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2540The whole genome expression analysis was performed on mutants lacking the nucleosome remodeling factor (NURF)-specific subunit NURF301 to determine in vivo gene targets of 'Drosophila' NURF. It is found that Null mutants do not undergo larval to pupal metamorphosis and also enhance dominant-negative mutations in ecdysone receptor moreover, purified NURF binds EcR in an ecdysone-dependent manner suggesting it is a direct effector of nuclear receptor activity.

Author(s): Yurii Sedkov [1, 5]; Elizabeth Cho [1, 5]; Svetlana Petruk [1]; Lucy Cherbas [2]; Sheryl T. Smith [1]; Richard S. Jones [3]; Peter Cherbas [2]; Eli Canaani [4]; James [...]

A linear octopole trap interface for an ion mobility time-of-fright mass spectrometer has been developed for focusing and accumulating continuous beams of ions produced by electrospray ionization. The interface improves experimental efficiencies by factors of ~50-200 compared with an analogous configuration that utilizes a three-dimensional Paul geometry trap (Hoaglund-Hyzer, C. S.; Lee, Y. J.; Counterman, A. E.; Clemmer, D. E. Anal. Chem. 2002, 74, 992-1006). With these improvements, it is possible to record nested drift (flight) time distributions for complex mixtures in fractions of a second. We demonstrate the approach for several well-defined peptide mixtures and an assessment of the detection limits is given. Additionally, we demonstrate the utility of the approach in the field of proteomics by an on-line, three-dimensional nano-LC-ion mobility-TOF separation of tryptic peptides from the Drosophila proteome.

Drosophila cells in culture can be transformed by introducing exogenous DNA carrying a selectable marker. Here we report on the fate of plasmids that contain an extended fragment of Drosophila DNA in addition to the selectable marker. A small minority of the resulting transformants appear to arise from homologous recombination at the chromosomal target. However, the majority of the insertions are the products of illegitimate events in the vicinity of the target DNA, and they often cause mutations in the targeted region. The efficiency of this process, its homology dependence, and the clustering of the products define a novel transformation pathway that we call 'parahomologous targeting.'

We describe an adaptation of фC31 integrase-mediated targeted cassette exchange for use in Drosophila cell lines. Single copies of an attP-bounded docking platform carrying a GFP-expression marker, with or without insulator elements flanking the attP sites, were inserted by P-element transformation into the Kc167 and Sg4 cell lines; each of the resulting docking-site lines carries a single mapped copy of one of the docking platforms. Vectors for targeted substitution contain a cloning cassette flanked by attB sites. Targeted substitution occurs by integrase-mediated substitution between the attP sites (integrated) and the attB sites (vector). We describe procedures for isolating cells carrying the substitutions and for eliminating the products of secondary off-target events. We demonstrate the technology by integrating a cassette containing a Cu2+-inducible mCherry marker, and we report the expression properties of those lines. When compared with clonal lines made by traditional transformation methods, which lead to the illegitimate insertion of tandem arrays, targeted insertion lines give more uniform expression, lower basal expression, and higher induction ratios. Targeted substitution, though intricate, affords results that should greatly improve comparative expression assays--a major emphasis of cell-based studies. [ABSTRACT FROM AUTHOR]

Highlights: [•] About 150 Drosophila cell lines from a variety of tissues sources exist in a community resource. [•] Extensive genomic and transcriptional data are available for many of these lines. [•] We review basic techniques for culture, transformation, and storage of cell lines. [•] We survey the wide range of applications for which Drosophila lines have been successfully used. [Copyright &y& Elsevier]

The ecdysone receptor is a heterodimer of two nuclear receptors, the Ecdysone receptor (EcR) and Ultraspiracle (USP). In Drosophila melanogaster, three EcR isoforms share common DNA and ligand-binding domains, but these proteins differ in their most N-terminal regions and, consequently, in the activation domains (AF1s) contained therein. The transcriptional coactivators for these domains, which impart unique transcriptional regulatory properties to the EcR isoforms, are unknown. Activating transcription factor 4 (ATF4) is a basic-leucine zipper transcription factor that plays a central role in the stress response of mammals. Here we show that Cryptocephal (CRC), the Drosophila homolog of ATF4, is an ecdysone receptor coactivator that is specific for isoform B2. CRC interacts with EcR-B2 to promote ecdysone-dependent expression of ecdysistriggering hormone (ETH), an essential regulator of insect molting behavior. We propose that this interaction explains some of the differences in transcriptional properties that are displayed by the EcR isoforms, and similar interactions may underlie the differential activities of other nuclear receptors with distinct AF1-coactivators. [ABSTRACT FROM AUTHOR]

Probes the transformation of Drosophila cells by introduction of exogenous DNA. Homologous or nonhomologous integration of host genome in transforming cells with exogenous DNA; Description of two targeted genes used in mutagenizing genes in the near diploid Drosophila cell line Kc; Definition and explanation of parahomologous targeting in Drosophila cells.

