Organization of the Euplotes crassus Micronuclear Genome1

Euplotes crassus, like other hypotrichous ciliated protozoa, eliminates most of its micronuclear chromosomal DNA in the process of forming the small linear DNA molecules that comprise the macronuclear genome. By characterizing randomly selected lambda phage clones of E. crassus micronuclear DNA, we have determined the distribution of repetitive and unique sequences and the arrangement of macronuclear genes relative to eliminated DNA. This allows us to compare the E. crassus micronuclear genome organization to that of another distantly related hypotrichous ciliate, Oxytricha nova. The clones from E. crassus segregate into three prevalent classes: those containing primarily eliminated repetitive DNA (Class I); those containing macronuclear genes in addition to repetitive sequences (Class II); and those containing only eliminated unique sequence DNA (Class III). All of the repetitive sequences in these clones belong to the same highly abundant repetitive element family. Our results demonstrate that the sequence organization of the E. crassus and O. nova micronuclear genomes is related in that the macronuclear genes are clustered together in the micronuclear genome and the eliminated unique sequences occur in long stretches that are uninterrupted by repetitive sequences. In both organisms a single repetitive element family comprises the majority of the eliminated interspersed middle repetitive DNA and appears to be preferentially associated with the macronuclear sequence clusters. The similarities in the sequence organization in these two organisms suggest that clustering of macronuclear genes plays a role in the chromosome fragmentation process.     

Coding properties of <Emphasis Type="Italic">Oxytricha trifallax</Emphasis> (<Emphasis Type="Italic">Sterkiella histriomuscorum</Emphasis>) macronuclear chromosomes: analysis of a pilot genome project

The macronuclear genomes of spirotrichous ciliates are almost entirely polyploid, single-gene chromosomes (nanochromosomes). We recently performed a pilot genome project for a member of this group, Oxytricha trifallax (Sterkiella histriomuscorum), in which 2000 nanochromosomes were cloned at random and end-sequenced. Here we describe the global properties of the coding regions predicted for these molecules, including nucleotide composition, codon usage, and intron properties. In identifying splice donor, acceptor and branch sites, we found that longer introns in Oxytricha have a stronger signal at the donor site than do smaller introns, as has been found for Caenorhabditis elegans and Drosophila, despite the overall small size of the introns. A systematic search for multi-gene chromosomes identified 11 candidate nanochromosomes. We compare the results from this large dataset with those obtained from earlier studies and with statistics recorded from ciliates and other eukaryotes.     

Evolution of the scrambled germline gene encoding α-telomere binding protein in three hypotrichous ciliates

The micronuclear genes encoding α-telomere-binding protein (αTP) in Oxytricha trifallax and Stylonychia mytilus contain multiple internal eliminated segments, or IESs, that divide the gene into multiple parts called macronuclear destined segments, or MDSs. The MDSs have become disordered, or scrambled, during evolution. The scrambled structures of the αTP genes in Oxytricha trifallax and S. mytilus have been compared with the previously published scrambled structure of the αTP gene in O. nova. The scrambled patterns of the αTP gene in the three species are similar but show significant differences. The micronuclear genes in O. nova and S. mytilus consist of 13 IESs and 14 MDSs, but the gene in O. trifallax is divided into three additional MDSs by the presence of three additional IESs, believed to have been inserted into the O. trifallaxαTP gene after divergence of O. trifallax from the other two species. Corresponding IESs among the three species have shifted along the DNA during evolution, presumably by a mutational mechanism that changes the short repeat sequences that flank IESs. The IESs also have changed markedly in length by insertion and/or deletion of nucleotides. Comparison of the putative αTP amino acid sequences in the three species reveals three conserved and three nonconserved domains. The 5′ nontranslated regions of the gene-sized molecules encoding αTP contain several conserved segments, and the 3′ nontranscribed trailer contains one conserved segment. Received: 29 May 1998; in revised form: 3 August 1998 / Accepted: 18 August 1998     

Organization of gene and non-gene sequences in micronuclear DNA of Oxytricha nova.

In order to study the derivation of the macronuclear genome from the micronuclear genome in Oxytricha nova micronuclear DNA was partially digested with EcoRI, size fractionated, and then cloned in the lambda phage Charon 8. Clones were selected a) at random b) by hybridization with macronuclear DNA or c) by hybridization with clones of macronuclear DNA. One group of these clones contains only unique sequence DNA, and all of these had sequences that were homologous to macronuclear sequences. The number of macronuclear genes with sequences homologous to these micronuclear clones indicates that macronuclear sequences are clustered in the micronuclear genome. Many micronuclear clones contain repetitive DNA sequences and hybridize to numerous EcoRI fragments of total micronuclear DNA, yielding similar but non-identical patterns. Some micronuclear clones containing these repetitive sequences also contained unique sequence DNA that hybridized to a macronuclear sequence. These clones define a major interspersed repetitive sequence family in the micronuclear genome that is eliminated during formation of the macronuclear genome.     

