scholarly journals Expanding the Zebrafish Genetic Code through Site-Specific Introduction of Azido-lysine, Bicyclononyne-lysine, and Diazirine-lysine

2019 ◽  
Author(s):  
Junetha Syed ◽  
Saravanan Palani ◽  
Scott T. Clarke ◽  
Zainab Asad ◽  
Andrew R. Bottrill ◽  
...  
Keyword(s):  
Genetic Code ◽  
Protein Function ◽  
Fluorescent Protein ◽  
Trna Synthetase ◽  
Glutathione S Transferase ◽  
Base Pairs ◽  
Site Specific ◽  
Green Fluorescent ◽  
Natural Amino Acids ◽  
Sense Codon

AbstractSite-specific incorporation of un-natural amino acids (UNAA) is a powerful approach to engineer and understand protein function [1-4]. Site-specific incorporation of UNAAs is achieved through repurposing the amber codon (UAG) as a sense codon for the UNAA, a tRNACUA that base pairs with an UAG codon in the mRNA and an orthogonal amino-acyl tRNA synthetase (aaRS) that charges the tRNACUA with the UNAA [5, 6]. Here, we report expansion of the zebrafish genetic code to incorporate the UNAAs, Azido-lysine (AzK), bicyclononyne-lysine (BCNK), and Diazirine-lysine (AbK) into green fluorescent protein (GFP) and Glutathione-S-transferase (GST). We also present proteomic evidence for UNAA incorporation into GFP. Our work sets the stage for the use of UNAA mutagenesis to investigate and engineer protein function in zebrafish.

scholarly journals Expanding the Zebrafish Genetic Code through Site-Specific Introduction of Azido-lysine, Bicyclononyne-lysine, and Diazirine-lysine

2019 ◽  
Vol 20 (10) ◽  
pp. 2577 ◽  
Author(s):  
Junetha Syed ◽  
Saravanan Palani ◽  
Scott T. Clarke ◽  
Zainab Asad ◽  
Andrew R. Bottrill ◽  
...  
Keyword(s):  
Genetic Code ◽  
Protein Function ◽  
Fluorescent Protein ◽  
Trna Synthetase ◽  
Glutathione S Transferase ◽  
Base Pairs ◽  
Site Specific ◽  
Green Fluorescent ◽  
Natural Amino Acids ◽  
Sense Codon

Site-specific incorporation of un-natural amino acids (UNAA) is a powerful approach to engineer and understand protein function. Site-specific incorporation of UNAAs is achieved through repurposing the amber codon (UAG) as a sense codon for the UNAA, using a tRNACUA that base pairs with an UAG codon in the mRNA and an orthogonal amino-acyl tRNA synthetase (aaRS) that charges the tRNACUA with the UNAA. Here, we report an expansion of the zebrafish genetic code to incorporate the UNAAs, azido-lysine (AzK), bicyclononyne-lysine (BCNK), and diazirine-lysine (AbK) into green fluorescent protein (GFP) and glutathione-s-transferase (GST). We also present proteomic evidence for UNAA incorporation into GFP. Our work sets the stage for the use of AzK, BCNK, and AbK introduction into proteins as a means to investigate and engineer their function in zebrafish.

Oligonucleotide Preparation Approach for Assembly of DNA Synthons

2019 ◽  
Vol 24 (6) ◽  
pp. 556-568
Author(s):  
Aleksei V. Yantsevich ◽  
Veronika V. Shchur ◽  
Sergey A. Usanov
Keyword(s):  
Fluorescent Protein ◽  
Solid Phase ◽  
Base Pairs ◽  
Egfp Gene ◽  
Standard Facility ◽  
Synthetic Dna ◽  
Pcr Product ◽  
Green Fluorescent ◽  
Inside Out ◽  
Spe Purification

