Ectopic mitotic recombination in Drosophila probed with bacterial beta-galactosidase gene-based reporter transgenes.

Plasmids were constructed to investigate homologous mitotic recombination in Drosophila cells. Heteroalleles containing truncated but overlapping segments of the bacterial beta-galactosidase gene (lacZ) were positioned either on separate plasmids or as direct repeats on the same chromosome. Recombination reconstituted a functional lacZgene leading to expression of LacZ+activity detectable by histochemical staining. High extrachromosomal recombination (ECR) frequencies between unlinked heteroalleles were observed upon transient co-transfection into Drosophila melanogaster Schneider line 2 (S2) cells. Stably transfected cells containing the lacZ heteroalleles linked on a chromosome exhibited intrachromosomal recombination (ICR) frequencies two orders of magnitude lower than ECR frequencies. Recombination was inducible by exposing the cells to ethyl methanesulphonate or mitomycin C. Recombination products were characterized by multiplex PCR analysis and unequal sister chromatid recombination was found as the predominant mechanism reconstituting the lacZ gene. To investigate recombination in vivo imaginal disc cells from transgenic larvae carrying the reporter gene on the X chromosome were isolated and stained for LacZ+ activity. The presence of a few LacZ+ clones indicated that mitotic recombination events occurred at frequencies two orders of magnitude lower than the corresponding event in cultured cells and late during larval development.

The pruned gene encodes the Drosophila serum response factor and regulates cytoplasmic outgrowth during terminal branching of the tracheal system.

We identified a Drosophila gene, pruned, that regulates formation of the terminal branches of the tracheal (respiratory) system. These branches arise by extension of long cytoplasmic processes from terminal tracheal cells towards oxygen-starved tissues, followed by formation of a lumen within the processes. The pruned gene is expressed in terminal cells throughout the period of terminal branching. pruned encodes the Drosophila homologue of serum response factor (SRF), which functions with an ETS domain ternary complex factor as a growth-factor-activated transcription complex in mammalian cells. In pruned loss of function mutants, terminal cells fail to extend cytoplasmic projections. A constitutively activated SRF drives formation of extra projections that grow out in an unregulated fashion. An activated ternary complex factor has a similar effect. We propose that the Drosophila SRF functions like mammalian SRF in an inducible transcription complex, and that activation of this complex by signals from target tissues induces expression of genes involved in cytoplasmic outgrowth.

The ETS domain protein pointed-P2 is a target of MAP kinase in the sevenless signal transduction pathway.

The fate of the R7 photoreceptor cell in the developing eye of Drosophila is controlled by the Sevenless (Sev) receptor tyrosine kinase. Sev activates a highly conserved signal transduction cascade involving the proteins Ras1 and Raf and the Rolled/mitogen-activated protein (Rl/MAP) kinase. Here we show that the ETS domain protein encoded by the P2 transcript of the pointed (pnt) gene is a nuclear target of this signalling cascade which acts downstream of Rl/MAP kinase. The PntP2 protein is phosphorylated by Rl/MAP kinase in vitro at a single site and this site is required for its function in vivo. Furthermore, we present genetic and biochemical data suggesting that MAP kinase controls neural development through phosphorylation of two antagonizing transcription factors of the ETS family, Yan and PntP2.