Shows that native ecydysone receptor (EcR) and Ultraspiricle (USP) are co-localized on ecdysone-responsive loci of polytene chromosomes. Promotion of physical association between EcR and USP by natural ecdysones; Requirement of EcR and USP in high-affinity altered by combinatorial actions among subunits and ligands.

Drosophila melanogaster cell lines are important resources for cell biologists. Here, we catalog the expression of exons, genes, and unannotated transcriptional signals for 25 lines. Unannotated transcription is substantial (typically 19% of euchromatic signal). Conservatively, we identify 1405 novel transcribed regions; 684 of these appear to be new exons of neighboring, often distant, genes. Sixty-four percent of genes are expressed detectably in at least one line, but only 21% are detected in all lines. Each cell line expresses, on average, 5885 genes, including a common set of 3109. Expression levels vary over several orders of magnitude. Major signaling pathways are well represented: most differentiation pathways are "off" and survival/growth pathways "on." Roughly 50% of the genes expressed by each line are not part of the common set, and these show considerable individuality. Thirty-one percent are expressed at a higher level in at least one cell line than in any single developmental stage, suggesting that each line is enriched for genes characteristic of small sets of cells. Most remarkable is that imaginal disc-derived lines can generally be assigned, on the basis of expression, to small territories within developing discs. These mappings reveal unexpected stability of even fine-grained spatial determination. No two cell lines show identical transcription factor expression. We conclude that each line has retained features of an individual founder cell superimposed on a common "cell line" gene expression pattern. [ABSTRACT FROM AUTHOR]

Steroid hormones induce cascades of gene activation and repression with transformative effects on cell fate . Steroid transduction plays a major role in the development and physiology of nearly all metazoan species, and in the progression of the most common forms of cancer. Despite the paramount importance of steroids in developmental and translational biology, a complete map of transcriptional response has not been developed for any hormone . In the case of 20-hydroxyecdysone (ecdysone) in Drosophila melanogaster, these trajectories range from apoptosis to immortalization. We mapped the ecdysone transduction network in a cohort of 41 cell lines, the largest such atlas yet assembled. We found that the early transcriptional response mirrors the distinctiveness of physiological origins: genes respond in restricted patterns, conditional on the expression levels of dozens of transcription factors. Only a small cohort of genes is constitutively modulated independent of initial cell state. Ecdysone-responsive genes tend to organize into directional same-stranded units, with consecutive genes induced from the same strand. Here, we identify half of the ecdysone receptor heterodimer as the primary rate-limiting step in the response, and find that initial receptor isoform levels modulate the activated cohort of target transcription factors. This atlas of steroid response reveals organizing principles of gene regulation by a model type II nuclear receptor and lays the foundation for comprehensive and predictive understanding of the ecdysone transduction network in the fruit fly. [ABSTRACT FROM AUTHOR]

A linear octopole trap interface for an ion mobility time of-flight mass spectrometer has been developed for focusing and accumulating continuous beams of ions produced by electrospray ionization. The interface improves experimental efficiencies by factors of ∼50-200 compared with an analogous configuration that utilizes a three-dimensional Paul geometry trap (Hoaglund-Hyzer, C. S.; Lee, Y. J.; Counterman, A. E.; Clemmer, D. E. Anal. Chem. 2002, 74, 992-1006). With these improvements, it is possible to record nested drift (flight) time distributions for complex mixtures in fractions of a second. We demonstrate the approach for several well-defined peptide mixtures and an assessment of the detection limits is given. Additionally, we demonstrate the utility of the approach in the field of proteomics by an on-line, three dimensional nano-LC-ion mobility-TOF separation of tryptic peptides from the Drosophila proteome. [ABSTRACT FROM AUTHOR]