Modeling Biological Networks

IV.1 Coordinators
IV.2 Participants
IV.3 Introduction
IV.4 Background and Significance
IV.5 Research Plan
IV.6 Specific Subprojects

• IV.6.vi Subproject 6 – Transcription Factor Responses and Regulatory Networks in Drosophila Development
  • IV.6.vi.a Introduction
  • IV.6.vi.b Advantages of the Drosophila Model
  • IV.6.vi.c Datasources
  • IV.6.vi.d Microarray Methodology
  • IV.6.vi.e Specific Aims
  • IV.6.vi.f Research Plan
    • IV.6.vi.f.1 Aim 1
    • IV.6.vi.f.2 Aim 2
    • IV.6.vi.f.3 Aim 3
  • IV.6.vi.g Significance
• [ Other Subprojects ]

IV.7 Connection to Specific Projects 2 (cytoskeleton) and 3 (organogenesis)
IV.8 Timeline

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IV.6.vi.e Specific Aims:
Aim 1. Design and manufacture a transcription factor microarray slide for distribution by the DGRC.
Aim 2. Analyze transcription factor changes during the metamorphosis of the Drosophila salivary gland.
Aim 3. Analyze transcription factor changes in hormone-treated Drosophila Kc cells and in mutant/modified Kc cells lacking elements of the regulatory network.

IV.6.vi.f Research Plan: IV.6.vi.f.1 Aim 1:

Producing the transcription factor microarray is straightforward. The latest edition of the annotation of the Drosophila genome identifies known transcription factors and DNA binding proteins as well as cognates of transcription factors from other organisms. Current tallies suggest the number will be ca. 1,000 (cf. about 14,500 total genes). The module analysis of Subproject 1 will also suggest other possibly significant genes of presently unknown function to include. We will cull Primers representing those genes from the total set and/or synthesize them de novo and amplify the fragments representing each gene by PCR. We will produce microarray slides using standard methods. All work will employ the LIMS system operating in the CGB using robots for high-throughput operations. We will test new primers by limited sequencing.

IV.6.vi.f.2 Aim 2:

Transcriptional changes during salivary gland metamorphosis have been described using a coarse timetable. We will study changes puff stage by puff stage - handing dissecting pairs of glands, using one for staging and preparing RNA from the other. We will linearly amplify the RNA and use it for microarray analysis. We will apply network analysis tools and correlate the results with puffing patterns and known genetic interactions.

IV.6.vi.f.3 Aim 3:

We will determine the transcriptional responses of Kc cells to the hormone ecdysone. We have in hand mutant Kc cells lacking ecdysone receptors. These stills can be "rescued" by transfection with particular wildtype and mutant EcR genes. Again, we will use network analysis to compare responses in all these settings.

IV.6.vi.g Significance:

These projects will be of educational significance. They will enable graduate students and post-docs from a variety of disciplines to learn the fundamentals of microarray technology and to learn how to harvest information from biological databases. The data produced will provide an excellent platform for network analysis and serve to introduce biologists to its methods. The transcription factor array design will be of considerable practical significance to Drosophila researchers; its low cost should catalyze the variety of biological studies and repetitions needed to make microarray results useful and reliable. Finally, this work will be of fundamental significance. Regulatory changes in the metamorphic Drosophila salivary gland have been studied intensively by genetic and cytogenetic methods and the Kc cell hormone response is an intensively-studied model. The data produced will be of immediate and widespread interest to those studying the salivary gland, metamorphosis, and nuclear hormone receptor responses.