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Daily rhythms of behavior are controlled by a circuit of circadian pacemaking neurons. In Drosophila, 150 pacemakers participate in this network, and recent observations suggest that the network is divisible into M and E oscillators, which normally interact and synchronize. Sixteen oscillator neurons (the small and large lateral neurons [LNvs]) express a neuropeptide called pigment-dispersing factor (PDF) whose signaling is often equated with M oscillator output. Given the significance of PDF signaling to numerous aspects of behavioral and molecular rhythms, determining precisely where and how signaling via the PDF receptor (PDFR) occurs is now a central question in the field. Here we show that GAL4-mediated rescue of pdfr phenotypes using a UAS-PDFR transgene is insufficient to provide complete behavioral rescue. In contrast, we describe a approximately 70-kB PDF receptor (pdfr) transgene that does rescue the entire pdfr circadian behavioral phenotype. The transgene is widely but heterogeneously expressed among pacemakers, and also among a limited number of non-pacemakers. Our results support an important hypothesis: the small LNv cells directly target a subset of the other crucial pacemaker neurons cells. Furthermore, expression of the transgene confirms an autocrine feedback signaling by PDF back to PDF-expressing cells. Finally, the results present an unexpected PDF receptor site: the large LNv cells appear to target a population of non-neuronal cells that resides at the base of the eye.
Pubmed ID: 20394051 RIS Download
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Recombineering (recombination-mediated genetic engineering) is a powerful method for fast and efficient construction of vectors for subsequent manipulation of the mouse genome or for use in cell culture experiments. It is also an efficient way of manipulating the bacterial genome directly. Recombineering is a method based on homologous recombination in E. Coli using recombination proteins provided from ? phage. Our bacterial strains contain a defective ? prophage inserted into the bacterial genome. The phage genes of interest, exo, bet, and gam, are transcribed from the ?PL promoter. This promoter is repressed by the temperature-sensitive repressor cI857 at 32C and derepressed (the repressor is inactive) at 42C. When bacteria containing this prophage are kept at 32C no recombination proteins are produced. However, after a brief (15 minutes) heat-shock at 42C a sufficient amount of recombination proteins are produced. exo is a 5''-3'' exonuclease that creates single-stranded overhangs on introduced linear DNA. bet protects these overhangs and assists in the subsequent recombination process. gam prevents degradation of linear DNA by inhibiting E. Coli RecBCD protein. Linear DNA (PCR product, oligo, etc.) with sufficient homology in the 5'' and 3'' ends to a target DNA molecule already present in the bacteria (plasmid, BAC, or the bacterial genome itself) can be introduced into heat-shocked and electrocompetent bacteria using electroporation. The introduced DNA will now be modified by exo and bet and undergo homologous recombination with the target molecule. The method is so efficient that co-electroporation of a supercoiled plasmid and a linear piece of DNA into heat-shocked, electrocompetent bacteria will work as well.
View all literature mentionsAn antibody supplier which banks and distributes hybridomas and monoclonal antibodies for use in research. The bank includes antibodies against targets such as GFP, transcription factors, stem cells, and human.
View all literature mentionsThis unknown targets
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View all literature mentionsThis monoclonal targets c-Myc
View all literature mentionsThis monoclonal targets Repo; Reversed polarity protein
View all literature mentionsThis polyclonal targets Myc Tag
View all literature mentionsThis monoclonal targets Pigment-dispersing factor neuropeptide
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