Publications by authors named "Ivan Liachko"

37 Publications

Genome sequence of Monilinia vaccinii-corymbosi sheds light on mummy berry disease infection of blueberry and mating type.

G3 (Bethesda) 2021 Feb;11(2)

Department of Horticultural Science, North Carolina State University, Raleigh, NC 27695, USA.

Mummy berry disease, caused by the fungal pathogen Monilinia vaccinii-corymbosi (Mvc), is one of the most economically important diseases of blueberries in North America. Mvc is capable of inducing two separate blighting stages during its life cycle. Infected fruits are rendered mummified and unmarketable. Genomic data for this pathogen is lacking, but could be useful in understanding the reproductive biology of Mvc and the mechanisms it deploys to facilitate host infection. In this study, PacBio sequencing and Hi-C interaction data were utilized to create a chromosome-scale reference genome for Mvc. The genome comprises nine chromosomes with a total length of 30 Mb, an N50 length of 4.06 Mb, and an average 413X sequence coverage. A total of 9399 gene models were predicted and annotated, and BUSCO analysis revealed that 98% of 1,438 searched conserved eukaryotic genes were present in the predicted gene set. Potential effectors were identified, and the mating-type (MAT) locus was characterized. Biotrophic effectors allow the pathogen to avoid recognition by the host plant and evade or mitigate host defense responses during the early stages of fruit infection. Following locule colonization, necrotizing effectors promote the mummification of host tissues. Potential biotrophic effectors utilized by Mvc include chorismate mutase for reducing host salicylate and necrotrophic effectors include necrosis-inducing proteins and hydrolytic enzymes for macerating host tissue. The MAT locus sequences indicate the potential for homothallism in the reference genome, but a deletion allele of the MAT locus, characterized in a second isolate, indicates heterothallism. Further research is needed to verify the roles of individual effectors in virulence and to determine the role of the MAT locus in outcrossing and population genotypic diversity.
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http://dx.doi.org/10.1093/g3journal/jkaa052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8022979PMC
February 2021

Chromosome-scale genome assembly of the sea louse Caligus rogercresseyi by SMRT sequencing and Hi-C analysis.

Sci Data 2021 02 11;8(1):60. Epub 2021 Feb 11.

Bond Life Sciences Center, University of Missouri, Columbia, USA.

Caligus rogercresseyi, commonly known as sea louse, is an ectoparasite copepod that impacts the salmon aquaculture in Chile, causing losses of hundreds of million dollars per year. In this study, we report a chromosome-scale assembly of the sea louse (C. rogercresseyi) genome based on single-molecule real-time sequencing (SMRT) and proximity ligation (Hi-C) analysis. Coding RNAs and non-coding RNAs, and specifically long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) were identified through whole transcriptome sequencing from different life stages. A total of 23,686 protein-coding genes and 12,558 non-coding RNAs were annotated. In addition, 6,308 lncRNAs and 5,774 miRNAs were found to be transcriptionally active from larvae to adult stages. Taken together, this genomic resource for C. rogercresseyi represents a valuable tool to develop sustainable control strategies in the salmon aquaculture industry.
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http://dx.doi.org/10.1038/s41597-021-00842-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7878743PMC
February 2021

Identifying the causes and consequences of assembly gaps using a multiplatform genome assembly of a bird-of-paradise.

Mol Ecol Resour 2021 Jan 10;21(1):263-286. Epub 2020 Oct 10.

Department of Ecology and Genetics-Evolutionary Biology, Science for Life Laboratories, Uppsala University, Uppsala, Sweden.

Genome assemblies are currently being produced at an impressive rate by consortia and individual laboratories. The low costs and increasing efficiency of sequencing technologies now enable assembling genomes at unprecedented quality and contiguity. However, the difficulty in assembling repeat-rich and GC-rich regions (genomic "dark matter") limits insights into the evolution of genome structure and regulatory networks. Here, we compare the efficiency of currently available sequencing technologies (short/linked/long reads and proximity ligation maps) and combinations thereof in assembling genomic dark matter. By adopting different de novo assembly strategies, we compare individual draft assemblies to a curated multiplatform reference assembly and identify the genomic features that cause gaps within each assembly. We show that a multiplatform assembly implementing long-read, linked-read and proximity sequencing technologies performs best at recovering transposable elements, multicopy MHC genes, GC-rich microchromosomes and the repeat-rich W chromosome. Telomere-to-telomere assemblies are not a reality yet for most organisms, but by leveraging technology choice it is now possible to minimize genome assembly gaps for downstream analysis. We provide a roadmap to tailor sequencing projects for optimized completeness of both the coding and noncoding parts of nonmodel genomes.
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http://dx.doi.org/10.1111/1755-0998.13252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7757076PMC
January 2021

Degradation of Recalcitrant Polyurethane and Xenobiotic Additives by a Selected Landfill Microbial Community and Its Biodegradative Potential Revealed by Proximity Ligation-Based Metagenomic Analysis.

Front Microbiol 2019 22;10:2986. Epub 2020 Jan 22.

Departamento de Bioquímica, Facultad de Química, Universidad Nacional Autónoma de México, Mexico City, Mexico.

