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m2 in the following generation and identified by their nonparental phenotypes: w If 1 Cy1 and w1 If Cy1. Combined with the eggshell phenotype of each single recombinant female, the SNP analysis allowed the linkage of each locus to 1 of 10 intervals defined by the centromere proximal w1 transgene of the FRT42B insertion, the nine SNPs, and the centromere distal If. Allele sequencing and molecular cloning: Preparation of genomic DNA and sequencing reactions were carried out as described. Each mutant sequence from P m/Df was aligned with that of the homozygous P chromosome using DNASTAR. To molecularly map the Plk1223 insertion, inverse PCR was carried out according to the Berkeley Drosophila Genome Project Resources website but using only the Sau3AI restriction enzyme. Sequences in the 39 and 59 ends of the P element obtained from two independent trials were compared with the Drosophila genome release 3, and the adjacent coding region CG17509 was sequenced in P dPds5cohiba/Df mutants as above. Immunostaining Drosophila ovaries: Ovaries were processed for immunofluorescence as described. The monoclonal anti-Grk antibody 1D12 and the rabbit polyclonal anti-CG antibody were diluted 1:50 and 1:1000, respectively. Cy3-conjugated and Alexa 488conjugated secondary antibodies were used at a dilution of 1:500. DNA was stained with either Oligreen or DAPI diluted 1:5000 and 0.3 mm, respectively, according to the company’s instructions. Ovaries were mounted in Vectashield and visualized with a Leica TCS NT confocal microscope. For visualization of mutant germ-line clones, 2- or 3-day-old adult females of the genotype y w P; P P/CyO hs-hid were heat-shocked on two consecutive days for 1 hr each. Heat-shocked adults were transferred to fresh food for five additional days, fattened on fresh yeast on the sixth day, and dissected on the seventh day as described. The autofluorescence of nuclear GFP was always preferred to its PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19816862 indirect immunolabeling using commercially available antibodies. To assess genetic interactions with mutants defective in DSB, the frequency between P m1/P m2; mei-P221/mei-P221 and P m1/P m2; mei-P221/TM3 flies was compared. For the LY3039478 alleles of each locus used in this study, see supplemental A screen for genes controlling meiosis and oocyte patterning: The Grk-mediated EGF receptor pathway is a sensitive readout for two fundamental processes of early oogenesis: meiosis and oocyte polarity. Abrogation of this pathway causes a characteristic ventralized eggshell phenotype that can be easily recognized by fusion or lack of the two dorsal appendages in the eggs of mutant females. We used this phenotype to identify new germ-line-specific genes on the right arm of chromosome 2 involved in meiotic progression and Grk ligand production. To isolate both lethal and viable EMS-derived mutations, we employed the FRT/ ovoD technique to produce germ-line clones homozygous for 2R in an otherwise heterozygous adult . Among 8179 independent lines, we isolated 310 potential mutations, of which 118 were kept for further analysis after a secondary screen. The final 118 lines were divided into four categories– strong, medium, weak, and small/collapsed –based on penetrance of the mutant phenotype. Only lines with the most penetrant phenotypes were used for complementation tests. This allowed the identification of 17 complementation groups with two or more alleles among 57 lines tested. Ten complementation groups were lethal or semilethal, suggesting th