An effective oligonucleotide preparation approach for the thermodynamically balanced, inside-out (TBIO) PCR-based assembly of long synthetic DNA molecules (synthons) is described in the current work. We replaced the necessity to purify individual oligonucleotides with just one purification procedure per approximately 500 base pairs (bp) of duplex DNA. So for an enhanced green fluorescent protein (EGFP) gene of 717 bp, we synthesized 24 oligonucleotides with a length of 50 bases and performed just two solid-phase extraction (SPE) purification procedures. It was found that the capacity of ZipTip microextractors, usually used for sample desalting in proteomics, perfectly corresponds to the gene synthesis scale (40–60 pmol). The robustness of the approach was validated with a 65-mer oligonucleotide design of the same gene. The modification of the oligonucleotide concentration gradient from the original TBIO scheme substantially increased the purity of the PCR product. We proposed a mechanism for the formation of supramolecular structures, which often occur during TBIO assembly. By using the proposed workflow, any laboratory with a standard facility for molecular biology manipulation, a 16-channel oligonucleotide synthesizer, and a conventional thermocycler has the ability to prepare one gene with a length of about 700 bp per day.

Munc-18-2 regulates exocytosis of H+-ATPase in rat inner medullary collecting duct cells

2004 ◽  
Vol 287 (5) ◽  
pp. C1366-C1374 ◽  
Author(s):  
Julie A. Nicoletta ◽  
Jonathan J. Ross ◽  
Guangmu Li ◽  
Qingzhang Cheng ◽  
Jonathon Schwartz ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Snare Complex ◽  
Glutathione S Transferase ◽  
Syntaxin 1A ◽  
Inner Medullary Collecting Duct ◽  
Medullary Collecting Duct ◽  
Reduced Rate ◽  
Green Fluorescent

Exocytic insertion of H+-ATPase into the apical membrane of inner medullary collecting duct (IMCD) cells is dependent on a soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein target receptor (SNARE) complex. In this study we determined the role of Munc-18 in regulation of IMCD cell exocytosis of H+-ATPase. We compared the effect of acute cell acidification (the stimulus for IMCD exocytosis) on the interaction of syntaxin 1A with Munc-18-2 and the 31-kDa subunit of H+-ATPase. Immunoprecipitation revealed that cell acidification decreased green fluorescent protein (GFP)-syntaxin 1A and Munc-18-2 interaction by 49 ± 7% and increased the interaction between GFP-syntaxin 1A and H+-ATPase by 170 ± 23%. Apical membrane Munc-18-2 decreased by 27.5 ± 4.6% and H+-ATPase increased by 246 ± 22%, whereas GP-135, an apical membrane marker, did not increase. Pretreatment of IMCD cells with a PKC inhibitor (GO-6983) diminished the previously described changes in Munc-18-2-syntaxin 1A interaction and redistribution of H+-ATPase. In a pull-down assay of H+-ATPase by glutathione S-transferase (GST)-syntaxin 1A bound to beads, preincubation of beads with an approximately twofold excess of His-Munc-18-2 decreased H+-ATPase pulled down by 64 ± 16%. IMCD cells that overexpress Munc-18-2 had a reduced rate of proton transport compared with control cells. We conclude that Munc-18-2 must dissociate from the syntaxin 1A protein for the exocytosis of H+-ATPase to occur. This dissociation leads to a conformational change in syntaxin 1A, allowing it to interact with H+-ATPase, synaptosome-associated protein (SNAP)-23, and vesicle-associated membrane protein (VAMP), forming the SNARE complex that leads to the docking and fusion of H+-ATPase vesicles.

21 SITE-SPECIFIC RECOMBINATION USING Dre-RECOMBINASE IN PORCINE CELLS AND EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 125
Author(s):  
S. Y. Yum ◽  
S. J. Kim ◽  
J. H. Moon ◽  
W. J. Choi ◽  
J. H. Lee ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Vector ◽  
Target Gene ◽  
Fluorescent Protein ◽  
Cellular Level ◽  
Cell Stage ◽  
Site Specific ◽  
Target Gene Expression ◽  
Green Fluorescent ◽  
Gateway Cloning System