Polyurethanes (PU) are the sixth most produced plastics with around 18-million tons in 2016, but since they are not recyclable, they are burned or landfilled, generating damage to human health and ecosystems. To elucidate the mechanisms that landfill microbial communities perform to attack recalcitrant PU plastics, we studied the degradative activity of a mixed microbial culture, selected from a municipal landfill by its capability to grow in a water PU dispersion (WPUD) as the only carbon source, as a model for the BP8 landfill microbial community. The WPUD contains a polyether-polyurethane-acrylate (PE-PU-A) copolymer and xenobiotic additives (-methylpyrrolidone, isopropanol and glycol ethers). To identify the changes that the BP8 microbial community culture generates to the WPUD additives and copolymer, we performed chemical and physical analyses of the biodegradation process during 25 days of cultivation. These analyses included Nuclear magnetic resonance, Fourier transform infrared spectroscopy, Thermogravimetry, Differential scanning calorimetry, Gel permeation chromatography, and Gas chromatography coupled to mass spectrometry techniques. Moreover, for revealing the BP8 community structure and its genetically encoded potential biodegradative capability we also performed a proximity ligation-based metagenomic analysis. The additives present in the WPUD were consumed early whereas the copolymer was cleaved throughout the 25-days of incubation. The analysis of the biodegradation process and the identified biodegradation products showed that BP8 cleaves esters, C-C, and the recalcitrant aromatic urethanes and ether groups by hydrolytic and oxidative mechanisms, both in the soft and the hard segments of the copolymer. The proximity ligation-based metagenomic analysis allowed the reconstruction of five genomes, three of them from novel species. In the metagenome, genes encoding known enzymes, and putative enzymes and metabolic pathways accounting for the biodegradative activity of the BP8 community over the additives and PE-PU-A copolymer were identified. This is the first study revealing the genetically encoded potential biodegradative capability of a microbial community selected from a landfill, that thrives within a WPUD system and shows potential for bioremediation of polyurethane- and xenobiotic additives-contamitated sites.
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http://dx.doi.org/10.3389/fmicb.2019.02986DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6987047PMC
January 2020

Divergent evolutionary trajectories following speciation in two ectoparasitic honey bee mites.

Commun Biol 2019 1;2:357. Epub 2019 Oct 1.

1Okinawa Institute of Science and Technology, 1919-1 Tancha Onna-son, 904-0495 Okinawa, Japan.

Multispecies host-parasite evolution is common, but how parasites evolve after speciating remains poorly understood. Shared evolutionary history and physiology may propel species along similar evolutionary trajectories whereas pursuing different strategies can reduce competition. We test these scenarios in the economically important association between honey bees and ectoparasitic mites by sequencing the genomes of the sister mite species and . These genomes were closely related, with 99.7% sequence identity. Among the 9,628 orthologous genes, 4.8% showed signs of positive selection in at least one species. Divergent selective trajectories were discovered in conserved chemosensory gene families (IGR, SNMP), and Halloween genes (CYP) involved in moulting and reproduction. However, there was little overlap in these gene sets and associated GO terms, indicating different selective regimes operating on each of the parasites. Based on our findings, we suggest that species-specific strategies may be needed to combat evolving parasite communities.
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http://dx.doi.org/10.1038/s42003-019-0606-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6773775PMC
May 2020

Assignment of virus and antimicrobial resistance genes to microbial hosts in a complex microbial community by combined long-read assembly and proximity ligation.

Genome Biol 2019 08 2;20(1):153. Epub 2019 Aug 2.

USDA-ARS U.S. Meat Animal Research Center, Clay Center, NE, 68933, USA.

We describe a method that adds long-read sequencing to a mix of technologies used to assemble a highly complex cattle rumen microbial community, and provide a comparison to short read-based methods. Long-read alignments and Hi-C linkage between contigs support the identification of 188 novel virus-host associations and the determination of phage life cycle states in the rumen microbial community. The long-read assembly also identifies 94 antimicrobial resistance genes, compared to only seven alleles in the short-read assembly. We demonstrate novel techniques that work synergistically to improve characterization of biological features in a highly complex rumen microbial community.
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http://dx.doi.org/10.1186/s13059-019-1760-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6676630PMC
August 2019

Linking the resistome and plasmidome to the microbiome.

ISME J 2019 10 30;13(10):2437-2446. Epub 2019 May 30.

Department of Biological Sciences, University of Idaho, Moscow, ID, 83844, USA.

The rapid spread of antibiotic resistance among bacterial pathogens is a serious human health threat. While a range of environments have been identified as reservoirs of antibiotic resistance genes (ARGs), we lack understanding of the origins of these ARGs and their spread from environment to clinic. This is partly due to our inability to identify the natural bacterial hosts of ARGs and the mobile genetic elements that mediate this spread, such as plasmids and integrons. Here we demonstrate that the in vivo proximity-ligation method Hi-C can reconstruct a known plasmid-host association from a wastewater community, and identify the in situ host range of ARGs, plasmids, and integrons by physically linking them to their host chromosomes. Hi-C detected both previously known and novel associations between ARGs, mobile genetic elements and host genomes, thus validating this method. We showed that IncQ plasmids and class 1 integrons had the broadest host range in this wastewater, and identified bacteria belonging to Moraxellaceae, Bacteroides, and Prevotella, and especially Aeromonadaceae as the most likely reservoirs of ARGs in this community. A better identification of the natural carriers of ARGs will aid the development of strategies to limit resistance spread to pathogens.
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http://dx.doi.org/10.1038/s41396-019-0446-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6776055PMC
October 2019

Repeat elements organise 3D genome structure and mediate transcription in the filamentous fungus Epichloë festucae.

PLoS Genet 2018 10 24;14(10):e1007467. Epub 2018 Oct 24.

Statistics and Bioinformatics Group, Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand.

