Disruption of Topoisomerase II Perturbs Pairing in Drosophila Cell Culture

Abstract

Homolog pairing refers to the alignment and physical apposition of homologous chromosomal segments. Although commonly observed during meiosis, homolog pairing also occurs in nonmeiotic cells of several organisms, including humans and Drosophila. The mechanism underlying nonmeiotic pairing, however, remains largely unknown. Here, we explore the use of established Drosophila cell lines for the analysis of pairing in somatic cells. Using fluorescent in situ hybridization (FISH), we assayed pairing at nine regions scattered throughout the genome of Kc 167 cells, observing high levels of homolog pairing at all six euchromatic regions assayed and variably lower levels in regions in or near centromeric heterochromatin. We have also observed extensive pairing in six additional cell lines representing different tissues of origin, different ploidies, and two different species, demonstrating homolog pairing in cell culture to be impervious to cell type or culture history. Furthermore, by sorting Kc 167 cells into G 1 , S, and G 2 subpopulations, we show that even progression through these stages of the cell cycle does not significantly change pairing levels. Finally, our data indicate that disrupting Drosophila topoisomerase II (Top2) gene function with RNAi and chemical inhibitors perturbs homolog pairing, suggesting Top2 to be a gene important for pairing.

A Genomewide Survey Argues That Every Zygotic Gene Product Is Dispensable for the Initiation of Somatic Homolog Pairing in Drosophila

“…For nonrepetitive euchromatic sequences, we generated fluorescently labeled probes via nick translation of a genomic DNA template according to the kit manufacturer's protocol (Vysis). For template DNA, we used genomic clones contained in P1 (Berkeley Drosophila Genome Project) or BAC (BACPAC Resource Center, Children's Hospital Oakland Research Institute) vectors or a pool of 10-12 1-kb PCR products that were generated from a span of 30-40 kb of a chromosomal locus (Williams et al 2007).…”

“…0.5-0.6 mm; for 28B and 62E, by 0.4-0.5 mm; and for the 359-bp repeat, by 1 mm. Importantly, these probespecific distances did not vary between wild-type and mutant embryos, permitting the direct comparison of pairing levels for each locus between different backgrounds.To verify our findings, we measured the distance between homologous loci for the HisC and the 62E regions in a subset of the wild-type and mutant cycle 14 embryos used in the study and arranged the data in percentile plots, which display all the distances between FISH signals in a single experiment regardless of the nucleus of origin (Williams et al 2007) (supplemental Figure 1). Using this stringent method, nuclei with a single FISH signal that was roughly spherical in shape were given a measurement of 0 mm, while overlapping FISH signals that appeared elongated or dumbbell-shaped were treated as separate foci, and a measurement was made between the presumed centers of the two overlapping signals.…”

“…Embryos were mounted in Vectashield with DAPI (Vector Laboratories). For each embryo, a single region was chosen wherein all visible nuclei could be easily assayed, and then high-resolution three-dimensional images were collected and deconvolved using a Deltavision imaging system and Softworx software as described (Williams et al 2007).…”

“…Embryos were mounted in Vectashield with DAPI (Vector Laboratories). For each embryo, a single region was chosen wherein all visible nuclei could be easily assayed, and then high-resolution three-dimensional images were collected and deconvolved using a Deltavision imaging system and Softworx software as described (Williams et al 2007).Staging and genotyping: The 2-to 3-hr collections used for most experiments should capture embryos in the final 10 min of cell cycle 13 and the first 50 min of cell cycle 14. Due to the time spent manipulating embryos during the dechorionation step, most embryos were aged 5-10 min longer before development was stopped during fixation, and so very few cell cycle 13 embryos were observed.…”

Effects of Chromosomal Rearrangements on Transvection at theyellowGene ofDrosophila melanogaster

“…This pairing was first noted by Nettie Stevens (Stevens 1908) and Charles Metz (Metz 1916) through the examination of mitotic nuclei. Somatic pairing of homologous chromosomes has now been observed in Drosophila interphase nuclei using DNA as well as RNA in situ hybridization techniques (reviewed by McKee 2004; for example, Kopczynski and Muskavitch 1992;Hiraoka et al 1993;Csink and Henikoff 1998;Fung et al 1998;Gemkow et al 1998;Sass and Henikoff 1999;Bantignies et al 2003;Ronshaugen and Levine 2004;Williams et al 2007;Hartl et al 2008), assessment of the frequency of site-specific FLP-mediated recombination (Golic and Golic 1996a), and methods that mark chromosomes with protein tags (Vazquez et al 2001(Vazquez et al , 2002(Vazquez et al , 2006. Here, we present our studies using transvection as our phenotypic assay for chromosomal pairing in Drosophila.…”