Unknown,Transcriptomics,Genomics,Proteomics

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Dmyc overexpression 7h and 14h

ABSTRACT: Fly strains: All transgenes are P[+] in w strains. w+;+;Act 5c > CD2 > GAL4 UAS-GFP (Neufeld et al. 1998 ); y w hs-FLP122; +; UAS-dMyc (Zaffran et al. 1998 ). y w hs-FLP122; +; +. Adult flies and larvae were raised in regular fly food consisting of cornmeal and molasses at 25Â°C. Larvae overexpressing either UAS-regulated dMyc;GFP or GFP alone transgenes were generated using the Flp/Gal4 method (Struhl and Basler 1993 ; Pignoni and Zipursky 1997 ; Neufeld et al. 1998 ). Larvae were staged from hatching and raised at a density of 50 per vial at 25Â°C. Third instar larvae (110 h after egg deposition, AED) were heat shocked at 37Â°C for 2 h, and larvae were collected 7 h after heat shock (~120 h AED). Total RNA was isolated using TRIzol reagent (Invitrogen) as described by manufacturer followed by RNeasy (Qiagen) clear up. cRNA targets were generated using a standard amino-allyl labeling protocol, where 30 Âµg each of "experimental" (dMyc;GFP: hs-FLP122; Act-GAL4, UAS-GFP; UAS-dMyc) and "reference" (GFP only :ywhs-FLP122; Act-GAL4, UAS-GFP; +) total RNAs were coupled to either Cy3 or Cy5 fluorophores. Paired labeled targets were processed on microarrays using protocols described elsewhere (Fazzio et al. 2001 ). Posthybridized arrays were scanned using a GenePix 4000 scanner (Axon Instruments). Data were generated from five independent replicates (two with one dye orientation and three with the reversed dye orientation) at 7h and 14h

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Bruce Edgar

PROVIDER: E-GEOD-2073 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Myc-dependent regulation of ribosomal RNA synthesis during Drosophila development.

Grewal Savraj S SS Li Ling L Orian Amir A Eisenman Robert N RN Edgar Bruce A BA

Nature cell biology 20050220 3

Regulating ribosome number is thought to control cellular growth. Synthesis of ribosomal RNA (rRNA) is a limiting step in ribosome biogenesis and rates of rRNA synthesis are generally altered depending on the growth status of a cell. Although studies in unicellular systems have addressed the mechanisms by which this occurs, few studies have applied a genetic approach to examine growth-dependent control of rRNA synthesis in metazoans. Here, we show that in Drosophila melanogaster Myc (dMyc) is a ...[more]

PMID: 15723055

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Project description:(This section will be revisited in good time prior to publication, for final approval) Research within the field of functional genomics provides a more profound understanding of living organisms, by elucidating the interaction between genes and gene products. This is of increasing importance in many fields, and especially within cancer research. With the use of microarray technology, the gene expression of a vast number of genes can be measured simultaneously. This allows us insight into tumor biology, and an opportunity to obtain knowledge of diagnostic and prognostic value, as well as hints towards the causes of cancer. In this study the gene expression in a selection of human neuroendocrine (NE) and non-neuroendocrine tumor cell lines were compared. Both qualitative and quantitative methods were applied to get an insight into the NE tumor biology. NE cells are found in practically every organ and tissue in the body and are assigned to synthesize peptide hormones and take up amino acids and transform these into biogenic amines by carboxylation. NE tumors arise after malignant transformation of neuroendocrine cells and are usually highly differentiated and have a low growth rate, which makes this a high prevalence tumor type. These rather rare tumors characteristically express NE markers, such as chromogranin A, and contains characteristic dense-secretory core granules. By using cDNA microarray analysis, the gene expression profiles of various NE tumor cell lines (e. g. carcinoid, neuroblastoma, medullary thyroid carcinoma and endocrine pancreatic tumor) were compared with those of non-NE tumor cells. As a reference the commercially available Universal human RNA reference (Stratagene) was used. Verification of the results were done by real time RT-PCR analysis after the NE nature of the cell lines was stated by immunohistochemical, electron microscopic and light microscopic examinations. A large number of genes were differentially expressed in NE cell lines vs. the non-NE cell lines, and were found to be of interest in NE tumor biology. Some of these genes were previously known in NE tumor biology, whereas the vast majority of the specified genes are novel in the context of NE tumor biology. Genes with little previous information were also identified and thought to be an asset in the search of new NE markers. A feasible prospective of the study would be expansion with additional samples cell lines and a more thorough verification with real time RT-PCR analysis. This would enhance the credibility and thus the utility of the findings. Future studies on the biological functions of the candidate genes and the mechanisms of their regulation, will hopefully enable suggestions of new NE markers of prognostic or diagnostic value, and eventually new drug targets. Keywords: neuroendocrine, neuroendocrine markers, gene expression profiling, cDNA microarray The hybridisation was performed by an experienced laboratory technician Eli Helge, (Dept. of Laboratory Medicine, Children`s and Women`s health, NTNU) at the microarray core facility. Three separate runs with 10, 11 and 3 slides was needed to obtain the 20 hybridisations (dyeswap x 10 cell lines) planned for this study. Of the 10 cell lines 6 were neuroendocrine and 4 were non-neuroendocrine. For comparison between these two groups the universal human reference RNA (Stratagene) was used as the "control" on each slide. An adapted version of the 3DNA Array 350 protocol (Genisphere) was used. The slides were scanned with a ScanArrayTMExpressHT (Packard BioScience) twice immediately after the hybridisation was completed to avoid photo bleaching of the fluorescent dyes.

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Dmyc overexpression 7h and 14h

Publications

Myc-dependent regulation of ribosomal RNA synthesis during Drosophila development.

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