polo Is Identified as a Suppressor of bubR1 Nondisjunction in a Deficiency Screen of the Third Chromosome in Drosophila melanogaster

Abstract

We have previously characterized an EMS-induced allele of the bubR1 gene (bubR1(D1326N)) that separates the two functions of BubR1, causing meiotic nondisjunction but retaining spindle assembly checkpoint activity during somatic cell division in Drosophila melanogaster. Using this allele, we demonstrate that bubR1 meiotic nondisjunction is dosage sensitive, occurs for both exchange and nonexchange homologous chromosomes, and is associated with decreased maintenance of sister chromatid cohesion and of the synaptonemal complex during prophase I progression. We took advantage of these features to perform a genetic screen designed to identify third chromosome deficiencies having a dominant effect on bubR1(D1326N)/bubR1(rev1) meiotic phenotypes. We tested 65 deficiencies covering 60% of the third chromosome euchromatin. Among them, we characterized 24 deficiencies having a dominant effect on bubR1(D1326N)/bubR1(rev1) meiotic phenotypes that we classified in two groups: (1) suppressor of nondisjunction and (2) enhancer of nondisjunction. Among these 24 deficiencies, our results show that deficiencies uncovering the polo locus act as suppressor of bubR1 nondisjunction by delaying meiotic prophase I progression and restoring chiasmata formation as observed by the loading of the condensin subunit SMC2. Furthermore, we identified two deficiencies inducing a lethal phenotype during embryonic development and thus affecting BubR1 kinase activity in somatic cells and one deficiency causing female sterility. Overall, our genetic screening strategy proved to be highly sensitive for the identification of modifiers of BubR1 kinase activity in both meiosis and mitosis.

Figure 1

Polo is a suppressor of bubR1 X NDJ. (A) Structure of SC in wild-type, in bubR1 mutant and in bubR1;polo mutant in region 3 of the germarium. C(3)G is in green, DNA is in blue, and Orb (as oocyte marker) is in red. Decreasing polo dosage in a bubR1 genetic background rescues the maintenance of the SC in the oocyte nucleus comparing to bubR1 mutant. (B) Sister chromatid cohesin subunit SMC1 in wild-type, in bubR1 mutant and in bubR1;polo mutant in region 3 of the germarium. SMC1 is in green, DNA is in blue, and Orb is in red. Decreasing polo dosage in bubR1 genetic background rescues the maintenance of the sister chromatid cohesion in the oocyte nucleus comparing to bubR1 mutant. (C) Condensin subunit SMC2 in wild-type, in bubR1 mutant and in bubR1;polo mutant at the pachytene/diplotene transition (stages 5-7). SMC2 is in green, DNA is in blue, and Orb is in red. Decreasing polo dosage in bubR1 genetic background rescues the loading of condensin subunit at stages 5–7 in the oocyte nucleus during chiasmata formation comparing to bubR1 mutant oocyte. Scale bars = 10 μm.

Figure 2

BubR1 kinase activity is essential during somatic cell cycle (A) The developmental arrest induced by Df(3L)BSC10 in BubR1 kinase dead embryos takes place after the syncitial division and prevents the initialization of gastrulation. (B) Embryonic phenotype at stage 5 after 250 μM colchicine treatment in wild-type and progeny from a cross made between bubR1^rev1/TSTL females to bubR1^D1326N/CyO;Df(3L)BSC10/TM3 males. Phospho-Histone 3 is in red and DAPI is in green. In contrast to wild-type embryos, replicated chromosomes in mutant embryos after colchicine treatment appear condensed and the presence of phospho-histone 3 epitope is detected. Scale bars = 10μm.