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STAR Protocols Dec 2023Translation is a fundamental process of cellular behavior. Here, we present a protocol for measuring translation in Drosophila epithelial tissues using...
Translation is a fundamental process of cellular behavior. Here, we present a protocol for measuring translation in Drosophila epithelial tissues using O-propargyl-puromycin (OPP), a puromycin derivative. We detail steps for larval dissection, OPP incorporation, fixation, OPP labeling, immunostaining, and imaging. We also provide details of quantification analysis. Significantly, OPP addition to methionine-containing media enables polypeptide labeling in living cells. Here, we study wing imaginal discs, an excellent model system for investigating growth, proliferation, pattern formation, differentiation, and cell death. For complete details on the use and execution of this protocol, please refer to Lee et al. (2018), Ji et al. (2019), and Kiparaki et al. (2022)..
Topics: Animals; Drosophila; Imaginal Discs; Larva; Puromycin
PubMed: 37862174
DOI: 10.1016/j.xpro.2023.102653 -
Stem Cell Research & Therapy Mar 2022Spinal interneurons (INs) relay sensory and motor control information between the brain and body. When this relay circuitry is disrupted from injury or disease, it is...
BACKGROUND
Spinal interneurons (INs) relay sensory and motor control information between the brain and body. When this relay circuitry is disrupted from injury or disease, it is devastating to patients due to the lack of native recovery in central nervous system (CNS) tissues. Obtaining a purified population of INs is necessary to better understand their role in normal function and as potential therapies in CNS. The ventral V0 (V0) INs are excitatory neurons involved in locomotor circuits and are thus of interest for understanding normal and pathological spinal cord function. To achieve scalable amounts of V0 INs, they can be derived from pluripotent sources, such as mouse embryonic stem cells (mESCs), but the resultant culture is heterogenous, obscuring the specific role of V0 INs. This study generated a transgenic mESC line to enrich V0 INs from induced cultures to allow for a scalable, enriched population for future in vitro and in vivo studies.
METHODS
The transgenic Evx1-PAC mESC line was created by CRISPR-Cas9-mediated insertion of puromycin-N-acetyltransferase (PAC) into the locus of V0 IN marker Evx1. Evx1 and PAC mRNA expression were measured by qPCR. Viability staining helped establish the selection protocol for V0 INs derived from Evx1-PAC mESCs inductions. Immunostaining was used to examine composition of selected inductions. Cultures were maintained up to 30 days to examine maturation by expression of mature/synaptic markers, determined by immunostaining, and functional activity in co-cultures with selected motor neurons (MNs) and V2a INs on microelectrode arrays (MEAs).
RESULTS
V0 IN inductions were best selected with 4 µg/mL puromycin on day 10 to 11 and showed reduction of other IN populations and elimination of proliferative cells. Long-term selected cultures were highly neuronal, expressing neuronal nuclear marker NeuN, dendritic marker MAP2, pre-synaptic marker Bassoon, and glutamatergic marker VGLUT2, with some cholinergic VAChT-expressing cells. Functional studies on MEAs showed that co-cultures with MNs or MNs plus V2a INs created neuronal networks with synchronized bursting.
CONCLUSIONS
Evx1-PAC mESCs can be used to purify V0 IN cultures for largely glutamatergic neurons that can be used in network formation studies or for rodent models requiring transplanted V0 INs.
Topics: Animals; Homeodomain Proteins; Humans; Interneurons; Mice; Mice, Transgenic; Motor Neurons; Mouse Embryonic Stem Cells; Puromycin
PubMed: 35346349
DOI: 10.1186/s13287-022-02801-7 -
Biochemistry Jun 2015In an earlier study, β³-puromycin was used for the selection of modified ribosomes, which were utilized for the incorporation of five different β-amino acids into...
In an earlier study, β³-puromycin was used for the selection of modified ribosomes, which were utilized for the incorporation of five different β-amino acids into Escherichia coli dihydrofolate reductase (DHFR). The selected ribosomes were able to incorporate structurally disparate β-amino acids into DHFR, in spite of the use of a single puromycin for the selection of the individual clones. In this study, we examine the extent to which the structure of the β³-puromycin employed for ribosome selection influences the regio- and stereochemical preferences of the modified ribosomes during protein synthesis; the mechanistic probe was a single suppressor tRNA(CUA) activated with each of four methyl-β-alanine isomers (1-4). The modified ribosomes were found to incorporate each of the four isomeric methyl-β-alanines into DHFR but exhibited a preference for incorporation of 3(S)-methyl-β-alanine (β-mAla; 4), i.e., the isomer having the same regio- and stereochemistry as the O-methylated β-tyrosine moiety of β³-puromycin. Also conducted were a selection of clones that are responsive to β²-puromycin and a demonstration of reversal of the regio- and stereochemical preferences of these clones during protein synthesis. These results were incorporated into a structural model of the modified regions of 23S rRNA, which included in silico prediction of a H-bonding network. Finally, it was demonstrated that incorporation of 3(S)-methyl-β-alanine (β-mAla; 4) into a short α-helical region of the nucleic acid binding domain of hnRNP LL significantly stabilized the helix without affecting its DNA binding properties.
