Monomethylation of histone H4-lysine 20 is involved in chromosome structure and stability and is essential for mouse development.
AuthorsOda H, Okamoto I, Murphy N, Chu J, Price SM, Shen MM, Torres-Padilla ME, Heard E, Reinberg D,
JournalMol Cell Biol
PubMed ID19223465
'PR-Set7/Set8/KMT5A is the sole enzyme known to catalyze monomethylation of histone H4 lysine 20 (H4K20) and is present only in multicellular organisms that compact a large fraction of their DNA. We found that mouse embryos that are homozygous null mutants for the gene PR-Set7 display early embryonic lethality prior to ... More
Cell type specific applicability of 5-ethynyl-2'-deoxyuridine (EdU) for dynamic proliferation assessment in flow cytometry.
AuthorsDiermeier-Daucher S, Clarke ST, Hill D, Vollmann-Zwerenz A, Bradford JA, Brockhoff G,
JournalCytometry A
PubMed ID19235202
'Using the nucleoside analogue EdU (5-ethynyl-2''-deoxyuridine) for thymidine substitution instead of BrdU (5-bromo-2''-deoxyuridine) in cell proliferation assays has recently been proposed. However, the effect of EdU on cell viability, DNA synthesis, and cell cycle progression and consequently its usability for dynamic cell proliferation analysis in vitro has not been explored. ... More
Imaging and analysis of 3D tumor spheroids enriched for a cancer stem cell phenotype.
AuthorsRobertson FM, Ogasawara MA, Ye Z, Chu K, Pickei R, Debeb BG, Woodward WA, Hittelman WN, Cristofanilli M, Barsky SH,
JournalJ Biomol Screen
PubMed ID20639504
'Tumors that display a highly metastatic phenotype contain subpopulations of cells that display characteristics similar to embryonic stem cells. These cells exhibit the ability to undergo self-renewal; slowly replicate to retain a nucleoside analog label, leading to their definition as ' ... More
APC/C-CCS52A complexes control meristem maintenance in the Arabidopsis root.
AuthorsVanstraelen M, Baloban M, Da Ines O, Cultrone A, Lammens T, Boudolf V, Brown SC, De Veylder L, Mergaert P, Kondorosi E,
JournalProc Natl Acad Sci U S A
PubMed ID19553203
'Plant organs originate from meristems where stem cells are maintained to produce continuously daughter cells that are the source of different cell types. The cell cycle switch gene CCS52A, a substrate specific activator of the anaphase promoting complex/cyclosome (APC/C), controls the mitotic arrest and the transition of mitotic cycles to ... More
Cell cycle synchronization of Escherichia coli using the stringent response, with fluorescence labeling assays for DNA content and replication.
AuthorsFerullo DJ, Cooper DL, Moore HR, Lovett ST,
JournalMethods
PubMed ID19245839
'We describe a method for synchronization of the cell cycle in the bacterium Escherichia coli. Treatment of asynchronous cultures with the amino acid analog, dl-serine hydroxamate, induces the stringent response, with concomitant arrest of DNA replication at initiation. Following release of the stringent response, cells initiate DNA replication in synchrony, ... More
Click-iT assay with improved DNA distribution histograms.
AuthorsHamelik RM, Krishan A,
JournalCytometry A
PubMed ID19658154
'The Click-iT Assay developed and commercialized by Invitrogen is based on incorporation of a new 5-bromo-2''-deoxyuridine analog, 5-ethynyl-2''-deoxyuridine (EdU) into newly synthesized DNA and its recognition by azide dyes via a copper mediated ' ... More
EdU, a new thymidine analogue for labelling proliferating cells in the nervous system.
AuthorsChehrehasa F, Meedeniya AC, Dwyer P, Abrahamsen G, Mackay-Sim A,
JournalJ Neurosci Methods
PubMed ID18996411
Labelling and identifying proliferating cells is central to understanding neurogenesis and neural lineages in vivo and in vitro. We present here a novel thymidine analogue, ethynyl deoxyuridine (EdU) for labelling dividing cells, detected with a fluorescent azide which forms a covalent bond via the ... More
Evaluation of 5-ethynyl-2'-deoxyuridine staining as a sensitive and reliable method for studying cell proliferation in the adult nervous system.
