2D and 3D Chromosome Painting in Malaria Mosquitoes

Abstract

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting chromosomal rearrangements, and studying three-dimensional (3D) organization of chromosomes in the cell nucleus. The advent of laser capture microdissection (LCM) and whole genome amplification (WGA) allows obtaining large quantities of DNA from single cells. The increased sensitivity of WGA kits prompted us to develop chromosome paints and to use them for exploring chromosome organization and evolution in non-model organisms. Here, we present a simple method for isolating and amplifying the euchromatic segments of single polytene chromosome arms from ovarian nurse cells of the African malaria mosquito Anopheles gambiae. This procedure provides an efficient platform for obtaining chromosome paints, while reducing the overall risk of introducing foreign DNA to the sample. The use of WGA allows for several rounds of re-amplification, resulting in high quantities of DNA that can be utilized for multiple experiments, including 2D and 3D FISH. We demonstrated that the developed chromosome paints can be successfully used to establish the correspondence between euchromatic portions of polytene and mitotic chromosome arms in An. gambiae. Overall, the union of LCM and single-chromosome WGA provides an efficient tool for creating significant amounts of target DNA for future cytogenetic and genomic studies.

Mapping Genomic Scaffolds to Chromosomes Using Laser Capture Microdissection in Application to Hawaiian Picture-Winged Drosophila

“…Intact chromosome arms were isolated from larvae of D. differens using a published LCM protocol for isolating and amplifying the euchromatic segments of individual polytene chromosome arms [George et al, 2014]. One pair of salivary glands was placed in 1 drop of Carnoy solution on a Zeiss 1.0 PET membrane slide.…”

“…Alternatively, relatively quick chromosome mapping information can be obtained through the isolation of euchromatic segments from distinct chromosome arms using laser capture microdissection (LCM), coupled with whole-genome amplification and NGS [George et al, 2014]. Here, we test this approach in application to polytene chromosomes of Hawaiian picture-winged Drosophila species, an iconic example of sequential colonization, speciation, and adaptive radiation [Carson and Clague, 1995;Carson, 1997].…”

“…Slides were dried again in a controlled sterile climate to avoid contamination. LCM of chromosomes was done with the PALM MicroBeam Laser Microdissection system and the PALMRobo software as described previously [George et al, 2014]. Microdissected chromosomes were lysed to release gDNA.…”

Three-dimensional Organization of Polytene Chromosomes in Somatic and Germline Tissues of Malaria Mosquitoes

“…Chromosome territories have been detected in numerous organisms, such as worms [8], fruit flies [9], humans [10], primates [11,12], mice [6,13], and plants [14,15]. These territories are microscopically visible by observing giant polytene chromosomes [16] or by painting non-polytene chromosomes with of 25 fluorescent labels [8,[17][18][19][20][21]. Second, neighboring territories facilitate chromosome-chromosome (Chr-Chr) interactions [22] while limiting chromosome entanglement, possibly by specific chromosome folding [23].…”

Heterochromatin, histone modifications, and nuclear architecture in disease vectors

“…Microdissected chromosome paints have been used to demonstrate chromosome territories in polytene and nonpolytene interphase nuclei of An. gambiae [42]. A very recent study applied chromosome region-specific microdissected paints to investigate tissue-specific features of chromosome architecture in An.…”