Biology subjects

Hawley, R. S.

Publications and source records attributed to Hawley, R. S..

5 recordsLinked to original sources

bioRxiv2018

Molecular analysis of Drosophila melanogaster B chromosomes reveals their origin, composition, and structure

The number of chromosomes carried by an individual species is one of its defining characteristics. Some species, however, can also carry supernumerary chromosomes referred to as B chromosomes. B chromosomes were recently identified in a laboratory stock of Drosophila melanogaster--an established model organism with a wealth of genetic and genomic resources--enabling us to subject them to extensive molecular analysis. We isolated the B chromosomes by pulsed-field gel electrophoresis and determined their composition through next-generation sequencing. Although these B chromosomes carry no known euchromatic sequence, they are rich in transposable elements and long arrays of short nucleotide repeats, the most abundant being the uncharacterized AAGAT satellite repeat. Fluorescent in-situ hybridization on metaphase chromosome spreads revealed this repeat is located on Chromosome 4, strongly suggesting the origin of the B chromosomes is Chromosome 4. Cytological and quantitative comparisons of signal intensity between Chromosome 4 and the B chromosomes supports the hypothesis that the structure of the B chromosome is an isochromosome. We also report the identification of a new B chromosome variant in a related laboratory stock. This B chromosome has a similar repeat signature as the original but is smaller and much less prevalent. We examined additional stocks with similar genotypes and did not find B chromosomes, but did find these stocks lacked the AAGAT satellite repeat. Our molecular characterization of D. melanogaster B chromosomes is the first step towards understanding how supernumerary chromosomes arise from essential chromosomes and what may be necessary for their stable inheritance.

genetics↗

bioRxiv2018

A nuclear hormone receptor and lipid metabolism axis are required for the maintenance and regeneration of reproductive organs

Understanding how stem cells and their progeny maintain and regenerate reproductive organs is of fundamental importance. The freshwater planarian Schmidtea mediterranea provides an attractive system to study these processes because its hermaphroditic reproductive system (RS) arises post-embryonically and when lost can be fully and functionally regenerated from the proliferation and regulation of experimentally accessible stem and progenitor cells. By controlling the function of a nuclear hormone receptor gene (nhr-1), we established conditions in which to study the formation, maintenance and regeneration of both germline and somatic tissues of the planarian RS. We found that nhr-1(RNAi) not only resulted in the gradual degeneration and complete loss of the adult hermaphroditic RS, but also in the significant downregulation of a large cohort of genes associated with lipid metabolism. One of these, Smed-acs-1, a homologue of Acyl-CoA synthetase, was indispensable for the development, maintenance and regeneration of the RS, but not for the homeostasis or regeneration of other somatic tissues. Remarkably, supplementing nhr-1(RNAi) animals with either bacterial Acyl-CoA synthetase or the lipid metabolite Acetyl-CoA rescued the phenotype restoring the maintenance and function of the hermaphroditic RS. Our findings uncovered a likely evolutionarily conserved role for nuclear hormone receptors and lipid metabolism in the regulation of stem and progenitor cells required for the long-term maintenance and regeneration of animal reproductive organs, tissues and cells.

developmental biology↗

bioRxiv2018

Synaptonemal complex architecture facilitates the chromosome-specific regulation of recombination in Drosophila

The synaptonemal complex (SC) is a conserved meiotic structure that regulates the repair of double strand breaks (DSBs) into crossovers or gene conversions. The removal of any central region SC component, such as the Drosophila melanogaster transverse filament protein C(3)G, causes a complete loss of SC structure and crossovers. To better understand the role of the SC in meiosis, we used CRISPR/Cas9 to construct three in-frame deletions within the predicted coiled-coil region of the C(3)G protein. These three deletion mutants disrupt SC maintenance at different times during pachytene and exhibit distinct defects in key meiotic processes, allowing us to define the stages of pachytene when the SC is necessary for homolog pairing and recombination. Our studies demonstrate that the X chromosome and the autosomes display substantially different defects in pairing and recombination when SC structure is disrupted, suggesting that the X chromosome is potentially regulated differently than the autosomes.

genetics↗

bioRxiv2018

High-quality genome assemblies of 15 Drosophila species generated using Nanopore sequencing

The Drosophila genus is a unique group containing a wide range of species that occupy diverse ecosystems. In addition to the most widely studied species, Drosophila melanogaster, many other members in this genus also possess a well-developed set of genetic tools. Indeed, high-quality genomes exist for several species within the genus, facilitating studies of the function and evolution of cis-regulatory regions and proteins by allowing comparisons across at least 50 million years of evolution. Yet, the available genomes still fail to capture much of the substantial genetic diversity within the Drosophila genus. We have therefore tested protocols to rapidly and inexpensively sequence and assemble the genome from any Drosophila species using single-molecule sequencing technology from Oxford Nanopore. Here, we use this technology to present high-quality genome assemblies of 15 Drosophila species: 10 of the 12 originally sequenced Drosophila species (ananassae, erecta, mojavensis, persimilis, pseudoobscura, sechellia, simulans, virilis, willistoni, and yakuba), four additional species that had previously reported assemblies (biarmipes, bipectinata, eugracilis, and mauritiana), and one novel assembly (triauraria). Genomes were generated from an average of 29x depth-of-coverage data that after assembly resulted in an average contig N50 of 4.4 Mb. Subsequent alignment of contigs from the published reference genomes demonstrates that our assemblies could be used to close over 60% of the gaps present in the currently published reference genomes. Importantly, the materials and reagents cost for each genome was approximately $1,000 (USD). This study demonstrates the power and cost-effectiveness of long-read sequencing for genome assembly in Drosophila and provides a framework for the affordable sequencing and assembly of additional Drosophila genomes.

genomics↗

bioRxiv2018

Rapid low-cost assembly of the Drosophila melanogaster reference genome using low-coverage, long-read sequencing

Accurate and comprehensive characterization of genetic variation is essential for deciphering the genetic basis of diseases and other phenotypes. A vast amount of genetic variation stems from large-scale sequence changes arising from the duplication, deletion, inversion, and translocation of sequences. In the past 10 years, high-throughput short reads have greatly expanded our ability to assay sequence variation due to single nucleotide polymorphisms. However, a recent de novo assembly of a second Drosophila melanogaster reference genome has revealed that short read genotyping methods miss hundreds of structural variants, including those affecting phenotypes. While genomes assembled using high-coverage long reads can achieve high levels of contiguity and completeness, concerns about cost, errors, and low yield have limited widespread adoption of such sequencing approaches. Here we resequenced the reference strain of D. melanogaster (ISO1) on a single Oxford Nanopore MinION flow cell run for 24 hours. Using only reads longer than 1 kb or with at least 30x coverage, we assembled a highly contiguous de novo genome. The addition of inexpensive paired reads and subsequent scaffolding using an optical map technology achieved an assembly with completeness and contiguity comparable to the D. melanogaster reference assembly. Comparison of our assembly to the reference assembly of ISO1 uncovered a number of structural variants (SVs), including novel LTR transposable element insertions and duplications affecting genes with developmental, behavioral, and metabolic functions. Collectively, these SVs provide a snapshot of the dynamics of genome evolution. Furthermore, our assembly and comparison to the D. melanogaster reference genome demonstrates that high-quality de novo assembly of reference genomes and comprehensive variant discovery using such assemblies are now possible by a single lab for under $1,000 (USD).

genomics↗