RESUMO
ADP-ribosyltransferases use NAD+ to catalyse substrate ADP-ribosylation1, and thereby regulate cellular pathways or contribute to toxin-mediated pathogenicity of bacteria2-4. Reversible ADP-ribosylation has traditionally been considered a protein-specific modification5, but recent in vitro studies have suggested nucleic acids as targets6-9. Here we present evidence that specific, reversible ADP-ribosylation of DNA on thymidine bases occurs in cellulo through the DarT-DarG toxin-antitoxin system, which is found in a variety of bacteria (including global pathogens such as Mycobacterium tuberculosis, enteropathogenic Escherichia coli and Pseudomonas aeruginosa)10. We report the structure of DarT, which identifies this protein as a diverged member of the PARP family. We provide a set of high-resolution structures of this enzyme in ligand-free and pre- and post-reaction states, which reveals a specialized mechanism of catalysis that includes a key active-site arginine that extends the canonical ADP-ribosyltransferase toolkit. Comparison with PARP-HPF1, a well-established DNA repair protein ADP-ribosylation complex, offers insights into how the DarT class of ADP-ribosyltransferases evolved into specific DNA-modifying enzymes. Together, our structural and mechanistic data provide details of this PARP family member and contribute to a fundamental understanding of the ADP-ribosylation of nucleic acids. We also show that thymine-linked ADP-ribose DNA adducts reversed by DarG antitoxin (functioning as a noncanonical DNA repair factor) are used not only for targeted DNA damage to induce toxicity, but also as a signalling strategy for cellular processes. Using M. tuberculosis as an exemplar, we show that DarT-DarG regulates growth by ADP-ribosylation of DNA at the origin of chromosome replication.
Assuntos
ADP-Ribosilação , Proteínas de Bactérias/metabolismo , DNA/química , DNA/metabolismo , Timina/química , Timina/metabolismo , Adenosina Difosfato Ribose/metabolismo , Antitoxinas , Proteínas de Bactérias/química , Toxinas Bacterianas , Sequência de Bases , Biocatálise , DNA/genética , Adutos de DNA/química , Adutos de DNA/metabolismo , Dano ao DNA , Reparo do DNA , Elementos de DNA Transponíveis/genética , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Modelos Moleculares , Mycobacterium/enzimologia , Mycobacterium/genética , Nitrogênio/química , Nitrogênio/metabolismo , Poli(ADP-Ribose) Polimerases/química , Origem de Replicação/genética , Especificidade por Substrato , Thermus/enzimologia , Timidina/química , Timidina/metabolismoRESUMO
Controlling the pairing strength of nucleobases in DNA through reactions with compounds found inside the cell is a formidable challenge. Here we report how a thiazolyl substituent turns a strongly pairing ethynylpyridone C-nucleoside into a reactive residue in oligonucleotides. The thiazolyl-bearing pyridone reacts with soft nucleophiles, such as glutathione, but not with hard nucleophiles like hydroxide or carbonate. The addition products pair much more weakly with adenine in a complementary strand than the starting material, and also change their fluorescence. This makes oligonucleotides containing the new deoxynucleoside interesting for controlled release. Due to its reactivity toward N, P, S, and Se-nucleophiles, and the visual signal accompanying chemical conversion, the fluorescent nucleotide reported here may also have applications in chemical biology, sensing and diagnostics.
