Mitochondrial Genomes in Perkinsus Decode Conserved Frameshifts in All Genes.

Mitochondrial genomes of apicomplexans, dinoflagellates, and chrompodellids that collectively make up the Myzozoa, encode only three proteins (Cytochrome b [COB], Cytochrome c oxidase subunit 1 [COX1], Cytochrome c oxidase subunit 3 [COX3]), contain fragmented ribosomal RNAs, and display extensive recombination, RNA trans-splicing, and RNA-editing. The early-diverging Perkinsozoa is the final major myzozoan lineage whose mitochondrial genomes remained poorly characterized. Previous reports of Perkinsus genes indicated independent acquisition of non-canonical features, namely the occurrence of multiple frameshifts. To determine both ancestral myzozoan and novel perkinsozoan mitochondrial genome features, we sequenced and assembled mitochondrial genomes of four Perkinsus species. These data show a simple ancestral genome with the common reduced coding capacity but disposition for rearrangement. We identified 75 frameshifts across the four species that occur as distinct types and that are highly conserved in gene location. A decoding mechanism apparently employs unused codons at the frameshift sites that advance translation either +1 or +2 frames to the next used codon. The locations of frameshifts are seemingly positioned to regulate protein folding of the nascent protein as it emerges from the ribosome. The cox3 gene is distinct in containing only one frameshift and showing strong selection against residues that are otherwise frequently encoded at the frameshift positions in cox1 and cob. All genes lack cysteine codons implying a reduction to 19 amino acids in these genomes. Furthermore, mitochondrion-encoded rRNA fragment complements are incomplete in Perkinsus spp. but some are found in the nuclear DNA suggesting import into the organelle. Perkinsus demonstrates further remarkable trajectories of organelle genome evolution including pervasive integration of frameshift translation into genome expression.

Development of the Myzozoan Aquatic Parasite Perkinsus marinus as A Versatile Experimental Genetic Model Organism.

The phylum Perkinsozoa is an aquatic parasite lineage that has devastating effects on commercial and natural mollusc populations, and also comprises parasites of algae, fish and amphibians. They are related to dinoflagellates and apicomplexans and thus offer excellent genetic models for both parasitological and evolutionary studies. Genetic transformation was previously achieved for Perkinsus spp. but with few tools for transgene expression and limited selection efficacy. We sought to expand the power of experimental genetic tools for Perkinsus using P. marinus as a model. We constructed a modular plasmid assembly system for expression of multiple genes simultaneously. We developed efficient selection systems for three drugs, puromycin, bleomycin and blasticidin, that are effective in as little as three weeks. We developed eleven new promoters of variable expression strength. Furthermore, we identified that genomic integration of transgenes is predominantly via non-homologous recombination but with transgene fragmentation including deletion of some elements. To counter these dynamic processes, we show that bi-cistronic transcripts using the viral 2A peptides can couple selection to the maintenance of the expression of a transgene of interest. Collectively, these new tools and insights provide great new capacity to genetically modify and study Perkinsus as an aquatic parasite and evolutionary model.

A Comprehensive Subcellular Atlas of the Toxoplasma Proteome via hyperLOPIT Provides Spatial Context for Protein Functions.

Apicomplexan parasites cause major human disease and food insecurity. They owe their considerable success to highly specialized cell compartments and structures. These adaptations drive their recognition, nondestructive penetration, and elaborate reengineering of the host's cells to promote their growth, dissemination, and the countering of host defenses. The evolution of unique apicomplexan cellular compartments is concomitant with vast proteomic novelty. Consequently, half of apicomplexan proteins are unique and uncharacterized. Here, we determine the steady-state subcellular location of thousands of proteins simultaneously within the globally prevalent apicomplexan parasite Toxoplasma gondii. This provides unprecedented comprehensive molecular definition of these unicellular eukaryotes and their specialized compartments, and these data reveal the spatial organizations of protein expression and function, adaptation to hosts, and the underlying evolutionary trajectories of these pathogens.

An essential pentatricopeptide repeat protein in the apicomplexan remnant chloroplast.

The malaria parasite Plasmodium and other apicomplexans such as Toxoplasma evolved from photosynthetic organisms and contain an essential, remnant plastid termed the apicoplast. Transcription of the apicoplast genome is polycistronic with extensive RNA processing. Yet little is known about the mechanism of apicoplast RNA processing. In plants, chloroplast RNA processing is controlled by multiple pentatricopeptide repeat (PPR) proteins. Here, we identify the single apicoplast PPR protein, PPR1. We show that the protein is essential and that it binds to RNA motifs corresponding with previously characterized processing sites. Additionally, PPR1 shields RNA transcripts from ribonuclease degradation. This is the first characterization of a PPR protein from a nonphotosynthetic plastid.

The Biochemistry and Evolution of the Dinoflagellate Nucleus.

Dinoflagellates are known to possess a highly aberrant nucleus-the so-called dinokaryon-that exhibits a multitude of exceptional biological features. These include: (1) Permanently condensed chromosomes; (2) DNA in a cholesteric liquid crystalline state, (3) extremely large DNA content (up to 200 pg); and, perhaps most strikingly, (4) a deficit of histones-the canonical building blocks of all eukaryotic chromatin. Dinoflagellates belong to the Alveolata clade (dinoflagellates, apicomplexans, and ciliates) and, therefore, the biological oddities observed in dinoflagellate nuclei are derived character states. Understanding the sequence of changes that led to the dinokaryon has been difficult in the past with poor resolution of dinoflagellate phylogeny. Moreover, lack of knowledge of their molecular composition has constrained our understanding of the molecular properties of these derived nuclei. However, recent advances in the resolution of the phylogeny of dinoflagellates, particularly of the early branching taxa; the realization that divergent histone genes are present; and the discovery of dinoflagellate-specific nuclear proteins that were acquired early in dinoflagellate evolution have all thrown new light nature and evolution of the dinokaryon.