Structural features of genomes, including the three-dimensional arrangement of DNA in the nucleus, are increasingly seen as key contributors to the regulation of gene expression. However, studies on how genome structure and nuclear organisation influence transcription have so far been limited to a handful of model species. This narrow focus limits our ability to draw general conclusions about the ways in which three-dimensional structures are encoded, and to integrate information from three-dimensional data to address a broader gamut of biological questions. Here, we generate a complete and gapless genome sequence for the filamentous fungus, Epichloë festucae. We use Hi-C data to examine the three-dimensional organisation of the genome, and RNA-seq data to investigate how Epichloë genome structure contributes to the suite of transcriptional changes needed to maintain symbiotic relationships with the grass host. Our results reveal a genome in which very repeat-rich blocks of DNA with discrete boundaries are interspersed by gene-rich sequences that are almost repeat-free. In contrast to other species reported to date, the three-dimensional structure of the genome is anchored by these repeat blocks, which act to isolate transcription in neighbouring gene-rich regions. Genes that are differentially expressed in planta are enriched near the boundaries of these repeat-rich blocks, suggesting that their three-dimensional orientation partly encodes and regulates the symbiotic relationship formed by this organism.
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http://dx.doi.org/10.1371/journal.pgen.1007467DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6218096PMC
October 2018

A near complete, chromosome-scale assembly of the black raspberry (Rubus occidentalis) genome.

Gigascience 2018 08 1;7(8). Epub 2018 Aug 1.

USDA-ARS National Clonal Germplasm Repository, 33447 Peoria Rd., Corvallis, OR, 97333, USA.

Background: The fragmented nature of most draft plant genomes has hindered downstream gene discovery, trait mapping for breeding, and other functional genomics applications. There is a pressing need to improve or finish draft plant genome assemblies.

Findings: Here, we present a chromosome-scale assembly of the black raspberry genome using single-molecule real-time Pacific Biosciences sequencing and high-throughput chromatin conformation capture (Hi-C) genome scaffolding. The updated V3 assembly has a contig N50 of 5.1 Mb, representing an ∼200-fold improvement over the previous Illumina-based version. Each of the 235 contigs was anchored and oriented into seven chromosomes, correcting several major misassemblies. Black raspberry V3 contains 47 Mb of new sequences including large pericentromeric regions and thousands of previously unannotated protein-coding genes. Among the new genes are hundreds of expanded tandem gene arrays that were collapsed in the Illumina-based assembly. Detailed comparative genomics with the high-quality V4 woodland strawberry genome (Fragaria vesca) revealed near-perfect 1:1 synteny with dramatic divergence in tandem gene array composition. Lineage-specific tandem gene arrays in black raspberry are related to agronomic traits such as disease resistance and secondary metabolite biosynthesis.

Conclusions: The improved resolution of tandem gene arrays highlights the need to reassemble these highly complex and biologically important regions in draft plant genomes. The updated, high-quality black raspberry reference genome will be useful for comparative genomics across the horticulturally important Rosaceae family and enable the development of marker assisted breeding in Rubus.
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http://dx.doi.org/10.1093/gigascience/giy094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6131213PMC
August 2018

Assembly of 913 microbial genomes from metagenomic sequencing of the cow rumen.

Nat Commun 2018 02 28;9(1):870. Epub 2018 Feb 28.

The Roslin Institute and R(D)SVS, University of Edinburgh, Easter Bush, EH25 9RG, UK.

The cow rumen is adapted for the breakdown of plant material into energy and nutrients, a task largely performed by enzymes encoded by the rumen microbiome. Here we present 913 draft bacterial and archaeal genomes assembled from over 800 Gb of rumen metagenomic sequence data derived from 43 Scottish cattle, using both metagenomic binning and Hi-C-based proximity-guided assembly. Most of these genomes represent previously unsequenced strains and species. The draft genomes contain over 69,000 proteins predicted to be involved in carbohydrate metabolism, over 90% of which do not have a good match in public databases. Inclusion of the 913 genomes presented here improves metagenomic read classification by sevenfold against our own data, and by fivefold against other publicly available rumen datasets. Thus, our dataset substantially improves the coverage of rumen microbial genomes in the public databases and represents a valuable resource for biomass-degrading enzyme discovery and studies of the rumen microbiome.
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http://dx.doi.org/10.1038/s41467-018-03317-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5830445PMC
February 2018

Chromosome-scale scaffolding of the black raspberry ( L.) genome based on chromatin interaction data.

Hortic Res 2018 7;5. Epub 2018 Feb 7.

The New Zealand Institute for Plant & Food Research Limited, Private Bag 11600, Palmerston North, 4474 New Zealand.

Black raspberry ( L.) is a niche fruit crop valued for its flavor and potential health benefits. The improvement of fruit and cane characteristics via molecular breeding technologies has been hindered by the lack of a high-quality reference genome. The recently released draft genome for black raspberry (ORUS 4115-3) lacks assembly of scaffolds to chromosome scale. We used high-throughput chromatin conformation capture (Hi-C) and Proximity-Guided Assembly (PGA) to cluster and order 9650 out of 11,936 contigs of this draft genome assembly into seven pseudo-chromosomes. The seven pseudo-chromosomes cover ~97.2% of the total contig length (~223.8 Mb). Locating existing genetic markers on the physical map resolved multiple discrepancies in marker order on the genetic map. Centromeric regions were inferred from recombination frequencies of genetic markers, alignment of 303 bp centromeric sequence with the PGA, and heat map showing the physical contact matrix over the entire genome. We demonstrate a high degree of synteny between each of the seven chromosomes of black raspberry and a high-quality reference genome for strawberry ( L.) assembled using only PacBio long-read sequences. We conclude that PGA is a cost-effective and rapid method of generating chromosome-scale assemblies from Illumina short-read sequencing data.
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http://dx.doi.org/10.1038/s41438-017-0013-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5802725PMC
February 2018

Identification of a novel interspecific hybrid yeast from a metagenomic spontaneously inoculated beer sample using Hi-C.

Yeast 2018 01 19;35(1):71-84. Epub 2017 Oct 19.

Department of Genome Sciences, University of Washington, Seattle, WA, USA.