AuthorsZeng C, Pan F, Jones LA, Lim MM, Griffin EA, Sheline YI, Mintun MA, Holtzman DM, Mach RH,
JournalBrain Res
PubMed ID20064490
Recently, a novel method for detection of DNA synthesis has been developed based on the incorporation of 5-ethynyl-2'-deoxyuridine (EdU), a thymidine analogue, into cellular DNA and the subsequent reaction of EdU with a fluorescent azide in a copper-catalyzed [3+2] cycloaddition ( ... More
EdU incorporation is an alternative non-radioactive assay to [(3)H]thymidine uptake for in vitro measurement of mice T-cell proliferations.
AuthorsYu Y, Arora A, Min W, Roifman CM, Grunebaum E,
JournalJ Immunol Methods
PubMed ID19647746
RATIONALE: T lymphocyte proliferations can be measured by [(3)H]thymidine incorporation. However, many labs avoid this technique because of the need to use radioactive substrates. In addition, [(3)H]thymidine incorporation method does not permit simultaneous characterization of the proliferating cells. We developed the 5-ethynyl-2'-deoxyuridine (EdU) and Cu(I)-catalyzed cycloaddition ... More
AuthorsCronin SJ, Nehme NT, Limmer S, Liegeois S, Pospisilik JA, Schramek D, Leibbrandt A, Simoes Rde M, Gruber S, Puc U, Ebersberger I, Zoranovic T, Neely GG, von Haeseler A, Ferrandon D, Penninger JM,
JournalScience
PubMed ID19520911
Innate immunity represents the first line of defense in animals. We report a genome-wide in vivo Drosophila RNA interference screen to uncover genes involved in susceptibility or resistance to intestinal infection with the bacterium Serratia marcescens. We first employed whole-organism gene suppression, followed by tissue-specific silencing in gut epithelium or ... More
A chemical method for fast and sensitive detection of DNA synthesis in vivo.
AuthorsSalic A, Mitchison TJ,
JournalProc Natl Acad Sci U S A
PubMed ID18272492
We have developed a method to detect DNA synthesis in proliferating cells, based on the incorporation of 5-ethynyl-2'-deoxyuridine (EdU) and its subsequent detection by a fluorescent azide through a Cu(I)-catalyzed [3 + 2] cycloaddition reaction ("click" chemistry). Detection of the EdU label is highly sensitive and can be accomplished in ... More
The sub-cellular localization of Sulfolobus DNA replication.
AuthorsGristwood T, Duggin IG, Wagner M, Albers SV, Bell SD,
JournalNucleic Acids Res
PubMed ID22402489
Analyses of the DNA replication-associated proteins of hyperthermophilic archaea have yielded considerable insight into the structure and biochemical function of these evolutionarily conserved factors. However, little is known about the regulation and progression of DNA replication in the context of archaeal cells. In the current work, we describe the generation ... More
Click chemistry and bioorthogonal reactions: unprecedented selectivity in the labeling of biological molecules.
AuthorsBest MD,
JournalBiochemistry
PubMed ID19485420
In recent years, a number of bioorthogonal reactions have been developed, exemplified by click chemistry, that enable the efficient formation of a specific product, even within a highly complex chemical environment. While the exquisite selectivity and reliability of these transformations have led to their broad application in diverse research areas, ... More
Detection of S-phase cell cycle progression using 5-ethynyl-2'-deoxyuridine incorporation with click chemistry, an alternative to using 5-bromo-2'-deoxyuridine antibodies.
The 5-bromo-2'-deoxyuridine (BrdU) labeling of cells followed by antibody staining has been the standard method for direct measurement of cells in the S-phase. Described is an improved method for the detection of S-phase cell cycle progression based upon the application of click chemistry, the copper(I)-catalyzed variant of the Huisgen [3+2] ... More
A rapid and robust assay for detection of S-phase cell cycle progression in plant cells and tissues by using ethynyl deoxyuridine.
AuthorsKotogány E, Dudits D, Horváth GV, Ayaydin F,
JournalPlant Methods
PubMed ID20181034
ABSTRACT: BACKGROUND: Progress in plant cell cycle research is highly dependent on reliable methods for detection of cells replicating DNA. Frequency of S-phase cells (cells in DNA synthesis phase) is a basic parameter in studies on the control of cell division cycle and the developmental events of plant cells. Here ... More
Dendritic cell-nerve clusters are sites of T cell proliferation in allergic airway inflammation.
