Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of Arabidopsis MM1 and MM2d synchronized suspensions for cell cycle studies

ABSTRACT: This experiment was provided by TAIR (http://arabidopsis.org). Effective analysis of gene expression during the cell cycle depends on achieving a good level of synchronisation. Until recently, analysis of cell cycle processes in plants has been hampered by the lack of synchronizable cell suspensions for Arabidopsis. We have recently developed a cell synchrony system for Arabidopsis cell suspensions MM1 and MM2d, and have developed two methods of synchronization. The first synchronizes cycling cells by blocking cells at the G1/S boundary using aphidicolin. The second uses sucrose removal and resupply to synchronize cells during re-entry into the cell cycle. Cell cycle synchrony in suspension cultured cells: cells can be reproducibly synchronized by blocking at the G1/S boundary or in early S phase using aphidicolin for 24 hr and then reversing the block by washing (Menges and Murray, 2002). On aphidicolin removal, the synchronous resumption of S phase and progression through the cell cycle occur and sequential RNA samples were taken at 2-3 hourly intervals over a 19 hour period. We have carried out a transcriptional profiling analysis with the aim to study gene expression during cell cycle progression after aphidicolin treatment of suspension-cultured cells using the near full genome ATH1 arrays (Menges et al., 2003). Experimenter name = Jim Murray; Experimenter phone = 44 1223 334166; Experimenter fax = 44 1223 334162; Experimenter department = J Murray Laboratory; Experimenter institute = University of Cambridge; Experimenter address = Institute of Biotechnology; Experimenter address = Tennis Court Road; Experimenter address = Cambridge; Experimenter zip/postal_code = CB2 1QT; Experimenter country = United Kingdom Experiment Overall Design: 10 samples were used in this experiment

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Nottingham Arabidopsis Stock Centre (NASC)

PROVIDER: E-GEOD-5747 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The growth of Arabidopsis cell cultures following their sub-culture into fresh media follows standard growth kinetics of a period of exponential increase associated with high rate of cell division, followed by a slowing of the rate of increase as cells approach stationary phase. For the analysis described here, MM2d cells were subcultured into fresh MSS-medium and samples were taken at day 1, day3, day 5 and day7. We have carried out transcriptional profiling analysis with the aim to follow growth stage specific gene expression during unperturbed growth using the near full genome ATH1 arrays (Menges et al., 2003). Journal Absract:; (Plant Molecular Biology: 53, 2003); Plant cell suspension cultures are invaluable models for the study of cellular processes. Here we develop the recently described Arabidopsis suspension culture MM2d as a transcript profiling platform by means of Affymetrix ATH1 microarrays. Analysis of gene expression profiles during normal culture growth, during synchronous cell cycle re-entry and during synchronous cell cycle progression provides a unique integrated view of gene expression responses in a higher-plant system. Particularly striking is that expression of over 14 000 genes belonging to all defined categories can be reliably detected, suggesting that integrated and comparative analysis of data sets derived from transcript profiling of cultures is a powerful approach to identify candidate components involved in a wide range of biological processes. Combinatorial analysis of independent cell cycle synchrony methods allows the identification of genes that are apparently cell-cycle-regulated but are most likely responding to the induction of synchrony. We thus present an integrated genome-wide view of the transcriptional profile of a plant suspension culture and identify a refined set of 1082 cell cycle regulated genes largely independent of synchrony method. Experimenter name = Jim Murray; Experimenter phone = 44 1223 334166; Experimenter fax = 44 1223 334162; Experimenter department = J Murray Laboratory; Experimenter address = University of Cambridge; Experimenter address = Institute of Biotechnology; Experimenter address = Tennis Court Road; Experimenter address = Cambridge; Experimenter zip/postal_code = CB2 1QT; Experimenter country = United Kingdom Experiment Overall Design: 4 samples were used in this experiment

Project description:Paramecium bursaria chlorella virus (PBCV-1), a member of the family Phycodnaviridae, is a large dsDNA, plaque-forming virus that infects the unicellular green alga Chlorella NC64A. The 331 kb PBCV-1 genome is predicted to encode 365 proteins and 11 tRNAs. To follow global transcription during PBCV-1 replication, a microarray containing 50-mer probes to the PBCV-1 365 protein-encoding genes (CDS) was constructed. Competitive hybridization experiments were conducted employing cDNA from poly A-containing RNA obtained from cells at seven time points after virus infection. The results led to the following conclusions: i) the PBCV-1 replication cycle is temporally programmed and regulated; ii) 360 (99%) of the arrayed PBCV-1 CDSs are expressed at some time in the virus life cycle in the laboratory; iii) 227 (62%) of the CDSs are expressed before virus DNA synthesis begins; iv) these 227 CDSs were grouped into two classes: 127 transcripts disappear prior to initiation of virus DNA synthesis (considered early) and 100 transcripts are still detected after virus DNA synthesis begins (considered early/late); v) 133 (36%) of the CDSs are expressed after virus DNA synthesis begins (considered late); vi) expression of most late CDSs is inhibited by adding the DNA replication inhibitor aphidicolin prior to virus infection. This study provides the first comprehensive evaluation of virus gene expression during the PBCV-1 lifecycle. Time course analysis of chlorella virus PBCV-1: cDNAs from poly A-containing RNAs isolated from cells at seven times after PBCV-1 infection (20, 40, 60, 90, 120, 240, 360 min p.i.) were competitively hybridized against a reference sample on the microarrays. Three independent biological replica, four technical replica of the genome per array. Time course analysis of chlorella virus PBCV-1 gene expression in the presence of aphidicolin: cDNAs from poly A-containing RNAs isolated from aphidicolin-treated cells at seven times after PBCV-1 infection (20, 40, 60, 90, 120, 240, 360 min p.i.) were competitively hybridized against non-treated, infected samples from the same time points. Three independent biological replica, four technical replica of the genome per array.

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Transcription profiling of Arabidopsis MM1 and MM2d synchronized suspensions for cell cycle studies

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