Identification and molecular characterization of new “spindle” genes

Vitor J. Barbosa, Frankie Kimm, Ruth Lehmann. Developmental Genetics, NYU, Skirball Inst, New York, NY.

The establishment of dorsal-ventral polarity in the Drosophila oocyte requires the activity of the germline Gurken/TGF α (GRK) ligand to be correctly controlled both in time and in space. We did a mutagenesis screen using the ventralization of the eggshell (the “spindle” phenotype) as readout of defective GRK signaling in order to understand how events such as recombinational double strand break repair, microtubule-based transport and cytoskeleton polarity influence oocyte organization. Ventralized eggs were derived from germ line clones homozygous for the mutagenized 2R chromosome arm. After screening 8179 lines we isolated 119 “spindle” mutants. These have been divided, thus far, into 16 groups by complementation analysis, which is still in progress. Preliminary analysis of the phenotype in 6 of these groups showed a consistent correlation between the ventralization of the eggshell and Gurken activity either by lack of Gurken protein or by its mislocalization within the oocyte. We also performed epistatic analysis with these groups and mutants of both the meiotic checkpoint and the pathway leading to the formation of double-strand DNA breaks prior to meiotic recombination. These tests showed that the activity of these genes is independent of the meiotic checkpoint or downstream of it. Curiously, two of these mutations also affected the architecture of the egg chamber: one leading to misplacement of the oocyte nucleus, which was identified as a mutation in the kinesin heavy chain (Khc) gene and the other to a misplaced oocyte within the egg chamber. Two other complementation groups appear to be involved in the process of oocyte specification. In parallel with the phenotypic studies we are currently in the process of mapping and molecularly identifying the genes isolated.

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