Topics: Alanine; Escherichia coli; Escherichia coli Proteins; Heterogeneous-Nuclear Ribonucleoprotein L; Humans; Hydrogen Bonding; Models, Molecular; Molecular Dynamics Simulation; Mutant Proteins; Nucleotide Motifs; Peptidyl Transferases; Protein Conformation; Protein Stability; Puromycin; RNA, Bacterial; RNA, Ribosomal; RNA, Ribosomal, 23S; RNA, Transfer, Amino Acyl; Recombinant Proteins; Ribosomes; Stereoisomerism; Substrate Specificity; Tetrahydrofolate Dehydrogenase
PubMed: 25982410
DOI: 10.1021/acs.biochem.5b00389 -
The Journal of Cell Biology Dec 1971The frequency of colony formation in monolayers of cultured frog cell lines treated with puromycin was compared in (a) haploid and heteroploid lines and (b)...
The frequency of colony formation in monolayers of cultured frog cell lines treated with puromycin was compared in (a) haploid and heteroploid lines and (b) mutagen-treated and nontreated haploid lines. Evidence that resistant colonies result from gene mutation was negative, since the colony frequency is independent of both ploidy and mutagen treatment. A study of five frog cell lines showed that colony formation in puromycin depends on (a) the concentration of puromycin, (b) preselection of the population with puromycin, and, particularly, (c) the capacity of the treated population to survive some exposure to puromycin. One haploid and one heteroploid strain showing stable resistance to puromycin have been isolated; comparison of those variants with sensitive populations has shown that resistance to puromycin is correlated with the cells' capacity to exclude the drug. The evidence for different levels of membrane permeability, combined with evidence for many degrees of resistance among and within cell populations, suggests a model of self-determining membrane units. The evolution of a resistant phenotype may result from changes in the proportion of specific units in the membrane population.
Topics: Animals; Anura; Cell Division; Cell Line; Cell Membrane Permeability; Cell Survival; Cells, Cultured; Clone Cells; Culture Media; Culture Techniques; Drug Resistance; Haploidy; Mutagens; Mutation; Phenotype; Polyploidy; Puromycin; Rana pipiens; Time Factors; Tritium
PubMed: 5316064
DOI: 10.1083/jcb.51.3.742 -
FEBS Letters Jan 2017The ubiquitin-like modifier, FAT10, is involved in proteasomal degradation and antigen processing. As ubiquitin and the ubiquitin-like modifier, ISG15, cotranslationally...
The ubiquitin-like modifier, FAT10, is involved in proteasomal degradation and antigen processing. As ubiquitin and the ubiquitin-like modifier, ISG15, cotranslationally modify proteins, we investigated whether FAT10 could also be conjugated to newly synthesized proteins. Indeed, we found that nascent proteins are modified with FAT10, but not with the same preference for newly synthesized proteins as observed for ISG15. Our data show that puromycin-labeled polypeptides are strongly modified by ISG15 and less intensely by ubiquitin and FAT10. Nevertheless, conjugates of all three modifiers copurify with ribosomes. Taken together, we show that unlike ISG15, ubiquitin and FAT10 are conjugated to a similar degree to newly translated and pre-existing proteins.
Topics: Cytokines; HEK293 Cells; Humans; Protein Biosynthesis; Puromycin; Ribosomes; Substrate Specificity; Ubiquitin; Ubiquitins
PubMed: 27926780
DOI: 10.1002/1873-3468.12512 -
Journal of Virology Aug 1979The in vivo primary and secondary transcription capabilities of wild-type snowshoe hare (SSH) virus and certain of its temperature-sensitive (ts) mutants have been...
In vivo transcription and protein synthesis capabilities of bunyaviruses: wild-type snowshoe hare virus and its temperature-sensitive group I, group II, and group I/II mutants.