Assuntos
Nucleosídeos , Nucleotídeos , Pareamento de Bases , Timidina/química , Nucleosídeos/química , Nucleotídeos/química , Oligonucleotídeos/química , CorantesRESUMO
Detection of thymidine analogues after their incorporation into replicating DNA represents a powerful tool for the study of cellular DNA synthesis, progression through the cell cycle, cell proliferation kinetics, chronology of cell division, and cell fate determination. Recent advances in the concurrent detection of multiple such analogues offer new avenues for the investigation of unknown features of these vital cellular processes. Combined with quantitative analysis, temporal discrimination of multiple labels enables elucidation of various aspects of stem cell life cycle in situ, such as division modes, differentiation, maintenance, and elimination. Data obtained from such experiments are critically important for creating descriptive models of tissue histogenesis and renewal in embryonic development and adult life. Despite the wide use of thymidine analogues in stem cell research, there are a number of caveats to consider for obtaining valid and reliable labeling results when marking replicating DNA with nucleotide analogues. Therefore, in this review, we describe critical points regarding dosage, delivery, and detection of nucleotide analogues in the context of single and multiple labeling, outline labeling schemes based on pulse-chase, cumulative and multilabel marking of replicating DNA for revealing stem cell proliferative behaviors, and determining cell cycle parameters, and discuss preconditions and pitfalls in conducting such experiments. The information presented in our review is important for rational design of experiments on tracking dividing stem cells by marking replicating DNA with thymidine analogues.
Assuntos
Ciclo Celular , Autorrenovação Celular , Rastreamento de Células , Replicação do DNA , Células-Tronco , Timidina , Animais , Humanos , Células-Tronco/citologia , Células-Tronco/metabolismo , Timidina/análogos & derivados , Timidina/química , Timidina/farmacologiaRESUMO
DNA synthesis is a fundamental requirement for cell proliferation and DNA repair, but no single method can identify the location, direction and speed of replication forks with high resolution. Mammalian cells have the ability to incorporate thymidine analogs along with the natural A, T, G and C bases during DNA synthesis, which allows for labeling of replicating or repaired DNA. Here, we demonstrate the use of the Oxford Nanopore Technologies MinION to detect 11 different thymidine analogs including CldU, BrdU, IdU as well as EdU alone or coupled to Biotin and other bulky adducts in synthetic DNA templates. We also show that the large adduct Biotin can be distinguished from the smaller analog IdU, which opens the possibility of using analog combinations to identify the location and direction of DNA synthesis. Furthermore, we detect IdU label on single DNA molecules in the genome of mouse pluripotent stem cells and using CRISPR/Cas9-mediated enrichment, determine replication rates using newly synthesized DNA strands in human mitochondrial DNA. We conclude that this novel method, termed Replipore sequencing, has the potential for on target examination of DNA replication in a wide range of biological contexts.
Assuntos
Bromodesoxiuridina/química , Sequenciamento por Nanoporos , Timidina/genética , Animais , Biotina/química , Biotina/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Replicação do DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Nanoporos , Timidina/químicaRESUMO
Chemically modified nucleic acids are essential for the therapeutic application of oligonucleotides. In this study, 6'-C-spiro-thymidine exhibiting a fixed torsion angle γ was designed, synthesized, and incorporated into oligonucleotides. The conformational analysis of the 6'-C-spiro-thymidine monomer revealed that its torsion angle γ was in the +synclinal range (approx. 60°), which is similar to that in a natural RNA duplex, as expected. On the other hand, the sugar conformation of the RNA duplex is known to be predominantly an N-type, whereas that of the synthesized monomer was an S-type. The results of the UV melting analysis demonstrated that the duplex-forming ability of 6'-C-spiro-thymidine was inferior to that of natural DNA. Contrarily, 6'-C-spiro-thymidine could enhance the stability of oligonucleotides toward nucleases. Particularly, the incorporation of 6'-C-spiro-thymidine on the 3'-ends of the oligonucleotides significantly increased the nuclease resistance of the oligonucleotides.
