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ras Genes in Drosophila melanogaster
Published in Juan Carlos Lacal, Frank McCormick, The ras Superfamily of GTPases, 2017
Two additional genes associated with the sevenlesslRasl signal transduction pathway have been recently identified. The first one is Gapl, a putative GTPase activated protein of Ras1.41Gap1 was isolated in an enhancer trap screen for enhancers that induce specific expression of a reporter gene in the eye imaginai disk. It was also isolated in a gentic screen for mutations that enhance the phenotype of a temperature-sensitive sevenless allele, and independently, in a genetic screen for viable recessive mutations that show rough eye phenotype. The predicted Gapl protein has significant homology with the catalytic domain of known GAPs. It is most similar to rasGAP and NF1 (39 and 31% identity along 167 amino acids, respectively). Furthermore, out of 15 residues conserved in the catalytic domains of 5 mammalian and yeast GAPs, 14 residues are conserved in the Drosophila Gapl. Genetic analysis showed that reduction in the Gapl gene activity increased the effectiveness of signalling by sevenless. Complete inactivation of Gapl induced supernumerary R7 cells and could rescue the lack of the sevenless function. Thus, Gapl is located downstream to sevenless in the signal transduction pathway that induces R7 differentiation. It is a negative regulator of this pathway, probably by enhancing the GTPase activity of Ras1. Since the phenotype of loss-of-function mutations in Gapl is opposite to the phenotype observed in loss-of-function mutations in Ras1, the Gapl protein may not serve as a positive effector target of Ras1.
Differential efficacy of genetically swapping GAL4
Published in Journal of Neurogenetics, 2019
Ying-Jun Chen, Hao-Hsin Chang, Shih-Han Lin, Tzi-Yang Lin, Ting-Han Wu, Hsin-Ju Lin, Nan-Fu Liou, Chi-Jen Yang, Yuh-Tarng Chen, Kai Hsiang Chang, Cen-You Li, Ya-Hui Chou
Intersectional methods have made tracing and manipulating a single or small subset of defined neurons possible in Drosophila. A few large enhancer trap collections have been established to allow swapping or insertion of desired components to particular loci, however, the feasibility and potential limiting factors for successful swapping remain to be fully explored. In this work, we took advantage of the genetic swap feature in InSITE GAL4 enhancer trap lines and replaced the GAL4 component with LexA, GAL80 or QF. We found different swap efficiencies among LexA, GAL80 and QF. The limiting factor on experimental utility appeared to be the reproducibility of the expression patterns of swapped components. We hope that our efforts to validate some of these swapped lines will be useful to the community.
Zebrafish expression reporters and mutants reveal that the IgSF cell adhesion molecule Dscamb is required for feeding and survival
Published in Journal of Neurogenetics, 2018
Donald P. Julien, Alex W. Chan, Joshua Barrios, Jaffna Mathiaparanam, Adam Douglass, Marc A. Wolman, Alvaro Sagasti
A plasmid used to generate the enhancer trap donor construct was provided by Shin-Ichi Higashijima (pBluescript-SK-Gbait-Hsp-Gal4FF-BGHpA) (Kimura, Hisano, Kawahara, & Higashijima, 2015). After modification, this plasmid was injected into Tg(UAS:GFP), dscambt2b heterozygous embryos at the 1-cell stage, along with 1–5 nl of an injection mix containing Cas9 mRNA (200–300 pg/nl), donor plasmid (10–20 pg/nl), gRNA-Et1/Et2 (20–40 pg/nl), and gRNA-Mbait (20–40 pg/nl). Injected fish were screened for Gal4 expression to established stable enhancer trap lines. PCR was used to confirm enhancer trap integration and orientation. See Supplemental Methods for details.