A gap-free genome assembly of Chlamydomonas reinhardtii and detection of translocations induced by CRISPR-mediated mutagenesis

Genomic assemblies of the unicellular green alga Chlamydomonas reinhardtii have provided important resources for researchers. However, assembly errors, large gaps, and unplaced scaffolds as well as strain-specific variants currently impede many types of analysis. By combining PacBio HiFi and Oxford Nanopore long-read technologies, we generated a de novo genome assembly for strain CC-5816, derived from crosses of strains CC-125 and CC-124. Multiple methods of evaluating genome completeness and base-pair error rate suggest that the final telomere-to-telomere assembly is highly accurate. The CC-5816 assembly enabled previously difficult analyses that include characterization of the 17 centromeres, rDNA arrays on three chromosomes, and 56 insertions of organellar DNA into the nuclear genome. Using Nanopore sequencing, we identified sites of cytosine (CpG) methylation, which are enriched at centromeres. We analyzed CRISPR-Cas9 insertional mutants in the PF23 gene. Two of the three alleles produced progeny that displayed patterns of meiotic inviability that suggested the presence of a chromosomal aberration. Mapping Nanopore reads from pf23-2 and pf23-3 onto the CC-5816 genome showed that these two strains each carry a translocation that was initiated at the PF23 gene locus on chromosome 11 and joined with chromosomes 5 or 3, respectively. The translocations were verified by demonstrating linkage between loci on the two translocated chromosomes in meiotic progeny. The three pf23 alleles display the expected short-cilia phenotype, and immunoblotting showed that pf23-2 lacks the PF23 protein. Our CC-5816 genome assembly will undoubtedly provide an important tool for the Chlamydomonas research community

The Chlamydomonas genome project: A decade on

The green alga Chlamydomonas reinhardtii is a popular unicellular organism for studying photosynthesis, cilia biogenesis, and micronutrient homeostasis. Ten years since its genome project was initiated an iterative process of improvements to the genome and gene predictions has propelled this organism to the forefront of the omics era. Housed at Phytozome, the plant genomics portal of the Joint Genome Institute (JGI), the most up-to-date genomic data include a genome arranged on chromosomes and high-quality gene models with alternative splice forms supported by an abundance of whole transcriptome sequencing (RNA-Seq) data. We present here the past, present, and future of Chlamydomonas genomics. Specifically, we detail progress on genome assembly and gene model refinement, discuss resources for gene annotations, functional predictions, and locus ID mapping between versions and, importantly, outline a standardized framework for naming genes

Design and Bolometer Characterization of the SPT-3G First-year Focal Plane

During the austral summer of 2016-17, the third-generation camera, SPT-3G, was installed on the South Pole Telescope, increasing the detector count in the focal plane by an order of magnitude relative to the previous generation. Designed to map the polarization of the cosmic microwave background, SPT-3G contains ten 6-in-hexagonal modules of detectors, each with 269 trichroic and dual-polarization pixels, read out using 68x frequency-domain multiplexing. Here we discuss design, assembly, and layout of the modules, as well as early performance characterization of the first-year array, including yield and detector properties.Comment: Conference proceeding for Low Temperature Detectors 2017. Accepted for publication: 27 August 201

SPT-3G: A Next-Generation Cosmic Microwave Background Polarization Experiment on the South Pole Telescope

We describe the design of a new polarization sensitive receiver, SPT-3G, for the 10-meter South Pole Telescope (SPT). The SPT-3G receiver will deliver a factor of ~20 improvement in mapping speed over the current receiver, SPTpol. The sensitivity of the SPT-3G receiver will enable the advance from statistical detection of B-mode polarization anisotropy power to high signal-to-noise measurements of the individual modes, i.e., maps. This will lead to precise (~0.06 eV) constraints on the sum of neutrino masses with the potential to directly address the neutrino mass hierarchy. It will allow a separation of the lensing and inflationary B-mode power spectra, improving constraints on the amplitude and shape of the primordial signal, either through SPT-3G data alone or in combination with BICEP-2/KECK, which is observing the same area of sky. The measurement of small-scale temperature anisotropy will provide new constraints on the epoch of reionization. Additional science from the SPT-3G survey will be significantly enhanced by the synergy with the ongoing optical Dark Energy Survey (DES), including: a 1% constraint on the bias of optical tracers of large-scale structure, a measurement of the differential Doppler signal from pairs of galaxy clusters that will test General Relativity on ~200 Mpc scales, and improved cosmological constraints from the abundance of clusters of galaxies.Comment: 21 pages, 9 figures. To be published in Proceedings of SPIE Volume 9153. Presented at SPIE Astronomical Telescopes + Instrumentation 2014, conference 915

Deficiency in origin licensing proteins impairs cilia formation: implications for the aetiology of meier-gorlin syndrome

Mutations in ORC1, ORC4, ORC6, CDT1, and CDC6, which encode proteins required for DNA replication origin licensing, cause Meier-Gorlin syndrome (MGS), a disorder conferring microcephaly, primordial dwarfism, underdeveloped ears, and skeletal abnormalities. Mutations in ATR, which also functions during replication, can cause Seckel syndrome, a clinically related disorder. These findings suggest that impaired DNA replication could underlie the developmental defects characteristic of these disorders. Here, we show that although origin licensing capacity is impaired in all patient cells with mutations in origin licensing component proteins, this does not correlate with the rate of progression through S phase. Thus, the replicative capacity in MGS patient cells does not correlate with clinical manifestation. However, ORC1-deficient cells from MGS patients and siRNA-mediated depletion of origin licensing proteins also have impaired centrosome and centriole copy number. As a novel and unexpected finding, we show that they also display a striking defect in the rate of formation of primary cilia. We demonstrate that this impacts sonic hedgehog signalling in ORC1-deficient primary fibroblasts. Additionally, reduced growth factor-dependent signaling via primary cilia affects the kinetics of cell cycle progression following cell cycle exit and re-entry, highlighting an unexpected mechanism whereby origin licensing components can influence cell cycle progression. Finally, using a cell-based model, we show that defects in cilia function impair chondroinduction. Our findings raise the possibility that a reduced efficiency in forming cilia could contribute to the clinical features of MGS, particularly the bone development abnormalities, and could provide a new dimension for considering developmental impacts of licensing deficiency

Microtubules in Bacteria: Ancient Tubulins Build a Five-Protofilament Homolog of the Eukaryotic Cytoskeleton

Microtubules play crucial roles in cytokinesis, transport, and motility, and are therefore superb targets for anti-cancer drugs. All tubulins evolved from a common ancestor they share with the distantly related bacterial cell division protein FtsZ, but while eukaryotic tubulins evolved into highly conserved microtubule-forming heterodimers, bacterial FtsZ presumably continued to function as single homopolymeric protofilaments as it does today. Microtubules have not previously been found in bacteria, and we lack insight into their evolution from the tubulin/FtsZ ancestor. Using electron cryomicroscopy, here we show that the tubulin homologs BtubA and BtubB form microtubules in bacteria and suggest these be referred to as “bacterial microtubules” (bMTs). bMTs share important features with their eukaryotic counterparts, such as straight protofilaments and similar protofilament interactions. bMTs are composed of only five protofilaments, however, instead of the 13 typical in eukaryotes. These and other results suggest that rather than being derived from modern eukaryotic tubulin, BtubA and BtubB arose from early tubulin intermediates that formed small microtubules. Since we show that bacterial microtubules can be produced in abundance in vitro without chaperones, they should be useful tools for tubulin research and drug screening