Site-specific recombinases (SSR), such as Cre and Flp recombinases, which enable DNA excision, insertion, and translocation, have been used for conditional target gene expression in mouse and other vertebrates. In this study, we evaluated another SSR, Dre-recombinase (Dre), which is functionally similar to Cre recombinase in porcine fibroblasts and embryos. For this study, 2 fragment DNA constructs (rox GFP-polyA and rox RFP-polyA) were combined with piggybac transposition expression vector (Kim et al. 2011 J. Vet. Med. Sci.) using a multisite gateway cloning system (MultiSite Gateway® Pro, Invitrogen, Carlsbad, CA, USA). The expression vector carrying rox-flanked green fluorescent protein (GFP) followed by red fluorescent protein (RFP) and transposase were transfected into kidney-derived porcine cells by nucleofection (Neon® Transfection System, Invitrogen). A GFP-expressing cell line, which was not expressing RFP, was established. And then rox-flanked GFP were removed by Dre transfection and RFP was expressed in the kidney cells. At the cellular level, this excision was confirmed by site-specific RT-PCR and sequencing. The rox-flanked GFP cells were reconstructed with enucleated oocytes and then the cloned embryos were cultured in porcine zygote medium-5. Dre was micro-injected into 1 of the 2-cell-stage blastomeres. After 6 days, RFP expression was observed on the part of embryos after microinjection. In conclusion, the data demonstrated that, like other SSR, Dre might be applied in conditional target gene expression for generating porcine biomedical models.

Optimization of the posttranslational click modification of proteins

2011 ◽  
Vol 76 (9) ◽  
pp. 1089-1101
Author(s):  
Milan Vrabel ◽  
Emine Kaya ◽  
Stefan Prill ◽  
Veronika Ehmke ◽  
Thomas Carell
Keyword(s):  
Mass Spectrometry ◽  
Amino Acids ◽  
Fluorescent Protein ◽  
Click Reaction ◽  
Yellow Fluorescent Protein ◽  
Trna Synthetase ◽  
Site Specific ◽  
Specific Manner ◽  
Modified Proteins ◽  
A Site

In order to develop efficient methods that would enable the synthesis of posttranslationaly modified proteins in a site-specific manner we have adopted the orthogonal pyrrolysyl-tRNA synthetase/tRNA pair to genetically encode various pyrrolysine analogs, which we were able to insert into the yellow fluorescent protein (YFP). These experiments showed that the alkene and alkyne containing amino acids 5 and 6 are superior substrates for the pyrrolysyl-tRNA synthetase and that they can be successfully incorporated into proteins. Using the Cu(I)-catalyzed Huisgen–Meldal–Sharpless click reaction, the alkyne containing YFP was finally glycosylated with various sugars. We confirmed the presence of the modified amino acids as well as the corresponding sugar modifications by HPLC-MS/MS mass spectrometry.

scholarly journals A novel nuclear genetic code alteration in yeasts and the evolution of codon reassignment in eukaryotes

2016 ◽  
Author(s):  
Stefanie Mühlhausen ◽  
Peggy Findeisen ◽  
Uwe Plessmann ◽  
Henning Urlaub ◽  
Martin Kollmar
Keyword(s):  
Genetic Code ◽  
Trna Synthetase ◽  
Amino Acid Sequences ◽  
Codon Reassignment ◽  
Proteomics Data ◽  
Pachysolen Tannophilus ◽  
Sequence Comparisons ◽  
Codon Capture ◽  
Cellular Translation ◽  
Sense Codon

AbstractThe genetic code is the universal cellular translation table to convert nucleotide into amino acid sequences. Changes to sense codons are expected to be highly detrimental. However, reassignments of single or multiple codons in mitochondria and nuclear genomes demonstrated that the code can evolve. Still, alterations of nuclear genetic codes are extremely rare leaving hypotheses to explain these variations, such as the ‘codon capture’, the ‘genome streamlining’ and the ‘ambiguous intermediate’ theory, in strong debate. Here, we report on a novel sense codon reassignment inPachysolen tannophilus, a yeast related to the Pichiaceae. By generating proteomics data and using tRNA sequence comparisons we show that inPachysolenCUG codons are translated as alanine and not as the universal leucine. The polyphyly of the CUG-decoding tRNAs in yeasts is best explained by atRNA loss driven codon reassignmentmechanism. Loss of the CUG-tRNA in the ancient yeast is followed by gradual decrease of respective codons and subsequent codon capture by tRNAs whose anticodon is outside the aminoacyl-tRNA synthetase recognition region. Our hypothesis applies to all nuclear genetic code alterations and provides several testable predictions. We anticipate more codon reassignments to be uncovered in existing and upcoming genome projects.

scholarly journals Analysis of the hypoxia-sensing pathway in Drosophila melanogaster