AuthorsVeres TZ, Shevchenko M, Krasteva G, Spies E, Prenzler F, Rochlitzer S, Tschernig T, Krug N, Kummer W, Braun A,
JournalAm J Pathol
PubMed ID19179611
Interactions between T cells and dendritic cells in the airway mucosa precede secondary immune responses to inhaled antigen. The purpose of this study was to identify the anatomical locations where dendritic cell-T cell interactions occur, resulting in T cells activation by dendritic cells. In a mouse model of allergic airway ... More
Distinct populations of quiescent and proliferative pancreatic beta-cells identified by HOTcre mediated labeling.
AuthorsHesselson D, Anderson RM, Beinat M, Stainier DY,
JournalProc Natl Acad Sci U S A
PubMed ID19706417
Pancreatic beta-cells are critical regulators of glucose homeostasis, and they vary dramatically in their glucose stimulated metabolic response and levels of insulin secretion. It is unclear whether these parameters are influenced by the developmental origin of individual beta-cells. Using HOTcre, a Cre-based genetic switch that uses heat-induction to precisely control ... More
Large T antigen promotes JC virus replication in G2-arrested cells by inducing ATM- and ATR-mediated G2 checkpoint signaling.
AuthorsOrba Y, Suzuki T, Makino Y, Kubota K, Tanaka S, Kimura T, Sawa H,
JournalJ Biol Chem
PubMed ID19903823
Large T antigen (TAg) of the human polyomavirus JC virus (JCV) possesses DNA binding and helicase activities, which, together with various cellular proteins, are required for replication of the viral genome. We now show that JCV-infected cells expressing TAg accumulate in the G(2) phase of the cell cycle as a ... More
Chick embryo proliferation studies using EdU labeling.
AuthorsWarren M, Puskarczyk K, Chapman SC,
JournalDev Dyn
PubMed ID19253396
Cell proliferation studies are an important experimental tool. The most commonly used thymidine analogues, tritiated thymidine and bromodeoxyuridine (BrdU) label cells during S-phase. Both methods have significant drawbacks: low sensitivity in the case of tritiated thymidine and a denaturation step during BrdU detection that destroys most cellular epitopes, requiring careful ... More
Nucleotide excision repair-induced H2A ubiquitination is dependent on MDC1 and RNF8 and reveals a universal DNA damage response.
Chromatin modifications are an important component of the of DNA damage response (DDR) network that safeguard genomic integrity. Recently, we demonstrated nucleotide excision repair (NER)-dependent histone H2A ubiquitination at sites of ultraviolet (UV)-induced DNA damage. In this study, we show a sustained H2A ubiquitination at damaged DNA, which requires dynamic ... More
A rapid non-radioactive technique for measurement of repair synthesis in primary human fibroblasts by incorporation of ethynyl deoxyuridine (EdU).
AuthorsLimsirichaikul S, Niimi A, Fawcett H, Lehmann A, Yamashita S, Ogi T,
JournalNucleic Acids Res
PubMed ID19179371
Xeroderma pigmentosum (XP) is an autosomal recessive genetic disorder. Afflicted patients show extreme sun-sensitivity and skin cancer predisposition. XP is in most cases associated with deficient nucleotide excision repair (NER), which is the process responsible for removing photolesions from DNA. Measuring NER activity by nucleotide incorporation into repair patches, termed ... More
METT-10, a putative methyltransferase, inhibits germ cell proliferative fate in Caenorhabditis elegans.
AuthorsDorsett M, Westlund B, Schedl T,
JournalGenetics
PubMed ID19596901
Germ-line stem cells are unique because they either self-renew through mitosis or, at a certain frequency, switch to meiosis and produce gametes. The switch from proliferation to meiosis is tightly regulated, and aberrations in switching result in either too little or too much proliferation. To understand the genetic basis of ... More
Mitochondrial DNA (mtDNA) Biogenesis: Visualization and Duel Incorporation of BrdU and EdU Into Newly Synthesized mtDNA In Vitro.
AuthorsLentz SI, Edwards JL, Backus C, McLean LL, Haines KM, Feldman EL,
JournalJ Histochem Cytochem
PubMed ID19875847
Mitochondria are key regulators of cellular energy and are the focus of a large number of studies examining the regulation of mitochondrial dynamics and biogenesis in healthy and diseased conditions. One approach for monitoring mitochondrial biogenesis is to measure the rate of mitochondrial DNA (mtDNA) replication. We developed a sensitive ... More