Genetic transformation of the dinoflagellate chloroplast.

Coral reefs are some of the most important and ecologically diverse marine environments. At the base of the reef ecosystem are dinoflagellate algae, which live symbiotically within coral cells. Efforts to understand the relationship between alga and coral have been greatly hampered by the lack of an appropriate dinoflagellate genetic transformation technology. By making use of the plasmid-like fragmented chloroplast genome, we have introduced novel genetic material into the dinoflagellate chloroplast genome. We have shown that the introduced genes are expressed and confer the expected phenotypes. Genetically modified cultures have been grown for 1 year with subculturing, maintaining the introduced genes and phenotypes. This indicates that cells continue to divide after transformation and that the transformation is stable. This is the first report of stable chloroplast transformation in dinoflagellate algae.

The Conformation of Yeast Chromosome III Is Mating Type Dependent and Controlled by the Recombination Enhancer.

Mating-type switching in yeast occurs through gene conversion between the MAT locus and one of two silent loci (HML or HMR) on opposite ends of the chromosome. MATa cells choose HML as template, whereas MATα cells use HMR. The recombination enhancer (RE) located on the left arm regulates this process. One long-standing hypothesis is that switching is guided by mating-type-specific and possibly RE-dependent chromosome folding. Here, we use Hi-C, 5C, and live-cell imaging to characterize the conformation of chromosome III in both mating types. We discovered a mating-type-specific conformational difference in the left arm. Deletion of a 1-kb subregion within the RE, which is not necessary during switching, abolished mating-type-dependent chromosome folding. The RE is therefore a composite element with one subregion essential for donor selection during switching and a separate region involved in modulating chromosome conformation.

Differential chromosome conformations as hallmarks of cellular identity revealed by mathematical polymer modeling.

Inherently dynamic, chromosomes adopt many different conformations in response to DNA metabolism. Models of chromosome organization in the yeast nucleus obtained from genome-wide chromosome conformation data or biophysical simulations provide important insights into the average behavior but fail to reveal features from dynamic or transient events that are only visible in a fraction of cells at any given moment. We developed a method to determine chromosome conformation from relative positions of three fluorescently tagged DNA in living cells imaged in 3D. Cell type specific chromosome folding properties could be assigned based on positional combinations between three loci on yeast chromosome 3. We determined that the shorter left arm of chromosome 3 is extended in MATα cells, but can be crumpled in MATa cells. Furthermore, we implemented a new mathematical model that provides for the first time an estimate of the relative physical constraint of three linked loci related to cellular identity. Variations in this estimate allowed us to predict functional consequences from chromatin structural alterations in asf1 and recombination enhancer deletion mutant cells. The computational method is applicable to identify and characterize dynamic chromosome conformations in any cell type.

Tracking of single and multiple genomic loci in living yeast cells.

Nuclear organization is involved in numerous aspects of cellular function. In yeast, analysis of the nuclear position and dynamics of the silent and active mating-type loci has allowed to gain insight into the mechanisms involved in directing mating-type switching. The fluorescent repressor operator systems (FROS) have proven to be a powerful technique to tag DNA sequences to investigate chromosome position and dynamics in living cells. FROS rely on the transgenic expression of a bacterial repressor fused to a fluorescent protein which can bind to its respective operator DNA sequence integrated as multicopy tandem arrays at a specific genomic site. Different FROS exist which facilitate the tagging of up to three different loci simultaneously. This chapter describes detailed protocols for FROS usage and analysis in the yeast Saccharomyces cerevisiae.

High-resolution profiling of gammaH2AX around DNA double strand breaks in the mammalian genome.

Chromatin acts as a key regulator of DNA-related processes such as DNA damage repair. Although ChIP-chip is a powerful technique to provide high-resolution maps of protein-genome interactions, its use to study DNA double strand break (DSB) repair has been hindered by the limitations of the available damage induction methods. We have developed a human cell line that permits induction of multiple DSBs randomly distributed and unambiguously positioned within the genome. Using this system, we have generated the first genome-wide mapping of gammaH2AX around DSBs. We found that all DSBs trigger large gammaH2AX domains, which spread out from the DSB in a bidirectional, discontinuous and not necessarily symmetrical manner. The distribution of gammaH2AX within domains is influenced by gene transcription, as parallel mappings of RNA Polymerase II and strand-specific expression showed that gammaH2AX does not propagate on active genes. In addition, we showed that transcription is accurately maintained within gammaH2AX domains, indicating that mechanisms may exist to protect gene transcription from gammaH2AX spreading and from the chromatin rearrangements induced by DSBs.

Lab-on-Chip for fast 3D particle tracking in living cells.

We present a novel Lab-on-Chip technology for 3D particle tracking in living cells based on V-shaped micro-mirrors, which are used to observe fluorescent specimens from multiple vantage points, providing simultaneous stereo-images that can be recombined for 3D reconstruction. Our technology can be readily used with standard fluorescence microscopes, and we apply it to study chromatin dynamics using yeast strains with one or two GFP-tagged gene loci. Using an Andor EMCCD camera, loci are followed in 3D with inter-frame intervals of up to 10 ms and with an error of 27 nm per axis, yielding quantitative information on their dynamics with exquisite temporal spatial resolution.