Assuntos
Ácidos Nucleicos , Oligonucleotídeos , Alcanos , DNA/química , Oligonucleotídeos/química , RNA , Esqueleto , Compostos de Espiro , Açúcares , Timidina/químicaRESUMO
O2- and O4-alkylated thymidine lesions are known to be poorly repaired and persist in mammalian tissues. To understand how mammalian cells sense the presence and regulate the repair of these lesions, we employed a quantitative proteomic method to discover regioisomeric O2- and O4-n-butylthymidine (O2- and O4-nBudT)-binding proteins. We were able to identify 21 and 74 candidate DNA damage recognition proteins for O2-nBudT- and O4-nBudT-bearing DNA probes, respectively. Among these proteins, DDB1 and DDB2 selectively bind to O2-nBudT-containing DNA, whereas three high-mobility group (HMG) proteins (i.e., HMGB1, HMGB2, and mitochondrial transcription factor A (TFAM)) exhibit preferential binding to O4-nBudT-bearing DNA. We further demonstrated that TFAM binds directly and selectively with O4-alkyldT-harboring DNA, and the binding capacity depends mainly on the HMG box-A domain of TFAM. We also found that TFAM promotes transcriptional mutagenesis of O4-nBudT and O4-pyridyloxobutylthymidine, which is a DNA adduct induced by tobacco-specific N-nitrosamines, in vitro and in human cells. Together, we explored, for the first time, the interaction proteomes of O-alkyldT lesions, and our study expanded the functions of TFAM by revealing its capability in the recognition of O4-alkyldT-bearing DNA and uncovering its modulation of transcriptional mutagenesis of these lesions in human cells.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas Mitocondriais/química , Timidina/análogos & derivados , Fatores de Transcrição/química , Sítios de Ligação , DNA/química , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteínas Mitocondriais/metabolismo , Estrutura Molecular , Mutação , Timidina/química , Timidina/genética , Timidina/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Selenium-modified nucleosides are powerful tools to study the structure and function of nucleic acids and their protein interactions. The widespread application of 2-selenopyrimidine nucleosides is currently limited by low yields in established synthetic routes. Herein, we describe the optimization of the synthesis of 2-Se-uridine and 2-Se-thymidine derivatives by thermostable nucleoside phosphorylases in transglycosylation reactions using natural uridine or thymidine as sugar donors. Reactions were performed at 60 or 80 °C and at pHâ 9 under hypoxic conditions to improve the solubility and stability of the 2-Se-nucleobases in aqueous media. To optimize the conversion, the reaction equilibria in analytical transglycosylation reactions were studied. The equilibrium constants of phosphorolysis of the 2-Se-pyrimidines were between 5 and 10, and therefore differ by an order of magnitude from the equilibrium constants of any other known case. Hence, the thermodynamic properties of the target nucleosides are inherently unfavorable, and this complicates their synthesis significantly. A tenfold excess of sugar donor was needed to achieve 40-48 % conversion to the target nucleoside. Scale-up of the optimized conditions provided four Se-containing nucleosides in 6-40 % isolated yield, which compares favorably to established chemical routes.
Assuntos
Nucleosídeos/biossíntese , Pentosiltransferases/metabolismo , Timidina/análogos & derivados , Biocatálise , Glicosilação , Estrutura Molecular , Compostos Organosselênicos/química , Termodinâmica , Timidina/biossíntese , Timidina/químicaRESUMO
A novel class of nucleotide analogues with a dioxane ring as central scaffold has been developed. Synthetic routes in two diastereomeric series were realized, and the final thymidine analogues were synthesized with common functionalities for the automated oligonucleotide synthesis. The chemical space of the initially derived nucleotides was expanded by changing the central dioxane to analogous morpholine derivatives. This opens up the possibility for further derivatization by attaching different substituents at the morpholine nitrogen. The novel nucleotide building blocks were incorporated into double-stranded RNA sequences, and their hybridization properties investigated by melting-temperature analysis. Both scaffolds, dioxanes and morpholines, had an equal impact on double-strand stability, but Tm values differed depending on the chirality in the six-membered ring.
Assuntos
Dioxanos/química , Morfolinos/metabolismo , RNA de Cadeia Dupla/metabolismo , Morfolinos/síntese química , Morfolinos/química , Hibridização de Ácido Nucleico , Estereoisomerismo , Timidina/química , Temperatura de TransiçãoRESUMO
We describe highly efficient interstrand photocrosslinking of a DNA duplex containing 5-chloro-2'-deoxy-4-thiouridine (ClSdU) in one strand, proceeding via a two-step photochemical cascade, involving the formation of a thermally reversible crosslink between ClSdU and thymidine in the target strand and its subsequent conversion to a thermally stable fluorescent crosslink. These results show that ClSdU has great potential to be a valuable DNA photo-crosslinking reagent for chemical biology applications.
