNA. The combination of mutant initiator tRNA with the CCU anticodon and the reporter system developed here provides the first example of missense suppression in mammalian cells.

scholarly journals Regulation of Ribosomal Protein Synthesis in Mycobacteria: The Autogenous Control of rpsO

2021 ◽  
Vol 22 (18) ◽  
pp. 9679
Author(s):  
Leonid V. Aseev ◽  
Ludmila S. Koledinskaya ◽  
Oksana S. Bychenko ◽  
Irina V. Boni
Keyword(s):  
Protein Synthesis ◽  
Ribosomal Protein ◽  
Shuttle Vector ◽  
Ectopic Expression ◽  
Egfp Expression ◽  
Reporter System ◽  
Fluorescent Reporter ◽  
E Coli ◽  
Ribosomal Protein Synthesis

The autogenous regulation of ribosomal protein (r-protein) synthesis plays a key role in maintaining the stoichiometry of ribosomal components in bacteria. In this work, taking the rpsO gene as a classic example, we addressed for the first time the in vivo regulation of r-protein synthesis in the mycobacteria M. smegmatis (Msm) and M. tuberculosis (Mtb). We used a strategy based on chromosomally integrated reporters under the control of the rpsO regulatory regions and the ectopic expression of Msm S15 to measure its impact on the reporter expression. Because the use of E. coli as a host appeared inefficient, a fluorescent reporter system was developed by inserting Msm or Mtb rpsO-egfp fusions into the Msm chromosome and expressing Msm S15 or E. coli S15 in trans from a novel replicative shuttle vector, pAMYC. The results of the eGFP expression measurements in Msm cells provided evidence that the rpsO gene in Msm and Mtb was feedback-regulated at the translation level. The mutagenic analysis showed that the folding of Msm rpsO 5′UTR in a pseudoknot appeared crucial for repression by both Msm S15 and E. coli S15, thus indicating a striking resemblance of the rpsO feedback control in mycobacteria and in E. coli.

Respiratory energy requirements and rate of protein turnover in vivo determined by the use of an inhibitor of protein synthesis and a probe to assess its effect

1994 ◽  
Vol 92 (4) ◽  
pp. 585-594 ◽  
Author(s):  
T. J. Bouma ◽  
R. De Visser ◽  
J. H. J. A. Janssen ◽  
M. J. De Kock ◽  
P H. Van Leeuwen ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Protein Turnover ◽  
Energy Requirements ◽  
Inhibitor Of Protein Synthesis

Rapid in vivo induction of leukocyte tissue factor mRNA and protein synthesis following low dose endotoxin administration to rabbits

2001 ◽  
Vol 2 (3) ◽  
pp. 188-195 ◽  
Author(s):  
Tara C Brutzki ◽  
Myron J Kulczycky ◽  
Leslie Bardossy ◽  
Bryan J Clarke ◽  
Morris A Blajchman
Keyword(s):  
Protein Synthesis ◽  
Tissue Factor ◽  
Low Dose ◽  
Endotoxin Administration ◽  
In Vivo Induction

scholarly journals Edelfosine nanoemulsions inhibit tumor growth of triple negative breast cancer in zebrafish xenograft model

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sofia M. Saraiva ◽  
Carlha Gutiérrez-Lovera ◽  
Jeannette Martínez-Val ◽  
Sainza Lores ◽  
Belén L. Bouzo ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Tumor Growth ◽  
Triple Negative Breast Cancer ◽  
Triple Negative ◽  
Xenograft Model ◽  
Skin Barrier ◽  
Zebrafish Embryos ◽  
Biorelevant Media

AbstractTriple negative breast cancer (TNBC) is known for being very aggressive, heterogeneous and highly metastatic. The standard of care treatment is still chemotherapy, with adjacent toxicity and low efficacy, highlighting the need for alternative and more effective therapeutic strategies. Edelfosine, an alkyl-lysophospholipid, has proved to be a promising therapy for several cancer types, upon delivery in lipid nanoparticles. Therefore, the objective of this work was to explore the potential of edelfosine for the treatment of TNBC. Edelfosine nanoemulsions (ET-NEs) composed by edelfosine, Miglyol 812 and phosphatidylcholine as excipients, due to their good safety profile, presented an average size of about 120 nm and a neutral zeta potential, and were stable in biorelevant media. The ability of ET-NEs to interrupt tumor growth in TNBC was demonstrated both in vitro, using a highly aggressive and invasive TNBC cell line, and in vivo, using zebrafish embryos. Importantly, ET-NEs were able to penetrate through the skin barrier of MDA-MB 231 xenografted zebrafish embryos, into the yolk sac, leading to an effective decrease of highly aggressive and invasive tumoral cells’ proliferation. Altogether the results demonstrate the potential of ET-NEs for the development of new therapeutic approaches for TNBC.

scholarly journals LOCALIZED MUTAGENESIS FOR THE ISOLATION OF TEMPERATURE-SENSITIVE MUTANTS OF ESCHERICHIA COLI AFFECTED IN PROTEIN SYNTHESIS

Genetics ◽  
1979 ◽  
Vol 91 (2) ◽  
pp. 215-227
Author(s):  
W Scott Champney
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Temperature Sensitive ◽  
Chromosomal Locus ◽  
Ribosomal Subunits ◽  
Prolonged Incubation ◽  
Temperature Sensitive Mutants ◽  
Inhibition Of Growth ◽  
Localized Mutagenesis

ABSTRACT Two variations of the method of localized mutagenesis were used to introduce mutations into the 72 min region of the Escherichia coli chromosome. Twenty temperature-sensitive mutants, with linkage to markers in this region, have been examined. Each strain showed an inhibition of growth in liquid medium at 44°, and 19 of the mutants lost viability upon prolonged incubation at this temperature. A reduction in the rate of in vivo RNA and protein synthesis was observed for each mutant at 44°, relative to a control strain. Eleven of the mutants were altered in growth sensitivity or resistance to one or more of three ribosomal antibiotics. The incomplete assembly of ribosomal subunits was detected in nine strains grown at 44°. The characteristics of these mutants suggest that many of them are altered in genes for translational or transcriptional components, consistent with the clustering of these genes at this chromosomal locus.

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