Interspecific hybridization is a common mechanism enabling genetic diversification and adaptation; however, the detection of hybrid species has been quite difficult. The identification of microbial hybrids is made even more complicated, as most environmental microbes are resistant to culturing and must be studied in their native mixed communities. We have previously adapted the chromosome conformation capture method Hi-C to the assembly of genomes from mixed populations. Here, we show the method's application in assembling genomes directly from an uncultured, mixed population from a spontaneously inoculated beer sample. Our assembly method has enabled us to de-convolute four bacterial and four yeast genomes from this sample, including a putative yeast hybrid. Downstream isolation and analysis of this hybrid confirmed its genome to consist of Pichia membranifaciens and that of another related, but undescribed, yeast. Our work shows that Hi-C-based metagenomic methods can overcome the limitation of traditional sequencing methods in studying complex mixtures of genomes. Copyright © 2017 John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/yea.3280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5771821PMC
January 2018

Improvement of the Threespine Stickleback Genome Using a Hi-C-Based Proximity-Guided Assembly.

J Hered 2017 Sep;108(6):693-700

Department of Genetics, University of Georgia, 120 Green Street, Athens, GA 30602

Scaffolding genomes into complete chromosome assemblies remains challenging even with the rapidly increasing sequence coverage generated by current next-generation sequence technologies. Even with scaffolding information, many genome assemblies remain incomplete. The genome of the threespine stickleback (Gasterosteus aculeatus), a fish model system in evolutionary genetics and genomics, is not completely assembled despite scaffolding with high-density linkage maps. Here, we first test the ability of a Hi-C based proximity-guided assembly (PGA) to perform a de novo genome assembly from relatively short contigs. Using Hi-C based PGA, we generated complete chromosome assemblies from a distribution of short contigs (20-100 kb). We found that 96.40% of contigs were correctly assigned to linkage groups (LGs), with ordering nearly identical to the previous genome assembly. Using available bacterial artificial chromosome (BAC) end sequences, we provide evidence that some of the few discrepancies between the Hi-C assembly and the existing assembly are due to structural variation between the populations used for the 2 assemblies or errors in the existing assembly. This Hi-C assembly also allowed us to improve the existing assembly, assigning over 60% (13.35 Mb) of the previously unassigned (~21.7 Mb) contigs to LGs. Together, our results highlight the potential of the Hi-C based PGA method to be used in combination with short read data to perform relatively inexpensive de novo genome assemblies. This approach will be particularly useful in organisms in which it is difficult to perform linkage mapping or to obtain high molecular weight DNA required for other scaffolding methods.
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http://dx.doi.org/10.1093/jhered/esx058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5892396PMC
September 2017

The dynamic three-dimensional organization of the diploid yeast genome.

Elife 2017 05 24;6. Epub 2017 May 24.

Department of Genome Sciences, University of Washington, Seattle, United States.

The budding yeast is a long-standing model for the three-dimensional organization of eukaryotic genomes. However, even in this well-studied model, it is unclear how homolog pairing in diploids or environmental conditions influence overall genome organization. Here, we performed high-throughput chromosome conformation capture on diverged hybrid diploids to obtain the first global view of chromosome conformation in diploid yeasts. After controlling for the Rabl-like orientation using a polymer model, we observe significant homolog proximity that increases in saturated culture conditions. Surprisingly, we observe a localized increase in homologous interactions between the alleles specifically under galactose induction and saturated growth. This pairing is accompanied by relocalization to the nuclear periphery and requires Nup2, suggesting a role for nuclear pore complexes. Together, these results reveal that the diploid yeast genome has a dynamic and complex 3D organization.
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http://dx.doi.org/10.7554/eLife.23623DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5476426PMC
May 2017

Single-molecule sequencing and chromatin conformation capture enable de novo reference assembly of the domestic goat genome.

Nat Genet 2017 Apr 6;49(4):643-650. Epub 2017 Mar 6.

US Meat Animal Research Center, ARS USDA, Clay Center, Nebraska, USA.

The decrease in sequencing cost and increased sophistication of assembly algorithms for short-read platforms has resulted in a sharp increase in the number of species with genome assemblies. However, these assemblies are highly fragmented, with many gaps, ambiguities, and errors, impeding downstream applications. We demonstrate current state of the art for de novo assembly using the domestic goat (Capra hircus) based on long reads for contig formation, short reads for consensus validation, and scaffolding by optical and chromatin interaction mapping. These combined technologies produced what is, to our knowledge, the most continuous de novo mammalian assembly to date, with chromosome-length scaffolds and only 649 gaps. Our assembly represents a ∼400-fold improvement in continuity due to properly assembled gaps, compared to the previously published C. hircus assembly, and better resolves repetitive structures longer than 1 kb, representing the largest repeat family and immune gene complex yet produced for an individual of a ruminant species.
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http://dx.doi.org/10.1038/ng.3802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5909822PMC
April 2017

Differential paralog divergence modulates genome evolution across yeast species.

PLoS Genet 2017 02 14;13(2):e1006585. Epub 2017 Feb 14.

Department of Genome Sciences, University of Washington, Seattle, Washington, United States of America.

Evolutionary outcomes depend not only on the selective forces acting upon a species, but also on the genetic background. However, large timescales and uncertain historical selection pressures can make it difficult to discern such important background differences between species. Experimental evolution is one tool to compare evolutionary potential of known genotypes in a controlled environment. Here we utilized a highly reproducible evolutionary adaptation in Saccharomyces cerevisiae to investigate whether experimental evolution of other yeast species would select for similar adaptive mutations. We evolved populations of S. cerevisiae, S. paradoxus, S. mikatae, S. uvarum, and interspecific hybrids between S. uvarum and S. cerevisiae for ~200-500 generations in sulfate-limited continuous culture. Wild-type S. cerevisiae cultures invariably amplify the high affinity sulfate transporter gene, SUL1. However, while amplification of the SUL1 locus was detected in S. paradoxus and S. mikatae populations, S. uvarum cultures instead selected for amplification of the paralog, SUL2. We measured the relative fitness of strains bearing deletions and amplifications of both SUL genes from different species, confirming that, converse to S. cerevisiae, S. uvarum SUL2 contributes more to fitness in sulfate limitation than S. uvarum SUL1. By measuring the fitness and gene expression of chimeric promoter-ORF constructs, we were able to delineate the cause of this differential fitness effect primarily to the promoter of S. uvarum SUL1. Our data show evidence of differential sub-functionalization among the sulfate transporters across Saccharomyces species through recent changes in noncoding sequence. Furthermore, these results show a clear example of how such background differences due to paralog divergence can drive changes in genome evolution.
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http://dx.doi.org/10.1371/journal.pgen.1006585DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5308817PMC
February 2017

Evolution of protein phosphorylation across 18 fungal species.

