Cell type-specific characterization of nuclear DNA contents within complex tissues and organs

Zhang, Changqing; Gong, Fang; Lambert, Georgina M.; Galbraith, David W.

doi:10.1186/1746-4811-1-7

Cited by 72 publications

(22 citation statements)

References 62 publications

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“…We next measured the amount of chromosomal DNA in each nucleus, using the fluorescence intensity of Hhf1-GFP as a proxy. In separate studies, we found that Hhf1-GFP fluorescence intensity is proportional to the amount of chromosomal DNA in nuclei (manuscript in review), consistent with previous studies that used GFP-tagged histone as a proxy for ploidy [22] . The fluorescence intensity corresponding to 2N and 4N nuclei was based on the fluorescence intensity of individual anaphase/telophase nuclei (2N) and late metaphase nuclei (4N) in “no drug” cells.…”

Section: Resultssupporting

confidence: 88%

A Tetraploid Intermediate Precedes Aneuploid Formation in Yeasts Exposed to Fluconazole

et al. 2014

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Section: Resultssupporting

confidence: 88%

A Tetraploid Intermediate Precedes Aneuploid Formation in Yeasts Exposed to Fluconazole

et al. 2014

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“…[ 17 ] used a practical approach in which the suspensionsof intact nuclei are prepared by chopping a small amount offresh tissue in a suitable isolation buffer. This method affords high quality DNA histograms and provides immediate release of nuclei from cells or tissues and is currently widely used for flow cytometry of unfixed nuclear samples of plant cells [ 18 , 19 ]. Formaldehyde fixation of samples offers convenience in experiments involving multiple samples and timepoints such as cell cycle synchronization.…”

Section: Resultsmentioning

confidence: 99%

A rapid and robust assay for detection of S-phase cell cycle progression in plant cells and tissues by using ethynyl deoxyuridine

et al. 2010

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BackgroundProgress 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 we extend the microscopy and flow cytometry applications of the recently developed EdU (5-ethynyl-2'-deoxyuridine)-based S-phase assay to various plant species and tissues. We demonstrate that the presented protocols insure the improved preservation of cell and tissue structure and allow significant reduction in assay duration. In comparison with the frequently used detection of bromodeoxyuridine (BrdU) and tritiated-thymidine incorporation, this new methodology offers several advantages as we discuss here.ResultsApplications of EdU-based S-phase assay in microscopy and flow cytometry are presented by using cultured cells of alfalfa, Arabidopsis, grape, maize, rice and tobacco. We present the advantages of EdU assay as compared to BrdU-based replication assay and demonstrate that EdU assay -which does not require plant cell wall digestion or DNA denaturation steps, offers reduced assay duration and better preservation of cellular, nuclear and chromosomal morphologies. We have also shown that fast and efficient EdU assay can also be an efficient tool for dual parameter flow cytometry analysis and for quantitative assessment of replication in thick root samples of rice.ConclusionsIn plant cell cycle studies, EdU-based S-phase detection offers a superior alternative to the existing S-phase assays. EdU method is reliable, versatile, fast, simple and non-radioactive and it can be readily applied to many different plant systems.

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“…Therefore, the effect of IR on development was analyzed in live seedlings carrying growth-associated markers. The lines included (i) cyclin AtCYCB1;1- green fluorescent protein (GFP), which marks cells arrested in late S through early M phases [64], [65], and therefore activation, persistence, and relaxation of IR-induced cell division arrest; (ii) histone AtH2B- yellow fluorescent protein ( YFP ), a marker of chromatin organization, DNA content, and nuclear morphology, allowing us to visualize the relative evolution of cell DNA content in the organ [66], [67]; and (iii) DR5-GFP, a marker of auxin response which typically can be used to reflect changes in auxin content and distribution which are key regulators of organ growth [68], [69].…”

Section: Resultsmentioning

confidence: 99%

ATM-Mediated Transcriptional and Developmental Responses to γ-rays in Arabidopsis

et al. 2007

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ATM (Ataxia Telangiectasia Mutated) is an essential checkpoint kinase that signals DNA double-strand breaks in eukaryotes. Its depletion causes meiotic and somatic defects in Arabidopsis and progressive motor impairment accompanied by several cell deficiencies in patients with ataxia telangiectasia (AT). To obtain a comprehensive view of the ATM pathway in plants, we performed a time-course analysis of seedling responses by combining confocal laser scanning microscopy studies of root development and genome-wide expression profiling of wild-type (WT) and homozygous ATM-deficient mutants challenged with a dose of γ-rays (IR) that is sublethal for WT plants. Early morphologic defects in meristematic stem cells indicated that AtATM, an Arabidopsis homolog of the human ATM gene, is essential for maintaining the quiescent center and controlling the differentiation of initial cells after exposure to IR. Results of several microarray experiments performed with whole seedlings and roots up to 5 h post-IR were compiled in a single table, which was used to import gene information and extract gene sets. Sequence and function homology searches; import of spatio-temporal, cell cycling, and mutant-constitutive expression characteristics; and a simplified functional classification system were used to identify novel genes in all functional classes. The hundreds of radiomodulated genes identified were not a random collection, but belonged to functional pathways such as those of the cell cycle; cell death and repair; DNA replication, repair, and recombination; and transcription; translation; and signaling, indicating the strong cell reprogramming and double-strand break abrogation functions of ATM checkpoints. Accordingly, genes in all functional classes were either down or up-regulated concomitantly with downregulation of chromatin deacetylases or upregulation of acetylases and methylases, respectively. Determining the early transcriptional indicators of prolonged S-G2 phases that coincided with cell proliferation delay, or an anticipated subsequent auxin increase, accelerated cell differentiation or death, was used to link IR-regulated hallmark functions and tissue phenotypes after IR. The transcription burst was almost exclusively AtATM-dependent or weakly AtATR-dependent, and followed two major trends of expression in atm: (i)-loss or severe attenuation and delay, and (ii)-inverse and/or stochastic, as well as specific, enabling one to distinguish IR/ATM pathway constituents. Our data provide a large resource for studies on the interaction between plant checkpoints of the cell cycle, development, hormone response, and DNA repair functions, because IR-induced transcriptional changes partially overlap with the response to environmental stress. Putative connections of ATM to stem cell maintenance pathways after IR are also discussed.

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Cell type-specific characterization of nuclear DNA contents within complex tissues and organs

Cited by 72 publications

References 62 publications

A Tetraploid Intermediate Precedes Aneuploid Formation in Yeasts Exposed to Fluconazole

A Tetraploid Intermediate Precedes Aneuploid Formation in Yeasts Exposed to Fluconazole

A rapid and robust assay for detection of S-phase cell cycle progression in plant cells and tissues by using ethynyl deoxyuridine

ATM-Mediated Transcriptional and Developmental Responses to γ-rays in Arabidopsis

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