The in vivo primary and secondary transcription capabilities of wild-type snowshoe hare (SSH) virus and certain of its temperature-sensitive (ts) mutants have been analyzed. The results obtained agree with in vitro studies (Bouloy et al., C.R. Acad. Sci. Paris 280:213-215, 1975; M. Bouloy and C. Hannoun, Virology 69:258-264, 1976; M. Ranki and R. Pettersson, J. Virol. 16:1420-1425, 1975) which have shown that bunyaviruses are negative-stranded RNA viruses with a virion RNA-directed RNA polymerase. The in vivo transcription studies have demonstrated that in the presence of protein synthesis inhibitors (puromycin or cycloheximide) SSH virus can synthesize viral complementary RNA (primary transcription) throughout the infection cycle. The increased levels of viral complementary RNA obtained in the absence of protein synthesis inhibitors (secondary transcription) were not markedly reduced if cells were pretreated with actinomycin D (5 mug/ml), alpha-amanitin (25 mug/ml), or rifampin (100 mug/ml), although progeny virus yields were reduced by up to 80% in the actinomycin D- and rifampin-treated cells. The in vivo transcription capabilities of SSH group I ts mutants at temperatures which were nonpermissive (40 degrees C) for virus replication gave values comparable to those obtained at permissive temperatures (33 degrees C). The SSH group I mutants appear, therefore, to be RNA-positive mutant types. When compared with their transcription capabilities at 33 degrees C, the in vivo transcription abilities of four SSH group II ts mutants (and one double group I/II ts mutant) were found to be more impaired at 40 degrees C than those of the SSH group I ts mutants or wild-type SSH virus at 40 degrees C, although the viral complementary RNA synthetic capabilities of these group II (and group I/II) mutants at 40 degrees C were significantly higher than their primary transcription capabilities (as measured at 33 degrees C in the presence of puromycin or cycloheximide). It was concluded, therefore, that these SSH group II (and double group I/II) ts mutants have an intermediate RNA phenotype. Hybridization studies using (32)P-labeled individual L, M, and S viral RNA species of SSH virus have demonstrated the presence of viral complementary RNA to all three species in extracts of cells infected with SSH ts II-30 and incubated at 33 degrees C (primary and secondary transcription) or 40 degrees C, a nonpermissive temperature for its replication. The results of pulse-labeled in vivo protein analyses indicated that greater quantities of intracellular N protein (coded for by S RNA [J. R. Gentsch and D. H. L. Bishop, J. Virol. 28:417-419, 1978]) than G1 and G2 polypeptides (coded for by M RNA [J. R. Gentsch and D. H. L. Bishop, J. Virol. 30:767-776, 1979]) were present in extracts of cells infected with wild-type SSH virus. In extracts of SSH group I, II, or I/II ts mutant-infected cells incubated at 33 degrees C, N and G1, and for the group II mutant-infected cells, G2, viral polypeptides were detected, whereas in extracts obtained from group I or II mutant virus-infected cells incubated at 40 degrees C, low levels of N and G1 polypeptides were evident.
Topics: Animals; Arboviruses; Bunyamwera virus; Cell Line; Cricetinae; Cycloheximide; Kidney; Mutation; Puromycin; RNA, Viral; Temperature; Transcription, Genetic; Viral Proteins
PubMed: 480477
DOI: 10.1128/JVI.31.2.426-436.1979 -
RNA (New York, N.Y.) May 2000The binding site of puromycin was probed chemically in the peptidyl-transferase center of ribosomes from Escherichia coli and of puromycin-hypersensitive ribosomes from...
The binding site of puromycin was probed chemically in the peptidyl-transferase center of ribosomes from Escherichia coli and of puromycin-hypersensitive ribosomes from the archaeon Haloferax gibbonsii. Several nucleotides of the 23S rRNAs showed altered chemical reactivities in the presence of puromycin. They include A2439, G2505, and G2553 for E. coli, and G2058, A2503, G2505, and G2553 for Hf. gibbonsii (using the E. coli numbering system). Reproducible enhanced reactivities were also observed at A508 and A1579 within domains I and III, respectively, of E. coli 23S rRNA. In further experiments, puromycin was shown to produce a major reduction in the UV-induced crosslinking of deacylated-(2N3A76)tRNA to U2506 within the P' site of E. coli ribosomes. Moreover, it strongly stimulated the putative UV-induced crosslink between a streptogramin B drug and m2A2503/psi2504 at an adjacent site in E. coli 23S rRNA. These data strongly support the concept that puromycin, along with other peptidyl-transferase antibiotics, in particular the streptogramin B drugs, bind to an RNA structural motif that contains several conserved and accessible base moieties of the peptidyl transferase loop region. This streptogramin motif is also likely to provide binding sites for the 3' termini of the acceptor and donor tRNAs. In contrast, the effects at A508 and A1579, which are located at the exit site of the peptide channel, are likely to be caused by a structural effect transmitted along the peptide channel.
Topics: Base Sequence; Binding Sites; Escherichia coli; Haloferax; Molecular Sequence Data; Peptidyl Transferases; Puromycin; RNA, Archaeal; RNA, Bacterial; RNA, Ribosomal; RNA, Transfer; Ribosomes; Substrate Specificity
PubMed: 10836795
DOI: 10.1017/s1355838200000091 -
Proceedings of the National Academy of... Dec 1971Evidence is presented that ribosomes active in protein synthesis and attached to messenger RNA on polysomes have a smaller diameter than free cytoplasmic single...