2005 ◽  
Vol 393 (2) ◽  
pp. 471-480 ◽  
Author(s):  
Nathalie Arquier ◽  
Paul Vigne ◽  
Eric Duplan ◽  
Tien Hsu ◽  
Pascal P. Therond ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Fusion Protein ◽  
Gene Transcription ◽  
Fluorescent Protein ◽  
Hypoxia Inducible Factor ◽  
Glutathione S Transferase ◽  
Reporter Protein ◽  
Α Subunit ◽  
Von Hippel Lindau ◽  
Green Fluorescent

The mechanism by which hypoxia induces gene transcription involves the inhibition of HIF-1α (hypoxia-inducible factor-1 α subunit) PHD (prolyl hydroxylase) activity, which prevents the VHL (von Hippel-Lindau)-dependent targeting of HIF-1α to the ubiquitin/proteasome pathway. HIF-1α thus accumulates and promotes gene transcription. In the present study, first we provide direct biochemical evidence for the presence of a conserved hypoxic signalling pathway in Drosophila melanogaster. An assay for 2-oxoglutarate-dependent dioxygenases was developed using Drosophila embryonic and larval homogenates as a source of enzyme. Drosophila PHD has a low substrate specificity and hydroxylates key proline residues in the ODD (oxygen-dependent degradation) domains of human HIF-1α and Similar, the Drosophila homologue of HIF-1α. The enzyme promotes human and Drosophila [35S]VHL binding to GST (glutathione S-transferase)–ODD-domain fusion protein. Hydroxylation is enhanced by proteasomal inhibitors and was ascertained using an anti-hydroxyproline antibody. Secondly, by using transgenic flies expressing a fusion protein that combined an ODD domain and the green fluorescent protein (ODD–GFP), we analysed the hypoxic cascade in different embryonic and larval tissues. Hypoxic accumulation of the reporter protein was observed in the whole tracheal tree, but not in the ectoderm. Hypoxic stabilization of ODD–GFP in the ectoderm was restored by inducing VHL expression in these cells. These results show that Drosophila tissues exhibit different sensitivities to hypoxia.

scholarly journals LBP Proteins Modulate SF1-Independent Expression of P450scc in Human Placental JEG-3 Cells

2005 ◽  
Vol 19 (2) ◽  
pp. 409-420 ◽  
Author(s):  
Ningwu Huang ◽  
Walter L. Miller
Keyword(s):  
Green Fluorescent Protein ◽  
Cell Lines ◽  
Transcriptional Repression ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Transient Transfection ◽  
Glutathione S Transferase ◽  
Negative Effects ◽  
Chain Cleavage ◽  
Green Fluorescent

Abstract The cholesterol side-chain cleavage enzyme, P450scc, initiates biosynthesis of all steroid hormones. Adrenal and gonadal P450scc expression requires steroidogenic factor-1 (SF1), but P450scc expression in human placental JEG-3 cells utilizes an SF1-independent element at −155/−131 that is inactive in adrenals and gonads. We previously cloned two transcription factors, long terminal repeat binding protein (LBP)-1b and LBP-9, from JEG-3 cells. In transient transfection assays, LBP-1b activated the −155/−131 element whereas LBP-9 suppressed its LBP-1b-stimulated expression. To assess the roles of these factors on the intact P450scc gene, we stably expressed LBP-1b or LBP-9 in JEG-3 cells. All cell lines stably expressing a fusion protein of LBP-1b and enhanced green fluorescent protein increased P450scc expression, but cell lines stably expressing LBP-9 fused to enhanced green fluorescent protein either increased or decreased P450scc expression. 8-Br-cAMP induced endogenous LBP-9, but not LBP-1b expression. Glutathione-S-transferase pull-down assays showed that LBP-1b and LBP-9 can dimerize with themselves and with each other; LBP-1b residues 300–540 and LBP-9 residues 300–479 were required for dimer formation. Glutathione-S-transferase pull-down assays, bandshifts, and transient transfection assays showed that TReP-132 (another factor that can bind to −155/−131) does not interact with either LBP-1b or LBP-9, or influence their ability to induce or suppress transcription from the −155/−131 element. Gal4 transactivation assays showed that transcriptional repression activity by LBP-9 requires residues 100–200. RNAi interference of either LBP-1b or LBP-9 mRNAs decreased P450scc expression. LBP-1b is an important SF1-independent transcriptional activator stimulating P450scc expression in human placental JEG-3 cells, whereas LBP-9 modulates the action of LBP-1b, exerting both positive and negative effects.