Science 2016 10;354(6309):229-232

Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA.

Living organisms have evolved protein phosphorylation, a rapid and versatile mechanism that drives signaling and regulates protein function. We report the phosphoproteomes of 18 fungal species and a phylogenetic-based approach to study phosphosite evolution. We observe rapid divergence, with only a small fraction of phosphosites conserved over hundreds of millions of years. Relative to recently acquired phosphosites, ancient sites are enriched at protein interfaces and are more likely to be functionally important, as we show for sites on H2A1 and eIF4E. We also observe a change in phosphorylation motif frequencies and kinase activities that coincides with the whole-genome duplication event. Our results provide an evolutionary history for phosphosites and suggest that rapid evolution of phosphorylation can contribute strongly to phenotypic diversity.
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http://dx.doi.org/10.1126/science.aaf2144DOI Listing
October 2016

Neocentromeres Provide Chromosome Segregation Accuracy and Centromere Clustering to Multiple Loci along a Candida albicans Chromosome.

PLoS Genet 2016 Sep 23;12(9):e1006317. Epub 2016 Sep 23.

Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota, United States of America.

Assembly of kinetochore complexes, involving greater than one hundred proteins, is essential for chromosome segregation and genome stability. Neocentromeres, or new centromeres, occur when kinetochores assemble de novo, at DNA loci not previously associated with kinetochore proteins, and they restore chromosome segregation to chromosomes lacking a functional centromere. Neocentromeres have been observed in a number of diseases and may play an evolutionary role in adaptation or speciation. However, the consequences of neocentromere formation on chromosome missegregation rates, gene expression, and three-dimensional (3D) nuclear structure are not well understood. Here, we used Candida albicans, an organism with small, epigenetically-inherited centromeres, as a model system to study the functions of twenty different neocentromere loci along a single chromosome, chromosome 5. Comparison of neocentromere properties relative to native centromere functions revealed that all twenty neocentromeres mediated chromosome segregation, albeit to different degrees. Some neocentromeres also caused reduced levels of transcription from genes found within the neocentromere region. Furthermore, like native centromeres, neocentromeres clustered in 3D with active/functional centromeres, indicating that formation of a new centromere mediates the reorganization of 3D nuclear architecture. This demonstrates that centromere clustering depends on epigenetically defined function and not on the primary DNA sequence, and that neocentromere function is independent of its distance from the native centromere position. Together, the results show that a neocentromere can form at many loci along a chromosome and can support the assembly of a functional kinetochore that exhibits native centromere functions including chromosome segregation accuracy and centromere clustering within the nucleus.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5035033PMC
http://dx.doi.org/10.1371/journal.pgen.1006317DOI Listing
September 2016

Characterization of a panARS-based episomal vector in the methylotrophic yeast Pichia pastoris for recombinant protein production and synthetic biology applications.

Microb Cell Fact 2016 Aug 11;15(1):139. Epub 2016 Aug 11.

Institute of Medical Biology (A-STAR), 8a Biomedical Grove, #06-06, Singapore, 138648, Singapore.

Background: Recombinant protein production in the methylotrophic yeast Pichia pastoris largely relies on integrative vectors. Although the stability of integrated expression cassettes is well appreciated for most applications, the availability of reliable episomal vectors for this host would represent a useful tool to expedite cloning and high-throughput screening, ameliorating also the relatively high clonal variability reported in transformants from integrative vectors caused by off-target integration in the P. pastoris genome. Recently, heterologous and endogenous autonomously replicating sequences (ARS) were identified in P. pastoris by genome mining, opening the possibility of expanding the available toolbox to include efficient episomal plasmids. The aim of this technical report is to validate a 452-bp sequence ("panARS") in context of P. pastoris expression vectors, and to compare their performance to classical integrative plasmids. Moreover, we aimed to test if such episomal vectors would be suitable to sustain in vivo recombination, using fragments for transformation, directly in P. pastoris cells.

Results: A panARS-based episomal vector was evaluated using blue fluorescent protein (BFP) as a reporter gene. Normalized fluorescence from colonies carrying panARS-BFP outperformed the level of signal obtained from integrative controls by several-fold, whereas endogenous sequences, identified from the P. pastoris genome, were not as efficient in terms of protein production. At the single cell level, panARS-BFP clones showed lower interclonal variability but higher intraclonal variation compared to their integrative counterparts, supporting the idea that heterologous protein production could benefit from episomal plasmids. Finally, efficiency of 2-fragment and 3-fragment in vivo recombination was tested using varying lengths of overlapping regions and molar ratios between fragments. Upon optimization, minimal background was obtained for in vivo assembled vectors, suggesting this could be a quick and efficient method to generate of episomal plasmids of interest.

Conclusions: An expression vector based on the panARS sequence was shown to outperform its integrative counterparts in terms of protein productivity and interclonal variability, facilitating recombinant protein expression and screening. Using optimized fragment lengths and ratios, it was possible to perform reliable in vivo recombination of fragments in P. pastoris. Taken together, these results support the applicability of panARS episomal vectors for synthetic biology approaches.
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http://dx.doi.org/10.1186/s12934-016-0540-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4981965PMC
August 2016

High Throughput Analyses of Budding Yeast ARSs Reveal New DNA Elements Capable of Conferring Centromere-Independent Plasmid Propagation.

