Competing activities of heterotrimeric G proteins in Drosophila wing maturation.

Drosophila genome encodes six alpha-subunits of heterotrimeric G proteins. The Galphas alpha-subunit is involved in the post-eclosion wing maturation, which consists of the epithelial-mesenchymal transition and cell death, accompanied by unfolding of the pupal wing into the firm adult flight organ. Here we show that another alpha-subunit Galphao can specifically antagonize the Galphas activities by competing for the Gbeta13F/Ggamma1 subunits of the heterotrimeric Gs protein complex. Loss of Gbeta13F, Ggamma1, or Galphas, but not any other G protein subunit, results in prevention of post-eclosion cell death and failure of the wing expansion. However, cell death prevention alone is not sufficient to induce the expansion defect, suggesting that the failure of epithelial-mesenchymal transition is key to the folded wing phenotypes. Overactivation of Galphas with cholera toxin mimics expression of constitutively activated Galphas and promotes wing blistering due to precocious cell death. In contrast, co-overexpression of Gbeta13F and Ggamma1 does not produce wing blistering, revealing the passive role of the Gbetagamma in the Galphas-mediated activation of apoptosis, but hinting at the possible function of Gbetagamma in the epithelial-mesenchymal transition. Our results provide a comprehensive functional analysis of the heterotrimeric G protein proteome in the late stages of Drosophila wing development.

Europium-labeled GTP as a general nonradioactive substitute for [(35)S]GTPgammaS in high-throughput G protein studies.

[(35)S]GTPgammaS, the nonhydrolyzable radioactive GTP analog, has been a powerful tool in G protein studies and has set the standards in this field of research. However, its radioactive nature imposes clear limitations to its use in regular laboratory practice and in high-throughput experimentation. The europium-labeled GTP analog (Eu-GTP) has been used as an alternative in the analysis of G protein activation by G protein-coupled receptors in cellular membrane preparations. Here we expand the usage of Eu-GTP and show that it can be applied in other types of assays where [(35)S]GTPgammaS has been previously utilized. We demonstrate the applicability of the modified Eu-GTP binding technology to analysis of heterotrimeric and monomeric G proteins of natural and recombinant sources, from different organisms, in assays with soluble proteins and membrane-containing assays of a high-throughput format. The deci-nanomolar K(D) of Eu-GTP for the tested G proteins is similar to that of other fluorescent-modified GTP analogs, while the sensitivity achieved in time-resolved fluorescence analysis of Eu-GTP exceeds that of the radioactive measurements. Overall, the results of our modified Eu-GTP binding assay present Eu-GTP as a general nonradioactive alternative for G protein studies, especially attractive in high-throughput experiments.

Drosophila GoLoco-protein Pins is a target of Galpha(o)-mediated G protein-coupled receptor signaling.

G protein-coupled receptors (GPCRs) transduce their signals through trimeric G proteins, inducing guanine nucleotide exchange on their Galpha-subunits; the resulting Galpha-GTP transmits the signal further inside the cell. GoLoco domains present in many proteins play important roles in multiple trimeric G protein-dependent activities, physically binding Galpha-subunits of the Galpha(i/o) class. In most cases GoLoco binds exclusively to the GDP-loaded form of the Galpha-subunits. Here we demonstrate that the poly-GoLoco-containing protein Pins of Drosophila can bind to both GDP- and GTP-forms of Drosophila Galpha(o). We identify Pins GoLoco domain 1 as necessary and sufficient for this unusual interaction with Galpha(o)-GTP. We further pinpoint a lysine residue located centrally in this domain as necessary for the interaction. Our studies thus identify Drosophila Pins as a target of Galpha(o)-mediated GPCR receptor signaling, e.g., in the context of the nervous system development, where Galpha(o) acts downstream from Frizzled and redundantly with Galpha(i) to control the asymmetry of cell divisions.

Significant enhancement of fluorescence on hybridization of a molecular beacon to a target DNA in the presence of a site-specific DNA nickase.

We have developed a simple isothermal (55 degrees C) reaction that permits detection of DNA targets using only two components: a molecular beacon and a site-specific DNA nickase without deoxyribonucleotide triphosphates and primers. The loop sequence of the molecular beacon should contain a DNA nickase recognition site. The nickase-molecular beacon (NMB) combination permits a 100-fold increase in fluorescent signal. The applications of the NMB assay for enhancement of fluorescent signal in some isothermal methods are discussed.

Viral RNA-directed RNA polymerases use diverse mechanisms to promote recombination between RNA molecules.

An earlier developed purified cell-free system was used to explore the potential of two RNA-directed RNA polymerases (RdRps), Qbeta phage replicase and the poliovirus 3Dpol protein, to promote RNA recombination through a primer extension mechanism. The substrates of recombination were fragments of complementary strands of a Qbeta phage-derived RNA, such that if aligned at complementary 3'-termini and extended using one another as a template, they would produce replicable molecules detectable as RNA colonies grown in a Qbeta replicase-containing agarose. The results show that while 3Dpol efficiently extends the aligned fragments to produce the expected homologous recombinant sequences, only nonhomologous recombinants are generated by Qbeta replicase at a much lower yield and through a mechanism not involving the extension of RNA primers. It follows that the mechanisms of RNA recombination by poliovirus and Qbeta RdRps are quite different. The data favor an RNA transesterification reaction catalyzed by a conformation acquired by Qbeta replicase during RNA synthesis and provide a likely explanation for the very low frequency of homologous recombination in Qbeta phage.