scholarly journals Tobacco mosaic virus (TMV) Replicase and Movement Protein Function Synergistically in Facilitating TMV Spread by Lateral Diffusion in the Plasmodesmal Desmotubule of Nicotiana benthamiana

2008 ◽  
Vol 21 (3) ◽  
pp. 335-345 ◽  
Author(s):  
Dana Guenoune-Gelbart ◽  
Michael Elbaum ◽  
Guy Sagi ◽  
Amit Levy ◽  
Bernard L. Epel
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Protein Function ◽  
Fluorescent Protein ◽  
Lateral Diffusion ◽  
Integral Membrane Protein ◽  
Movement Proteins ◽  
Green Fluorescent ◽  
Cell Spread ◽  
Er Membrane

Virus spread through plasmodesmata (Pd) is mediated by virus-encoded movement proteins (MPs) that modify Pd structure and function. The MP of Tobacco mosaic virus (TMVMP) is an endoplasmic reticulum (ER) integral membrane protein that binds viral RNA (vRNA), forming a vRNA:MP:ER complex. It has been hypothesized that TMVMP causes Pd to dilate, thus potentiating a cytoskeletal mediated sliding of the vRNA:MP:ER complex through Pd; in the absence of MP, by contrast, the ER cannot move through Pd. An alternate model proposes that cell-to-cell spread takes place by diffusion of the MP:vRNA complex in the ER membranes which traverse Pd. To test these models, we measured the effect of TMVMP and replicase expression on cell-to-cell spread of several green fluorescent protein-fused probes: a soluble cytoplasmic protein, two ER lumen proteins, and two ER membrane-bound proteins. Our data support the diffusion model in which a complex that includes ER-embedded MP, vRNA, and other components diffuses in the ER membrane within the Pd driven by the concentration gradient between an infected cell and adjacent noninfected cells. The data also suggest that the virus replicase and MP function together in altering Pd conductivity.

scholarly journals Dynamin Regulates Focal Exocytosis in Phagocytosing Macrophages

2003 ◽  
Vol 14 (5) ◽  
pp. 2016-2028 ◽  
Author(s):  
Anke Di ◽  
Deborah J. Nelson ◽  
Vytautas Bindokas ◽  
Mary E. Brown ◽  
Frances Libunao ◽  
...  
Keyword(s):  
Cell Surface ◽  
Actin Polymerization ◽  
Kinase Inhibitors ◽  
Fluorescent Protein ◽  
Glutathione S Transferase ◽  
Particle Uptake ◽  
Cytoplasmic Vesicles ◽  
Intracellular Ca ◽  
Particle Ingestion ◽  
Green Fluorescent

Phagocytosis in macrophages is thought to involve insertion of cytoplasmic vesicles at sites of membrane expansion before particle ingestion (“focal” exocytosis). Capacitance (Cm) measurements of cell surface area were biphasic, with an initial rise indicative of exocytosis followed by a fall upon phagocytosis. Unlike other types of regulated exocytosis, the Cm rise was insensitive to intracellular Ca2+, but was inhibited by guanosine 5′-O-(2-thio)diphosphate. Particle uptake, but not Cm rise, was affected by phosphatidylinositol 3-kinase inhibitors. Inhibition of actin polymerization eliminated the Cm rise, suggesting possible coordination between actin polymerization and focal exocytosis. Introduction of anti-pan-dynamin IgG blocked Cm changes, suggesting that dynamin controls focal exocytosis and thereby phagocytosis. Similarly, recombinant glutathione S-transferase•amphiphysin-SH3 domain, but not a mutated form that cannot bind to dynamin, inhibited both focal exocytosis and phagocytosis. Immunochemical analysis of endogenous dynamin distribution in macrophages revealed a substantial particulate pool, some of which localized to a presumptive endosomal compartment. Expression of enhanced green fluorescent protein•dynamin-2 showed a motile dynamin pool, a fraction of which migrated toward and within the phagosomal cup. These results suggest that dynamin is involved in the production and/or movement of vesicles from an intracellular organelle to the cell surface to support membrane expansion around the engulfed particle.

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