Assuntos
Reagentes de Ligações Cruzadas/química , DNA/química , Oligodesoxirribonucleotídeos/química , Tiouridina/análogos & derivados , Reagentes de Ligações Cruzadas/efeitos da radiação , Temperatura , Tiouridina/efeitos da radiação , Timidina/química , Raios UltravioletaRESUMO
Thymidine derivatives bearing spiroacetal moieties on the C4'-position (5'R-spiro-thymidine and 5'S-spiro-thymidine) were synthesized and incorporated into oligonucleotides. The duplex- and triplex-forming abilities of both the oligonucleotides were evaluated from UV melting experiments. Oligonucleotides with the 5'S-spiro modifications could form thermally stable duplexes with complementary RNA and DNA; however, the 5'R-spiro modification significantly decreased the thermal stabilities of the duplexes and triplexes. Oligonucleotides with these spiro-thymidines showed significantly high resistance towards enzymatic degradation.
Assuntos
Oligonucleotídeos/química , Compostos de Espiro/química , Timidina/química , Estrutura Molecular , Oligonucleotídeos/síntese químicaRESUMO
The population and depopulation mechanisms leading to the lowest-lying triplet states of 2-Se-Thymine were studied at the MS-CASPT2/cc-pVDZ level of theory. Several critical points on different potential energy hypersurfaces were optimized, including minima, conical intersections, and singlet-triplet crossings. The accessibility of all relevant regions on the potential energy hypersurfaces was investigated by means of minimum energy paths and linear interpolation in internal coordinates techniques. Our analysis indicates that, after the population of the bright S2 state in the Franck-Condon region, the first photochemical event is a barrierless evolution towards one of its two minima. After that, three viable photophysical deactivation paths can take place. In one of them, the population in the S2 state is transferred to the T2 state via intersystem crossing and subsequently to the T1 state by internal conversion. Alternatively, the S1 state could be accessed by internal conversion through two distinct conical intersections with S2 state followed by singlet-triplet crossing with the T2 state. The absence of a second minimum on the T1 state and a small energy barrier on pathway along the potential energy surface towards the ground state from the lowest triplet state are attributed as potential reasons to explain why the lifetime of the triplet state of 2-Se-Thymine might be reduced in comparison with its thio-analogue.
Assuntos
Compostos Organosselênicos/química , Timidina/análogos & derivados , Cinética , Modelos Moleculares , Conformação Molecular , Oxirredução , Processos Fotoquímicos , Termodinâmica , Timidina/químicaRESUMO
Oxazinomycin is a C-nucleoside antibiotic that is produced by Streptomyces hygroscopicus and closely resembles uridine. Here, we show that the oxazinomycin triphosphate is a good substrate for bacterial and eukaryotic RNA polymerases (RNAPs) and that a single incorporated oxazinomycin is rapidly extended by the next nucleotide. However, the incorporation of several successive oxazinomycins or a single oxazinomycin in a certain sequence context arrested a fraction of the transcribing RNAP. The addition of Gre RNA cleavage factors eliminated the transcriptional arrest at a single oxazinomycin and shortened the nascent RNAs arrested at the polythymidine sequences suggesting that the transcriptional arrest was caused by backtracking of RNAP along the DNA template. We further demonstrate that the ubiquitous C-nucleoside pseudouridine is also a good substrate for RNA polymerases in a triphosphorylated form but does not inhibit transcription of the polythymidine sequences. Our results collectively suggest that oxazinomycin functions as a Trojan horse substrate and its inhibitory effect is attributable to the oxygen atom in the position corresponding to carbon five of the uracil ring.