G3 (Bethesda) 2016 04 7;6(4):993-1012. Epub 2016 Apr 7.

Department of Biomolecular Chemistry, School of Medicine and Public Health, University of Wisconsin, Madison, Wisconsin 53706

The ability of plasmids to propagate in Saccharomyces cerevisiae has been instrumental in defining eukaryotic chromosomal control elements. Stable propagation demands both plasmid replication, which requires a chromosomal replication origin (i.e., an ARS), and plasmid distribution to dividing cells, which requires either a chromosomal centromere for segregation or a plasmid-partitioning element. While our knowledge of yeast ARSs and centromeres is relatively advanced, we know less about chromosomal regions that can function as plasmid partitioning elements. The Rap1 protein-binding site (RAP1) present in transcriptional silencers and telomeres of budding yeast is a known plasmid-partitioning element that functions to anchor a plasmid to the inner nuclear membrane (INM), which in turn facilitates plasmid distribution to daughter cells. This Rap1-dependent INM-anchoring also has an important chromosomal role in higher-order chromosomal structures that enhance transcriptional silencing and telomere stability. Thus, plasmid partitioning can reflect fundamental features of chromosome structure and biology, yet a systematic screen for plasmid partitioning elements has not been reported. Here, we couple deep sequencing with competitive growth experiments of a plasmid library containing thousands of short ARS fragments to identify new plasmid partitioning elements. Competitive growth experiments were performed with libraries that differed only in terms of the presence or absence of a centromere. Comparisons of the behavior of ARS fragments in the two experiments allowed us to identify sequences that were likely to drive plasmid partitioning. In addition to the silencer RAP1 site, we identified 74 new putative plasmid-partitioning motifs predicted to act as binding sites for DNA binding proteins enriched for roles in negative regulation of gene expression and G2/M-phase associated biology. These data expand our knowledge of chromosomal elements that may function in plasmid partitioning and suggest underlying biological roles shared by such elements.
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http://dx.doi.org/10.1534/g3.116.027904DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4825667PMC
April 2016

The fitness consequences of aneuploidy are driven by condition-dependent gene effects.

PLoS Biol 2015 May 26;13(5):e1002155. Epub 2015 May 26.

Department of Genome Sciences, University of Washington, Seattle, Washington, United States of America.

Aneuploidy is a hallmark of tumor cells, and yet the precise relationship between aneuploidy and a cell's proliferative ability, or cellular fitness, has remained elusive. In this study, we have combined a detailed analysis of aneuploid clones isolated from laboratory-evolved populations of Saccharomyces cerevisiae with a systematic, genome-wide screen for the fitness effects of telomeric amplifications to address the relationship between aneuploidy and cellular fitness. We found that aneuploid clones rise to high population frequencies in nutrient-limited evolution experiments and show increased fitness relative to wild type. Direct competition experiments confirmed that three out of four aneuploid events isolated from evolved populations were themselves sufficient to improve fitness. To expand the scope beyond this small number of exemplars, we created a genome-wide collection of >1,800 diploid yeast strains, each containing a different telomeric amplicon (Tamp), ranging in size from 0.4 to 1,000 kb. Using pooled competition experiments in nutrient-limited chemostats followed by high-throughput sequencing of strain-identifying barcodes, we determined the fitness effects of these >1,800 Tamps under three different conditions. Our data revealed that the fitness landscape explored by telomeric amplifications is much broader than that explored by single-gene amplifications. As also observed in the evolved clones, we found the fitness effects of most Tamps to be condition specific, with a minority showing common effects in all three conditions. By integrating our data with previous work that examined the fitness effects of single-gene amplifications genome-wide, we found that a small number of genes within each Tamp are centrally responsible for each Tamp's fitness effects. Our genome-wide Tamp screen confirmed that telomeric amplifications identified in laboratory-evolved populations generally increased fitness. Our results show that Tamps are mutations that produce large, typically condition-dependent changes in fitness that are important drivers of increased fitness in asexually evolving populations.
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http://dx.doi.org/10.1371/journal.pbio.1002155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4444335PMC
May 2015

Accurate identification of centromere locations in yeast genomes using Hi-C.

Nucleic Acids Res 2015 Jun 4;43(11):5331-9. Epub 2015 May 4.

Department of Genome Sciences, University of Washington, 3720 15th Ave NE, Seattle, WA 98195, USA Department of Computer Science and Engineering, University of Washington, 185 Stevens Way, Seattle, WA 98195, USA

Centromeres are essential for proper chromosome segregation. Despite extensive research, centromere locations in yeast genomes remain difficult to infer, and in most species they are still unknown. Recently, the chromatin conformation capture assay, Hi-C, has been re-purposed for diverse applications, including de novo genome assembly, deconvolution of metagenomic samples and inference of centromere locations. We describe a method, Centurion, that jointly infers the locations of all centromeres in a single genome from Hi-C data by exploiting the centromeres' tendency to cluster in three-dimensional space. We first demonstrate the accuracy of Centurion in identifying known centromere locations from high coverage Hi-C data of budding yeast and a human malaria parasite. We then use Centurion to infer centromere locations in 14 yeast species. Across all microbes that we consider, Centurion predicts 89% of centromeres within 5 kb of their known locations. We also demonstrate the robustness of the approach in datasets with low sequencing depth. Finally, we predict centromere coordinates for six yeast species that currently lack centromere annotations. These results show that Centurion can be used for centromere identification for diverse species of yeast and possibly other microorganisms.
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http://dx.doi.org/10.1093/nar/gkv424DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4477656PMC
June 2015

An underlying mechanism for the increased mutagenesis of lagging-strand genes in Bacillus subtilis.

