PgmNr D1079: A new toolbox for the fly Sciara - a new/old model system that disobeys the rules for chromosome movement on spindles.

Authors:
S. A. Gerbi; Y. Yamamoto; J. Urban; J. Bliss


Institutes
Brown Univ BioMed Division, Providence, RI, USA.


Keyword: mitosis

Abstract:

     The fly Sciara has long been recognized as an outstanding model system to elucidate questions of chromosome mechanics. Sciara offers many unique biological features, several of which impact chromosome movement on spindles:

(a) chromosome imprinting; (b) a monopolar spindle in male meiosis I;  (c) non-disjunction of the X chromosome in male meiosis II; (d)  chromosome elimination in early embryogenesis; (e) sex determination; (f) evolution towards parthenogenesis; (g) germ line limited (L) chromosomes;  (h)  DNA amplification in salivary gland polytene chromosomes; (i) high resistance to radiation.

     We have now developed a toolbox to enable Sciara research, and we welcome new investigators [http://brown.edu/go/sciara-stocks]. We are completing the Sciara genome with cutting edge approaches for assembly using long reads from the PacBio RSII and Oxford Nanopore MinION sequencing platforms, using Illumina reads for polishing, and using BioNano Irys optical maps for scaffolding. Genome annotation used RNA-seq data from the Sciara transcriptome interrogating both sexes at multiple stages. We have used the genomic data to identify sequences of “DNA puffs” that represent sites of DNA amplification in salivary gland polytene chromosomes regulated by ecdysone.

     We have developed methods for transformation of Sciara to manipulate its genome. DNA has been introduced into ectopic sites in the Sciara genome using piggyBac. We present here a new method  for site-specific integration of large DNA into the Sciara genome. Previously, others have accomplished this by homologous recombination (HR). However, HR is inefficient; instead, the preferred pathway in most cells is non-homologous end-joining (NHEJ), but its primary previous application has been to create small indels. We have coupled NHEJ with obligate ligation-gated recombination (ObLiGaRe) for high efficiency precise insertion of large DNA into a unique double-strand break. This approach is easily applicable to a broad range of organisms, including those where a transformation system has not been available.

     With the new toolbox of the genome assembly and transformation methodology, the time is now ripe to elucidate many canonical processes using the unique biological features of Sciara.