Assuntos
RNA Polimerases Dirigidas por DNA/química , RNA/química , Transcrição Gênica/efeitos dos fármacos , Uridina/análogos & derivados , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/genética , Oxigênio/química , Pseudomonas/química , RNA/genética , Clivagem do RNA/efeitos dos fármacos , Streptomyces/química , Especificidade por Substrato , Timidina/química , Timidina/genética , Transcrição Gênica/genética , Fatores de Elongação da Transcrição/genética , Uracila/química , Uridina/síntese química , Uridina/química , Uridina/farmacologiaRESUMO
Certain viruses of bacteria (bacteriophages) enzymatically hypermodify their DNA to protect their genetic material from host restriction endonuclease-mediated cleavage. Historically, it has been known that virion DNAs from the Delftia phage ΦW-14 and the Bacillus phage SP10 contain the hypermodified pyrimidines α-putrescinylthymidine and α-glutamylthymidine, respectively. These bases derive from the modification of 5-hydroxymethyl-2'-deoxyuridine (5-hmdU) in newly replicated phage DNA via a pyrophosphorylated intermediate. Like ΦW-14 and SP10, the Pseudomonas phage M6 and the Salmonella phage ViI encode kinase homologs predicted to phosphorylate 5-hmdU DNA but have uncharacterized nucleotide content [Iyer et al. (2013) Nucleic Acids Res 41:7635-7655]. We report here the discovery and characterization of two bases, 5-(2-aminoethoxy)methyluridine (5-NeOmdU) and 5-(2-aminoethyl)uridine (5-NedU), in the virion DNA of ViI and M6 phages, respectively. Furthermore, we show that recombinant expression of five gene products encoded by phage ViI is sufficient to reconstitute the formation of 5-NeOmdU in vitro. These findings point to an unexplored diversity of DNA modifications and the underlying biochemistry of their formation.
Assuntos
Bactérias/metabolismo , Infecções Bacterianas/microbiologia , Proteínas de Bactérias/metabolismo , Bacteriófagos/genética , DNA Viral/biossíntese , Timidina/química , Uridina/química , Bacteriófagos/crescimento & desenvolvimento , Bacteriófagos/metabolismo , Genoma ViralRESUMO
DNA reacts directly with UV light with a wavelength shorter than 300 nm. Although ground surface sunlight includes little of this short-wavelength UV light due to its almost complete absorption by the atmosphere, sunlight is the primary cause of skin cancer. Photosensitization by endogenous substances must therefore be involved in skin cancer development mechanisms. Uric acid is the final metabolic product of purines in humans, and is present at relatively high concentrations in cells and fluids. When a neutral mixed solution of 2'-deoxycytidine, 2'-deoxyguanosine, thymidine, and 2'-deoxyadenosine was irradiated with UV light with a wavelength longer than 300 nm in the presence of uric acid, all the nucleosides were consumed in a uric acid dose-dependent manner. These reactions were inhibited by the addition of radical scavengers, ethanol and sodium azide. Two products from 2'-deoxycytidine were isolated and identified as N4-hydroxy-2'-deoxycytidine and N4,5-cyclic amide-2'-deoxycytidine, formed by cycloaddition of an amide group from uric acid. A 15N-labeled uric acid, uric acid-1,3-15N2, having two 14N and two 15N atoms per molecule, produced N4,5-cyclic amide-2'-deoxycytidine containing both 14N and 15N atoms from uric acid-1,3-15N2. Singlet oxygen, hydroxyl radical, peroxynitrous acid, hypochlorous acid, and hypobromous acid generated neither N4-hydroxy-2'-deoxycytidine nor N4,5-cyclic amide-2'-deoxycytidine in the presence of uric acid. These results indicate that uric acid is a photosensitizer for the reaction of nucleosides by UV light with a wavelength longer than 300 nm, and that an unidentified radical derived from uric acid with a delocalized unpaired electron may be generated.
