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James W Posakony

Publications and source records attributed to James W Posakony.

5 recordsLinked to original sources
PubMed2004

GenePalette: a universal software tool for genome sequence visualization and analysis.

To make effective use of the growing host of complete genome sequences, biologists must have easy-to-use software tools that allow them to visualize, analyze, and modify genome data in an interactive and generalized manner. In an effort to bridge the gap between genome and researcher, we have created GenePalette (www.genepalette.org), a desktop application that can access any genome sequence and display the positions of various features [e.g., transcription factor binding sites (TFBSs)] relative to the introns and exons of annotated genes. Written in Java, GenePalette can run on all Java-supporting operating systems (Mac, PC, Unix, Linux). Annotated sequence encompassing the majority of public genome data is rapidly retrieved from GenBank or Ensembl. The software provides intuitive access to the selected genomic region through three interface components: a colorful graphical display showing a schematic of genes and features; an annotated sequence view in which features and genes are highlighted directly on the sequence; and the selectable raw sequence. The three interface components are fully integrated and presented on one page, permitting the user to move easily between representations at different levels of resolution, ranging from kilobases to individual nucleotides. GenePalette is a particularly powerful platform for analyzing the organization of cis-regulatory elements and designing wet-lab experiments to investigate them.

Computational Biology↗
PubMed2004

New Drosophila transgenic reporters: insulated P-element vectors expressing fast-maturing RFP.

In vivo green fluorescent protein (GFP)/red fluorescent protein (RFP) double-labeling studies have been hampered by several inconvenient properties of DsRed, the first described RFP. These disadvantages include a very slow (> 24 h) maturation time, emission of contaminating green light, and low solubility. A recently developed variant of DsRed, called DsRed.T4, has a much shorter maturation time, no significant green emission, and improved solubility. We have constructed Drosophila P-element transformation vectors encoding DsRed.T4 for promoter/enhancer analysis, labeling of living cells, or RFP tagging of proteins. These new vectors have all of the features of the widely used Pelican/Stinger GFP vectors, including insulator sequences to reduce position effects, an extensive polylinker, and both cytoplasmic and nuclear-localized forms of the reporter. We have also constructed an upstream activating sequence (UAS)-DsRed.T4 vector, for GAL4 activation of the reporter. We find that DsRed.T4 is very easily detected in transgenic flies without contamination of the GFP signal and that it matures to its fluorescent form nearly simultaneously with GFP. This advance in Drosophila reporter technology makes timed double-labeling experiments in developing transgenic animals possible for the first time.

Animals↗
PubMed2002

Default repression and Notch signaling: Hairless acts as an adaptor to recruit the corepressors Groucho and dCtBP to Suppressor of Hairless.

The DNA-binding transcription factor Suppressor of Hairless [Su(H)] functions as an activator during Notch (N) pathway signaling, but can act as a repressor in the absence of signaling. Hairless (H), a novel Drosophila protein, binds to Su(H) and has been proposed to antagonize N signaling by inhibiting DNA binding by Su(H). Here we show that, in vitro, H directly binds two corepressor proteins, Groucho (Gro) and dCtBP. Reduction of gro or dCtBP function enhances H mutant phenotypes and suppresses N phenotypes in the adult mechanosensory bristle. This activity of gro is surprising, because it is directed oppositely to its traditionally defined role as a neurogenic gene. We find that Su(H)-H complexes can bind to DNA with high efficiency in vitro. Furthermore, a H-VP16 fusion protein causes dominant-negative phenotypes in vivo, a result consistent with the proposal that H functions in transcriptional repression. Taken together, our findings indicate that "default repression" of N pathway target genes by an unusual adaptor/corepressor complex is essential for proper cell fate specification during Drosophila peripheral nervous system development.

Alcohol Oxidoreductases↗
PubMed2002

SCORE: a computational approach to the identification of cis-regulatory modules and target genes in whole-genome sequence data. Site clustering over random expectation.

A large fraction of the information content of metazoan genomes resides in the transcriptional and posttranscriptional cis-regulatory elements that collectively provide the blueprint for using the protein-coding capacity of the DNA, thus guiding the development and physiology of the entire organism. As successive whole-genome sequencing projects--including those of mice and humans--are completed, we have full access to the regulatory genome of yet another species. But our ability to decipher the cis-regulatory code, and hence to link genes into regulatory networks on a global scale, is currently very limited. Here we describe SCORE (Site Clustering Over Random Expectation), a computational method for identifying transcriptional cis-regulatory modules based on the fact that they often contain, in statistically improbable concentrations, multiple binding sites for the same transcription factor. We have carried out a Drosophila genomewide inventory of predicted binding sites for the Notch-regulated transcription factor Suppressor of Hairless [Su(H)] and found that the fly genome contains highly nonrandom clusterings of Su(H) sites over a broad range of sequence intervals. We found that the most statistically significant clusters are very heavily enriched in both known and logical targets of Su(H) binding and regulation. The utility of the SCORE approach was validated by in vivo experiments showing that proper expression of the novel gene Him in adult muscle precursor cells depends both on Su(H) gene activity and sequences that include a previously unstudied cluster of four Su(H) sites, indicating that Him is a likely direct target of Su(H).

Animals↗