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.
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•Robust methodology for single-cell analysis of the cell surface repertoire•Comprehensive single-cell analysis for the mouse hematopoietic system•Lineage progression analysis and network construction using single-cell data•Characterization of the unique cellular hierarchy in a mouse AML model
Single-cell analysis of the cell surface repertoire enables mapping of cellular heterogeneity and differentiation hierarchy, applied here to the hematopoietic system but with broad applicability.
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.
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•Single-cell mRNA-seq analysis reveals heterogeneity in ESCs cultured in serum•Gene expression variability is associated with distinct chromatin characteristics•Computational analysis identifies an ESC-priming pathway•External culture system affects gene expression variability of ESCs
Guo et al. investigate mechanisms of gene expression variation and cellular heterogeneity in mouse embryonic stem cells using single-cell mRNA-seq analysis. They find a differentiation-priming pathway in the cell culture system and show that serum provokes gene expression variability in mouse embryonic stem cells.
α-l-Threofuranosyl nucleoside triphosphates (tNTPs) are tetrafuranose nucleoside derivatives and potential progenitors of present-day β-d-2‘-deoxyribofuranosyl nucleoside triphosphates (dNTPs). ...Therminator DNA polymerase, a variant of the 9°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 MnCl2. 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.
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 ∼17-fold. Upon photolysis at near UV wavelengths, an ∼15-fold increase in activity is observed, representing an ∼85−90% yield of uncaged product with a quantum yield φP = 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.
Oestradiol regulates reproductive physiology and cardiovascular health in women. In the endometrium of ovariectomized ewes, previous work demonstrated that a single dose of oestradiol (50
μg) ...up-regulates oestrogen receptor-
α (ER) and progesterone receptor (PR) gene expression within 24
h. Here we compared responses to different doses of oestradiol and different dosing regimens in two diverse tissues: endometrium and liver. ER, c-fos, cyclophilin and glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA concentrations were analyzed on replicate RNA slot blots in both tissues, while PR and apolipoprotein AI (apo AI) mRNA concentrations were only analyzed in endometrium or liver, respectively. Along with ER mRNA, oestradiol strongly up-regulated GAPDH and cyclophilin mRNA concentrations in endometrium. In liver, however, oestradiol down-regulated them, along with apo AI mRNA. Responses to different doses and dose regimens, including repeated 50
μg doses, were similar to those evoked by a single 50
μg dose of oestradiol. Thus, oestradiol appears to have all-or-none effects which include up-regulation of ER, cyclophilin and GAPDH gene expression in endometrium and down-regulation of ER, apo AI, cyclophilin and GAPDH gene expression in liver. These results illustrate the sharp contrast between two mammalian tissues in their responses to physiological levels of oestradiol.
An in Vitro Selection System for TNA Ichida, Justin K; Zou, Keyong; Horhota, Allen ...
Journal of the American Chemical Society,
03/2005, Letnik:
127, Številka:
9
Journal Article
Recenzirano
Odprti dostop
(3‘-2‘)-α-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.
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.
The α-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 α-l-threofuranosyl nucleoside triphosphates, despite being one carbon shorter than the native 2‘-deoxyfuranosyl nucleoside triphosphates, are effective substrates for selected DNA polymerases.
Caged Thiophosphotyrosine Peptides Zou, Keyong; Miller, W. Todd; Givens, Richard S. ...
Angewandte Chemie (International ed.),
August 17, 2001, Letnik:
40, Številka:
16
Journal Article
Recenzirano
Caged but not trapped! Caged reagents are molecules from which biological effectors are released by photolysis. Here, p‐hydroxyphenacyl bromide is used to derivatize a model peptide containing a ...thiophosphotyrosine residue in order to mimic phosphorylation at tyrosine. Upon photolysis, a biologically active peptide is released. Peptides and proteins caged on thiophosphotyrosine will be useful in signal transduction research.
An SH2 binding peptide containing the YEEI motif, and a PKA catalytic subunit were chosen to explore the caging of signaling polypeptides at crucial tyrosine or threonine phosphorylation sites. The ...interaction between phosphorylated tyrosine residue and SH2 domain (pTyr-SH2) mediates countless protein-protein interactions. Phosphorylation can be mimicked by thiophosphorylation. To use light to control the pTyr-SH2 association, a thiophosphate group was introduced into peptide EPQYEEIPILG at the tyrosine residue by kinase Hck and ATP(γ)S. The peptide was then caged on the thiophosphate group with 2-nitrobenzyl bromide (NBB) or 4-hydroxyphenacyl bromide (HPB). Photolysis was performed by irradiation at 312 nm. EPQYp SEEIPILG was released from EPQYpS(NB)EEIPILG in 50–60% yield. The t1/2 values were 5.5 min at pH5.8 and 8.0 min at pH7.3, which correspond to quantum yields ( p) of 0.37 and 0.25. EPQYp,EEIPILG was produced from EPQYps(HP)EEIPILG in a yield of 50–70% at both pH 5.8 and pH 7.3. The t1/2 values were 0.55 min at pH 5.8 and 0.52 min at pH 7.3, which correspond to p values of 0.65 and 0.56. When the binding ability of caged 35S-labeled peptides to immobilized SH2 was examined, only 1 to 4% of the radioactivity was bound, which largely represents non-specific binding. Upon photolysis, in both cases, EPQYpS(NB)EEIPILG and EPQYpS(HP)EEIPILG the binding ability of the peptide was restored. Phosphothreonine 197 is crucial for the activity of the PKA catalytic subunit. Nonphosphorylated, inactive protein H6-C199A/C343A was obtained by expression in the presence of H89, a specific inhibitor of PKA, to prevent autophosphorylation. A thiophosphate group was introduced at Thr 197 by the kinase PDK1 and ATP(γ)S, resulting in an active protein. The thiophosphorylated protein was purified by TNB-thiol agarose. Caging of the protein with HPB led to inactivation with a 20-fold reduction in activity. Upon photolysis, the protein was released with a t1/2 of 1.5 min and p of 0.2. Regeneration of the thiophosphate group was determined by the increase in coupling ability to TNB-thiol gel, an activated disulfide-containing resin. Concomitant with the increased binding was a 15–18 fold increase in catalytic activity.
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