Assuntos
DNA/química , Desoxiadenosinas/química , Desoxirribonucleosídeos/química , Fármacos Fotossensibilizantes/química , Ácido Úrico/química , Bromatos/química , Desoxicitidina/química , Desoxiguanosina/química , Etanol/química , Sequestradores de Radicais Livres/química , Ácido Hipocloroso/química , Cinética , Ácido Peroxinitroso/química , Processos Fotoquímicos , Oxigênio Singlete/química , Azida Sódica/química , Timidina/química , Raios UltravioletaRESUMO
We compared the formation of γH2AX foci (marker of DNA double-strand breaks) in human lung fibroblasts (MRC-5 line) during their 24-h incubation in a medium containing 3H-labeled thymidine or amino acids (glycine, alanine, and proline) with specific radioactivity from 100 to 400 MBq/liter. A linear dependence of changes in the number of γH2AX foci on the specific radioactivity of the medium was revealed. The quantitative yield of DNA double-strand breaks under the influence of 3H-thymidine was more than 2-fold higher than under the influence of 3H-labeled amino acids. Comparative analysis of the yields of DNA double-strand breaks during cell incubation with 3H-labeled amino acids showed that 3H-alanine produced more pronounced effect that 3H-proline, which is consistent with the data on the content of their non-radioactive analogs in chromatin proteins.
Assuntos
Quebras de DNA de Cadeia Dupla , Fibroblastos , Histonas/genética , Pulmão , Trítio/farmacologia , Aminoácidos/química , Aminoácidos/farmacologia , Células Cultivadas , Meios de Cultura/química , Meios de Cultura/farmacologia , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibroblastos/efeitos da radiação , Histonas/metabolismo , Humanos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Pulmão/efeitos da radiação , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/efeitos da radiação , Timidina/química , Timidina/farmacologia , Trítio/químicaRESUMO
Base J replaces 1% of thymine in most kinetoplastid flagellates, and is implicated in transcription regulation. Base J is synthesized in two steps: first, a thymine base in DNA is converted to 5-hydroxymethyluracil by J-binding proteins (JBP1, JBP2); secondly, a glucosyl transferase glycosylates the 5-hydroxymethyluracil to form base J. Here, we present a highly sensitive and selective LC-MS/MS method to quantify the in vitro JBP1 activity on synthetic oligonucleotide substrates. The method demonstrated successful to support biochemical studies of JBPs and can be used as a template for additional JBP activity studies or for inhibitor screening in the future.
Assuntos
Cromatografia Líquida de Alta Pressão , Proteínas de Ligação a DNA/metabolismo , Proteínas de Protozoários/metabolismo , Espectrometria de Massas em Tandem , Timidina/análogos & derivados , Leishmania/metabolismo , Especificidade por Substrato , Timidina/análise , Timidina/química , Timidina/metabolismoRESUMO
In this study, we designed 5'-amino-5'-deoxy-5'-hydroxymethylthymidine as a new oligonucleotide modification with an amino group directly attached to the 5'-carbon atom. We successfully synthesized two isomers of 5'-amino-5'-deoxy-5'-hydroxymethylthymidine via dihydroxylation of the 5'-vinyl group incorporated into 5'-deoxy-5'-C-methenylthymidine derivative. Moreover, it was found that the nuclease resistance, binding selectivity to single-stranded RNA, and triplex-forming ability of an oligonucleotide containing RT residues of the new compound were higher than those of the unmodified oligonucleotide.
