Meiotic recombination begins with the induction of programmed double-strand breaks (DSBs).
Meiotic recombination begins with the induction of programmed double-strand breaks (DSBs). best defined by the presence of additional punctate SC staining throughout the euchromatin in up to four nuclei. As the cyst progresses in region 2A, it enters into pachytene where full-length 956154-63-5 SC is usually created. In Drosophila, meiotic DSBs are created by the Spo11 homolog, Mei-W68, after the SC is usually fully created (McKim and Hayashi-Hagihara, 1998; Mehrotra and McKim, 2006). DSBs can be visualized in Drosophila by the quick phosphorylation of the histone 2A variant (H2AV) at DSB sites that occur in all 16 nuclei within the cyst (in both the pro-oocytes and surrounding nurse cells) in region 2A (Mehrotra and McKim, 2006). As the cyst progresses into region 2B (early/mid-pachytene), only two nuclei have total SC, and DSB figures are reduced from those found in early pachytene. By region 3, or mid-pachytene, the oocyte nucleus has been selected and most of the H2AV staining at 956154-63-5 DSB sites is usually removed, indicating that repair is usually either in progress or complete. Identification of the mutant A germline clone screen for EMS-induced meiotic mutations around the that caused high levels of nondisjunction at the first meiotic division (Collins et al., 2012). This fully recessive mutation resulted in a CCT transition within a previously uncharacterized gene known as (Physique 1figure product 1A) and is predicted to truncate the protein 24 amino acids from the end (R213STOP) (Materials and methods) (Physique 1figure product 1B). We have named this gene and have therefore subsequently renamed the mutant, gene (denoted transheterozygotes, suggesting that is predicted to encode a protein with several identifiable domains. In the N-terminal region there is a Cys3HisCys4 Really Interesting New Gene (RING) domain name (Physique 1figure product 1B,C). RING domains are structural domains that bind two zinc cations and are typically found in E3 ligases (Metzger et al., 2014). In the middle of the protein there is a predicted coiled-coil domain name (Physique 1figure product 1D) (Lupas et al., 1991). Coiled-coil domains are often involved in proteinCprotein interactions and are commonly found in proteins that localize to the SC (Sym et al., 1993; Page and Hawley, 2004; Smolikov et al., 2009; Collins et al., 2014). Additionally, the C-terminal region of Vilya 956154-63-5 is usually serine rich, with the last quarter of the protein being approximately 25% serines (Physique 1figure product 1E). These characteristics are common of members of the Zip3 protein family (Reynolds et al., 2013) (observe Discussion). is required for programmed DSB formation in early pachytene Since causes very high levels of chromosome missegregation and encodes a protein with a potential coiled-coil domain name, we asked whether this mutant was disrupting the early events in meiotic prophase. Specifically, we wondered if it affected SC formation or two processes that depend around the SC: the pairing and clustering of centromeres and the pairing of homologous chromosomes. We first assayed the processes of centromere clustering (Physique 1figure product 2A) and homolog pairing (Physique 1figure product 2B) in early pachytene nuclei. Unlike mutants that fail to pair and/or cluster their centromeres properly and thus display greater than three centromere foci (Takeo et al., 2011), oocytes homozygous for showed no defects in centromere pairing/clustering when compared to wild type. Similarly, was not defective in euchromatic homolog pairing as assayed for the chromosome by fluorescence in situ hybridization (FISH). Moreover, immunofluorescence analysis of JIP2 early pachytene nuclei did not reveal defects in the ability of the SC protein Corolla to localize properly in germaria?(Physique 1DCa,b). As well, we did not detect defects in timing or localization of the TF SC protein, C(3)G, or Orb, a cytoplasmic marker for oocyte determination (Physique 1figure product 3). Taken together, we were unable to detect significant defects in any of the processes that occur prior to the 956154-63-5 initiation of DSBs. However, the formation of DSBs, as assayed with an antibody realizing H2AV, was greatly reduced in and homozygotes and and and and and oocytes are defective in DSB formation. Immunofluorescence analysis of oocytes was also.