Evidence is presented that ribosomes active in protein synthesis and attached to messenger RNA on polysomes have a smaller diameter than free cytoplasmic single ribosomes. Measurements have been made on these two types of ribosomes of differences in sedimentation velocity and diffusion constant. Differences in these quantities suggest about a 20-A decrease in the diameter of the ribosomes from chick embryo muscles when they are attached to messenger RNA. Similar differences are also observed in rabbit reticulocytes and mouse ascites tumor cells. These two ribosomal states have different sensitivity to Pronase digestion and dissociate into ribosomal subunits at different KCl concentrations. This size difference is not associated with a significant difference in overall ribosomal mass and appears not to be dependent upon the presence of nascent polypeptide chains.
Topics: Animals; Carbon Isotopes; Carcinoma, Krebs 2; Cell Line; Centrifugation, Density Gradient; Chick Embryo; Cytoplasm; In Vitro Techniques; Mice; Muscles; Peptides; Pronase; Puromycin; RNA, Messenger; RNA, Ribosomal; Rabbits; Reticulocytes; Ribonucleases; Ribosomes
PubMed: 5289247
DOI: 10.1073/pnas.68.12.3021 -
ELife Aug 2020Puromycin is an amino-acyl transfer RNA analog widely employed in studies of protein synthesis. Since puromycin is covalently incorporated into nascent polypeptide...
Puromycin is an amino-acyl transfer RNA analog widely employed in studies of protein synthesis. Since puromycin is covalently incorporated into nascent polypeptide chains, anti-puromycin immunofluorescence enables visualization of nascent protein synthesis. A common assumption in studies of local messenger RNA translation is that the anti-puromycin staining of puromycylated nascent polypeptides in fixed cells accurately reports on their original site of translation, particularly when ribosomes are stalled with elongation inhibitors prior to puromycin treatment. However, when we attempted to implement a proximity ligation assay to detect ribosome-puromycin complexes, we found no evidence to support this assumption. We further demonstrated, using biochemical assays and live cell imaging of nascent polypeptides in mammalian cells, that puromycylated nascent polypeptides rapidly dissociate from ribosomes even in the presence of elongation inhibitors. Our results suggest that attempts to define precise subcellular translation sites using anti-puromycin immunostaining may be confounded by release of puromycylated nascent polypeptide chains prior to fixation.
Topics: Animals; Cell Line, Tumor; Mice; Peptide Chain Elongation, Translational; Protein Synthesis Inhibitors; Proteins; Puromycin; RNA, Messenger; RNA, Transfer, Amino Acyl; Ribosomes
PubMed: 32844746
DOI: 10.7554/eLife.60048 -
European Journal of Biochemistry Dec 1998In a cell-free system derived from Escherichia coli, various analogues of spermine were used to study their effect on the binding of AcPhe-tRNA to poly (U)-programmed...
In a cell-free system derived from Escherichia coli, various analogues of spermine were used to study their effect on the binding of AcPhe-tRNA to poly (U)-programmed ribosomes and on the puromycin reaction carried out at 6 mM Mg2+ (Ac, acetyl). In the absence of factors washable from ribosomes (FWR fraction), mono-acylated or di-acylated analogues of spermine stimulate the binding of AcPhe-tRNA to a lesser degree than spermine, in the order: N1-acetylspermine > N1,N12-diacetylspermine approximately = N1,N12-dipivaloylspermine. Also, the above analogues do not show any sparing effect on Mg2+ requirements for AcPhe-tRNA binding to ribosomes, in contrast to spermine. The presence of FWR fraction during the binding or acetylation of the secondary amines of spermine moderates or abolishes the stimulatory effect. In addition, all analogues tested enhance the stability of the ternary complex AcPhe-tRNA-poly(U)-ribosome and the extent of AcPhe-puromycin synthesis, particularly in the absence of the FWR fraction. At the kinetic phase of AcPhe-puromycin synthesis, the analogues display both stimulatory and inhibitory effects, depending on the absence (partial noncompetitive inhibition) or the presence of the FWR fraction (nonessential activation in concert with partial noncompetitive inhibition). Detailed kinetic analysis shows that the analogues tested can mimic the behaviour of spermine, however, the potency to affect the peptidyltransferase activity depends on their degree of acylation, acyl-substituent size, charge distribution and on their chain flexibility.
Topics: Escherichia coli; Kinetics; Magnesium; Molecular Structure; Peptidyl Transferases; Poly U; Protein Binding; Puromycin; RNA, Transfer, Amino Acyl; Ribosomes; Spermine; Structure-Activity Relationship
PubMed: 9874209
DOI: 10.1046/j.1432-1327.1998.2580437.x