Assuntos
Oligonucleotídeos/síntese química , Timidina/química , Modelos Moleculares , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Timidina/análogos & derivadosRESUMO
Absolute cross sections (CSs) for electronic excitation by low-energy electron (LEE) scattering, from condensed thymidine (dT) in the 6-18 eV incident energy range, were measured by high-resolution electron energy loss spectroscopy (HREELS). Various electron energy loss (EEL) spectra were acquired using 1 ML of dT condensed on a multilayer film of Ar held at about 20 K under ultra-high vacuum (â¼1 × 10-11 Torr). dT is one of the most complex DNA constituents to be studied by HREELS and these spectra provide the first LEE energy-loss data for electronic excitation of a nucleoside. CSs for transitions to the states 13A', 13A'', 23A', 21A', 33A', 23A'', 43A', 33A'', 53A' and 51A' of dT were extracted from the EEL spectra. These states correlate to those previously measured for the thymine moiety. Two broad resonances are observed in the energy dependence of the CSs at around 8 and 10 eV; these energies are close to those found in earlier gas- and solid-phase studies on the interaction of LEEs with dT, thymine and related molecules. A quantitative comparison between the electronic CSs of dT and those of thymine and tetrahydrofuran indicates that no variation is induced in the electronic CSs of thymine upon chemically binding to a deoxyribose group.
Assuntos
Análise Espectral , Timidina/químicaRESUMO
BACKGROUND: In the poultry industry, quantitative analysis of chicken T cell proliferation is important in many biological applications such as drug screening, vaccine production, and cytotoxicity assessment. Several assays have been established to evaluate this immunological response in chicken cells. However, these assays have some disadvantages including use of radioactive labels ([3H]-Thymidine assay), necessity of DNA denaturation or digestion (BrdU incorporation assay), lack of sensitivity and underestimation of anti-proliferative effects (MTT assay), and modulation of activation molecules and cell viability reduction (CFSE assay). Overcoming these limitations, the EdU proliferation assay is sensitive and advantageous compared to [3H]-Thymidine radioactive labels in studies on cell proliferation in vitro and allows simultaneous identification of T cell populations. However, this assay has not been established using primary chicken cells to evaluate T cell proliferation by flow cytometry. RESULTS: Here, we established an assay to evaluate the proliferation of primary chicken splenocytes based on the incorporation of a thymidine analog (EdU) and a click reaction with a fluorescent azide, detected by a flow cytometer. We also established a protocol that combines EdU incorporation and immunostaining to detect CD4+ and CD8+ proliferating T cells. By inducing cell proliferation with increasing concentrations of a mitogen (Concanavalin A), we observed a linear increase in EdU positive cells, indicating that our protocol does not present any deficiency in the quantity and quality of reagents that were used to perform the click reaction. CONCLUSIONS: In summary, we established a reliable protocol to evaluate the proliferation of CD4+ and CD8+ chicken T cells by flow cytometry. Moreover, as this is an in-house protocol, the cost per sample using this protocol is low, allowing its implementation in laboratories that process a large number of samples.
Assuntos
Galinhas , Citometria de Fluxo/veterinária , Linfócitos T/citologia , Animais , Proliferação de Células , Citometria de Fluxo/métodos , Citometria de Fluxo/normas , Timidina/análogos & derivados , Timidina/químicaRESUMO
Binding reactions of HgII and AgI to pyrimidine-pyrimidine mismatches in duplex DNA were characterized using fluorescent nucleobase analogs, thermal denaturation and 1H NMR. Unlike AgI, HgII exhibited stoichiometric, site-specific binding of C-T mismatches. The on- and off-rates of HgII binding were approximately 10-fold faster to C-T mismatches (kon ≈ 105 M-1 s-1, koff ≈ 10-3 s-1) as compared to T-T mismatches (kon ≈ 104 M-1 s-1, koff ≈ 10-4 s-1), resulting in very similar equilibrium binding affinities for both types of 'all natural' metallo base pairs (Kd ≈ 10-150 nM). These results are in contrast to thermal denaturation analyses, where duplexes containing T-T mismatches exhibited much larger increases in thermal stability upon addition of HgII (ΔTm = 6-19°C), as compared to those containing C-T mismatches (ΔTm = 1-4°C). In addition to revealing the high thermodynamic and kinetic stabilities of C-HgII-T base pairs, our results demonstrate that fluorescent nucleobase analogs enable highly sensitive detection and characterization of metal-mediated base pairs - even in situations where metal binding has little or no impact on the thermal stability of the duplex.