RESUMEN
Therminator DNA polymerase is an efficient DNA-dependent TNA polymerase capable of polymerizing TNA oligomers of at least 80 nt in length. In order for Therminator to be useful for the in vitro selection of functional TNA sequences, its TNA synthesis fidelity must be high enough to preserve successful sequences. We used sequencing to examine the fidelity of Therminator-catalyzed TNA synthesis at different temperatures, incubation times, tNTP ratios and primer/template combinations. TNA synthesis by Therminator exhibits high fidelity under optimal conditions; the observed fidelity is sufficient to allow in vitro selection with TNA libraries of at least 200 nt in length.
Asunto(s)
ADN Polimerasa Dirigida por ADN/metabolismo , Ácidos Nucleicos/biosíntesis , Ácidos Nucleicos/química , Tetrosas/química , Oligonucleótidos/biosíntesis , Oligonucleótidos/química , Análisis de Secuencia de ADN , Moldes GenéticosRESUMEN
Variation in gene expression is an important feature of mouse embryonic stem cells (ESCs). However, the mechanisms responsible for global gene expression variation in ESCs are not fully understood. We performed single-cell mRNA-seq analysis of mouse ESCs and uncovered significant heterogeneity in ESCs cultured in serum. We define highly variable gene clusters with distinct chromatin states and show that bivalent genes are prone to expression variation. At the same time, we identify an ESC-priming pathway that initiates the exit from the naive ESC state. Finally, we provide evidence that a large proportion of intracellular network variability is due to the extracellular culture environment. Serum-free culture reduces cellular heterogeneity and transcriptome variation in ESCs.
Asunto(s)
Ensamble y Desensamble de Cromatina , Células Madre Embrionarias/metabolismo , Análisis de la Célula Individual/métodos , Animales , Técnicas de Cultivo de Célula/métodos , Línea Celular , Medios de Cultivo/química , Células Madre Embrionarias/citología , Ratones , Suero , TranscriptomaRESUMEN
[structure: see text] The alpha-l-threofuranosyl nucleoside triphosphates of T, G, and D (tTTP, tGTP, and tDTP) were synthesized from the described 2'-O-DMT-protected derivatives using the Eckstein method, while the corresponding C derivative (tCTP) was prepared from the 2'-O-acetyl derivative. The prepared alpha-l-threofuranosyl nucleoside triphosphates, despite being one carbon shorter than the native 2'-deoxyfuranosyl nucleoside triphosphates, are effective substrates for selected DNA polymerases.
Asunto(s)
ADN Polimerasa Dirigida por ADN/metabolismo , Nucleótidos/química , Nucleótidos/síntesis química , Estructura Molecular , EstereoisomerismoRESUMEN
Stem cell differentiation pathways are most often studied at the population level, whereas critical decisions are executed at the level of single cells. We have established a highly multiplexed, quantitative PCR assay to profile in an unbiased manner a panel of all commonly used cell surface markers (280 genes) from individual cells. With this method, we analyzed over 1,500 single cells throughout the mouse hematopoietic system and illustrate its utility for revealing important biological insights. The comprehensive single cell data set permits mapping of the mouse hematopoietic stem cell differentiation hierarchy by computational lineage progression analysis. Further profiling of 180 intracellular regulators enabled construction of a genetic network to assign the earliest differentiation event during hematopoietic lineage specification. Analysis of acute myeloid leukemia elicited by MLL-AF9 uncovered a distinct cellular hierarchy containing two independent self-renewing lineages with different clonal activities. The strategy has broad applicability in other cellular systems.
Asunto(s)
Antígenos de Superficie/análisis , Antígenos de Superficie/genética , Análisis de la Célula Individual , Animales , Diferenciación Celular/genética , Linaje de la Célula/genética , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Sistema Hematopoyético/citología , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Ratones , Reacción en Cadena de la PolimerasaRESUMEN
(3'-2')-alpha-l-Threose nucleic acid (TNA) is an unnatural polymer that possesses the rare ability to base-pair with RNA, DNA, and itself. This feature, coupled with its chemical simplicity, makes TNA of interest as a possible progenitor of RNA during the early history of life. To evaluate the functional potential of TNA, we have developed a system for the in vitro selection of TNA. We identified the Therminator DNA polymerase as a remarkably efficient DNA-dependent TNA polymerase capable of polymerizing more than 50 tNTPs. We have also developed a method of covalently linking a DNA template to the TNA strand that it encodes, thus obviating the need for a TNA-dependent DNA polymerase during cycles of selection.
Asunto(s)
ADN de Cadena Simple/química , ADN/química , Tetrosas/química , Proteínas Arqueales/química , Secuencia de Bases , ADN/síntesis química , ADN Polimerasa Dirigida por ADN/química , Datos de Secuencia Molecular , Conformación de Ácido NucleicoRESUMEN
alpha-l-Threofuranosyl nucleoside triphosphates (tNTPs) are tetrafuranose nucleoside derivatives and potential progenitors of present-day beta-d-2'-deoxyribofuranosyl nucleoside triphosphates (dNTPs). Therminator DNA polymerase, a variant of the 9 degrees N DNA polymerase, is an efficient DNA-directed threosyl nucleic acid (TNA) polymerase. Here we report a detailed kinetic comparison of Therminator-catalyzed TNA and DNA syntheses. We examined the rate of single-nucleotide incorporation for all four tNTPs and dNTPs from a DNA primer-template complex and carried out parallel experiments with a chimeric DNA-TNA primer-DNA template containing five TNA residues at the primer 3'-terminus. Remarkably, no drop in the rate of TNA incorporation was observed in comparing the DNA-TNA primer to the all-DNA primer, suggesting that few primer-enzyme contacts are lost with a TNA primer. Moreover, comparison of the catalytic efficiency of TNA synthesis relative to DNA synthesis at the downstream positions reveals a difference of no greater than 5-fold in favor of the natural DNA substrate. This disparity becomes negligible when the TNA synthesis reaction mixture is supplemented with 1.25 mM MnCl(2). These results indicate that Therminator DNA polymerase can recognize both a TNA primer and tNTP substrates and is an effective catalyst of TNA polymerization despite changes in the geometry of the reactants.
Asunto(s)
ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Ácidos Nucleicos/biosíntesis , Ácidos Nucleicos/síntesis química , Secuencia de Bases , ADN/biosíntesis , ADN/química , Cinética , Tetrosas/química , Tetrosas/metabolismoRESUMEN
Caged reagents are photoactivatable molecules with applications in biological research. While a great deal of work has been carried out on small caged molecules, less has been done on caged macromolecules, such as proteins. Caged proteins would be especially useful in signal transduction research. Since most proteins involved in cell signaling are regulated by phosphorylation, a means to cage phosphorylated proteins would be generally applicable. Here we show that the catalytic subunit of protein kinase A can be activated by thiophosphorylation at Thr-197. The modified protein can then be caged with 4-hydroxyphenacyl bromide to yield a derivative with a specific catalytic activity that is reduced by approximately 17-fold. Upon photolysis at near UV wavelengths, an approximately 15-fold increase in activity is observed, representing an approximately 85-90% yield of uncaged product with a quantum yield phi(P) = 0.21. Because protein kinases belong to a superfamily with structurally related catalytic domains, the protein chemistry demonstrated here should be applicable to a wide range of signaling proteins.