Proc Natl Acad Sci U S A 2015 Mar 23;112(10):E1096-105. Epub 2015 Feb 23.

Departments of Microbiology and

We previously reported that lagging-strand genes accumulate mutations faster than those encoded on the leading strand in Bacillus subtilis. Although we proposed that orientation-specific encounters between replication and transcription underlie this phenomenon, the mechanism leading to the increased mutagenesis of lagging-strand genes remained unknown. Here, we report that the transcription-dependent and orientation-specific differences in mutation rates of genes require the B. subtilis Y-family polymerase, PolY1 (yqjH). We find that without PolY1, association of the replicative helicase, DnaC, and the recombination protein, RecA, with lagging-strand genes increases in a transcription-dependent manner. These data suggest that PolY1 promotes efficient replisome progression through lagging-strand genes, thereby reducing potentially detrimental breaks and single-stranded DNA at these loci. Y-family polymerases can alleviate potential obstacles to replisome progression by facilitating DNA lesion bypass, extension of D-loops, or excision repair. We find that the nucleotide excision repair (NER) proteins UvrA, UvrB, and UvrC, but not RecA, are required for transcription-dependent asymmetry in mutation rates of genes in the two orientations. Furthermore, we find that the transcription-coupling repair factor Mfd functions in the same pathway as PolY1 and is also required for increased mutagenesis of lagging-strand genes. Experimental and SNP analyses of B. subtilis genomes show mutational footprints consistent with these findings. We propose that the interplay between replication and transcription increases lesion susceptibility of, specifically, lagging-strand genes, activating an Mfd-dependent error-prone NER mechanism. We propose that this process, at least partially, underlies the accelerated evolution of lagging-strand genes.
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http://dx.doi.org/10.1073/pnas.1416651112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4364195PMC
March 2015

Origin replication complex binding, nucleosome depletion patterns, and a primary sequence motif can predict origins of replication in a genome with epigenetic centromeres.

mBio 2014 Sep 2;5(5):e01703-14. Epub 2014 Sep 2.

Unlabelled: Origins of DNA replication are key genetic elements, yet their identification remains elusive in most organisms. In previous work, we found that centromeres contain origins of replication (ORIs) that are determined epigenetically in the pathogenic yeast Candida albicans. In this study, we used origin recognition complex (ORC) binding and nucleosome occupancy patterns in Saccharomyces cerevisiae and Kluyveromyces lactis to train a machine learning algorithm to predict the position of active arm (noncentromeric) origins in the C. albicans genome. The model identified bona fide active origins as determined by the presence of replication intermediates on nondenaturing two-dimensional (2D) gels. Importantly, these origins function at their native chromosomal loci and also as autonomously replicating sequences (ARSs) on a linear plasmid. A "mini-ARS screen" identified at least one and often two ARS regions of ≥100 bp within each bona fide origin. Furthermore, a 15-bp AC-rich consensus motif was associated with the predicted origins and conferred autonomous replicating activity to the mini-ARSs. Thus, while centromeres and the origins associated with them are epigenetic, arm origins are dependent upon critical DNA features, such as a binding site for ORC and a propensity for nucleosome exclusion.

Importance: DNA replication machinery is highly conserved, yet the definition of exactly what specifies a replication origin differs in different species. Here, we utilized computational genomics to predict origin locations in Candida albicans by combining locations of binding sites for the conserved origin replication complex, necessary for replication initiation, together with chromatin organization patterns. We identified predicted sequences that exhibited bona fide origin function and developed a linear plasmid assay to delimit the DNA fragments necessary for origin function. Additionally, we found that a short AC-rich motif, which is enriched in predicted origins, is required for origin function. Thus, we demonstrated a new machine learning paradigm for identification of potential origins from a genome with no prior information. Furthermore, this work suggests that C. albicans has two different types of origins: "hard-wired" arm origins that rely upon specific sequence motifs and "epigenetic" centromeric origins that are recruited to kinetochores in a sequence-independent manner.
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http://dx.doi.org/10.1128/mBio.01703-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4173791PMC
September 2014

Species-level deconvolution of metagenome assemblies with Hi-C-based contact probability maps.

G3 (Bethesda) 2014 May 22;4(7):1339-46. Epub 2014 May 22.

Department of Genome Sciences, University of Washington, Seattle, Washington 98195-5065

Microbial communities consist of mixed populations of organisms, including unknown species in unknown abundances. These communities are often studied through metagenomic shotgun sequencing, but standard library construction methods remove long-range contiguity information; thus, shotgun sequencing and de novo assembly of a metagenome typically yield a collection of contigs that cannot readily be grouped by species. Methods for generating chromatin-level contact probability maps, e.g., as generated by the Hi-C method, provide a signal of contiguity that is completely intracellular and contains both intrachromosomal and interchromosomal information. Here, we demonstrate how this signal can be exploited to reconstruct the individual genomes of microbial species present within a mixed sample. We apply this approach to two synthetic metagenome samples, successfully clustering the genome content of fungal, bacterial, and archaeal species with more than 99% agreement with published reference genomes. We also show that the Hi-C signal can secondarily be used to create scaffolded genome assemblies of individual eukaryotic species present within the microbial community, with higher levels of contiguity than some of the species' published reference genomes.
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http://dx.doi.org/10.1534/g3.114.011825DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4455782PMC
May 2014

GC-rich DNA elements enable replication origin activity in the methylotrophic yeast Pichia pastoris.

PLoS Genet 2014 Mar 6;10(3):e1004169. Epub 2014 Mar 6.

Department of Genome Sciences, University of Washington, Seattle, Washington, United States of America.

The well-studied DNA replication origins of the model budding and fission yeasts are A/T-rich elements. However, unlike their yeast counterparts, both plant and metazoan origins are G/C-rich and are associated with transcription start sites. Here we show that an industrially important methylotrophic budding yeast, Pichia pastoris, simultaneously employs at least two types of replication origins--a G/C-rich type associated with transcription start sites and an A/T-rich type more reminiscent of typical budding and fission yeast origins. We used a suite of massively parallel sequencing tools to map and dissect P. pastoris origins comprehensively, to measure their replication dynamics, and to assay the global positioning of nucleosomes across the genome. Our results suggest that some functional overlap exists between promoter sequences and G/C-rich replication origins in P. pastoris and imply an evolutionary bifurcation of the modes of replication initiation.
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http://dx.doi.org/10.1371/journal.pgen.1004169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3945215PMC
March 2014

An autonomously replicating sequence for use in a wide range of budding yeasts.

FEMS Yeast Res 2014 Mar 2;14(2):364-7. Epub 2013 Dec 2.

Department of Genome Sciences, University of Washington, Seattle, WA, USA.

The initiation of DNA replication at replication origins is essential for the duplication of genomes. In yeast, the autonomously replicating sequence (ARS) property of replication origins is necessary for the stable maintenance of episomal plasmids. However, because the sequence determinants of ARS function differ among yeast species, current ARS modules are limited for use to a subset of yeasts. Here, we describe a short ARS sequence that functions in at least 10 diverse species of budding yeast. These include, but are not limited to members of the Saccharomyces, Lachancea, Kluyveromyces, and Pichia (Komagataella) genera spanning over 500 million years of evolution. In addition to its wide species range, this ARS and an optimized derivative confer improved plasmid stability relative to other currently used ARS modules.
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http://dx.doi.org/10.1111/1567-1364.12123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3959236PMC
March 2014

High-resolution mapping, characterization, and optimization of autonomously replicating sequences in yeast.

Genome Res 2013 Apr 12;23(4):698-704. Epub 2012 Dec 12.

Department of Genome Sciences, University of Washington, Seattle, Washington 98105, USA.

DNA replication origins are necessary for the duplication of genomes. In addition, plasmid-based expression systems require DNA replication origins to maintain plasmids efficiently. The yeast autonomously replicating sequence (ARS) assay has been a valuable tool in dissecting replication origin structure and function. However, the dearth of information on origins in diverse yeasts limits the availability of efficient replication origin modules to only a handful of species and restricts our understanding of origin function and evolution. To enable rapid study of origins, we have developed a sequencing-based suite of methods for comprehensively mapping and characterizing ARSs within a yeast genome. Our approach finely maps genomic inserts capable of supporting plasmid replication and uses massively parallel deep mutational scanning to define molecular determinants of ARS function with single-nucleotide resolution. In addition to providing unprecedented detail into origin structure, our data have allowed us to design short, synthetic DNA sequences that retain maximal ARS function. These methods can be readily applied to understand and modulate ARS function in diverse systems.
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http://dx.doi.org/10.1101/gr.144659.112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3613586PMC
April 2013

Novel features of ARS selection in budding yeast Lachancea kluyveri.

BMC Genomics 2011 Dec 28;12:633. Epub 2011 Dec 28.

School of Mathematics and Statistics, University of Sydney, Sydney, Australia.

Background: The characterization of DNA replication origins in yeast has shed much light on the mechanisms of initiation of DNA replication. However, very little is known about the evolution of origins or the evolution of mechanisms through which origins are recognized by the initiation machinery. This lack of understanding is largely due to the vast evolutionary distances between model organisms in which origins have been examined.

Results: In this study we have isolated and characterized autonomously replicating sequences (ARSs) in Lachancea kluyveri - a pre-whole genome duplication (WGD) budding yeast. Through a combination of experimental work and rigorous computational analysis, we show that L. kluyveri ARSs require a sequence that is similar but much longer than the ARS Consensus Sequence well defined in Saccharomyces cerevisiae. Moreover, compared with S. cerevisiae and K. lactis, the replication licensing machinery in L. kluyveri seems more tolerant to variations in the ARS sequence composition. It is able to initiate replication from almost all S. cerevisiae ARSs tested and most Kluyveromyces lactis ARSs. In contrast, only about half of the L. kluyveri ARSs function in S. cerevisiae and less than 10% function in K. lactis.

Conclusions: Our findings demonstrate a replication initiation system with novel features and underscore the functional diversity within the budding yeasts. Furthermore, we have developed new approaches for analyzing biologically functional DNA sequences with ill-defined motifs.
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http://dx.doi.org/10.1186/1471-2164-12-633DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3306766PMC
December 2011

A comprehensive genome-wide map of autonomously replicating sequences in a naive genome.

PLoS Genet 2010 May 13;6(5):e1000946. Epub 2010 May 13.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, United States of America.

Eukaryotic chromosomes initiate DNA synthesis from multiple replication origins. The machinery that initiates DNA synthesis is highly conserved, but the sites where the replication initiation proteins bind have diverged significantly. Functional comparative genomics is an obvious approach to study the evolution of replication origins. However, to date, the Saccharomyces cerevisiae replication origin map is the only genome map available. Using an iterative approach that combines computational prediction and functional validation, we have generated a high-resolution genome-wide map of DNA replication origins in Kluyveromyces lactis. Unlike other yeasts or metazoans, K. lactis autonomously replicating sequences (KlARSs) contain a 50 bp consensus motif suggestive of a dimeric structure. This motif is necessary and largely sufficient for initiation and was used to dependably identify 145 of the up to 156 non-repetitive intergenic ARSs projected for the K. lactis genome. Though similar in genome sizes, K. lactis has half as many ARSs as its distant relative S. cerevisiae. Comparative genomic analysis shows that ARSs in K. lactis and S. cerevisiae preferentially localize to non-syntenic intergenic regions, linking ARSs with loci of accelerated evolutionary change.
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http://dx.doi.org/10.1371/journal.pgen.1000946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2869322PMC
May 2010