Organization of the Euplotes crassus micronuclear genome

Euplotes crassus, like other hypotrichous ciliated protozoa, eliminates most of its micronuclear chromosomal DNA in the process of forming the small linear DNA molecules that comprise the macronuclear genome. By characterizing randomly selected lambda phage clones of E. crassus micronuclear DNA, we have determined the distribution of repetitive and unique sequences and the arrangement of macronuclear genes relative to eliminated DNA. This allows us to compare the E. crassus micronuclear genome organization to that of another distantly related hypotrichous ciliate, Oxytricha nova. The clones from E. crassus segregate into three prevalent classes: those containing primarily eliminated repetitive DNA (Class I); those containing macronuclear genes in addition to repetitive sequences (Class II); and those containing only eliminated unique sequence DNA (Class III). All of the repetitive sequences in these clones belong to the same highly abundant repetitive element family. Our results demonstrate that the sequence organization of the E. crassus and O. nova micronuclear genomes is related in that the macronuclear genes are clustered together in the micronuclear genome and the eliminated unique sequences occur in long stretches that are uninterrupted by repetitive sequences. In both organisms a single repetitive element family comprises the majority of the eliminated interspersed middle repetitive DNA and appears to be preferentially associated with the macronuclear sequence clusters. The similarities in the sequence organization in these two organisms suggest that clustering of macronuclear genes plays a role in the chromosome fragmentation process.     

Transposon Domestication versus Mutualism in Ciliate Genome Rearrangements

Ciliated protists rearrange their genomes dramatically during nuclear development via chromosome fragmentation and DNA deletion to produce a trimmer and highly reorganized somatic genome. The deleted portion of the genome includes potentially active transposons or transposon-like sequences that reside in the germline. Three independent studies recently showed that transposase proteins of the DDE/DDD superfamily are indispensible for DNA processing in three distantly related ciliates. In the spirotrich Oxytricha trifallax, high copy-number germline-limited transposons mediate their own excision from the somatic genome but also contribute to programmed genome rearrangement through a remarkable transposon mutualism with the host. By contrast, the genomes of two oligohymenophorean ciliates, Tetrahymena thermophila and Paramecium tetraurelia, encode homologous PiggyBac-like transposases as single-copy genes in both their germline and somatic genomes. These domesticated transposases are essential for deletion of thousands of different internal sequences in these species. This review contrasts the events underlying somatic genome reduction in three different ciliates and considers their evolutionary origins and the relationships among their distinct mechanisms for genome remodeling.     

Comparative Genome Analyses Highlight Transposon-Mediated Genome Expansion and the Evolutionary Architecture of 3D Genomic Folding in Cotton

Transposable element (TE) amplification has been recognized as a driving force mediating genome size expansion and evolution, but the consequences for shaping 3D genomic architecture remains largely unknown in plants. Here, we report reference-grade genome assemblies for three species of cotton ranging 3-fold in genome size, namely Gossypium rotundifolium (K₂), G. arboreum (A₂), and G. raimondii (D₅), using Oxford Nanopore Technologies. Comparative genome analyses document the details of lineage-specific TE amplification contributing to the large genome size differences (K₂, 2.44 Gb; A₂, 1.62 Gb; D₅, 750.19 Mb) and indicate relatively conserved gene content and synteny relationships among genomes. We found that approximately 17% of syntenic genes exhibit chromatin status change between active (“A”) and inactive (“B”) compartments, and TE amplification was associated with the increase of the proportion of A compartment in gene regions (∼7,000 genes) in K₂ and A₂ relative to D₅. Only 42% of topologically associating domain (TAD) boundaries were conserved among the three genomes. Our data implicate recent amplification of TEs following the formation of lineage-specific TAD boundaries. This study sheds light on the role of transposon-mediated genome expansion in the evolution of higher-order chromatin structure in plants.     

Micronuclear DNA sequences of Oxytricha fallax homologous to the macronuclear inverted terminal repeat.

The macronucleus of the protozoan Oxytricha fallax is generated from a micronucleus following conjugation. While the micronucleus contains high molecular weight DNA, the macronucleus contains only short linear DNA molecules which all end in the same 20 bp inverted terminal repeat (Ma-ITR). The Ma-ITR was radioactively labeled and purified for use as a probe in hybridizations to micronuclear and macronuclear DNA. Sequences homologous to the Ma-ITR were detected in micronuclear DNA. The copy number of the repeat in the micronuclear genome is approximately that required to encode the macronuclear DNA termini. The micronuclear copies are found embedded in repeated long sequence blocks.     

Genome Wide Association for Addiction: Replicated Results and Comparisons of Two Analytic Approaches

Background

Vulnerabilities to dependence on addictive substances are substantially heritable complex disorders whose underlying genetic architecture is likely to be polygenic, with modest contributions from variants in many individual genes. “Nontemplate” genome wide association (GWA) approaches can identity groups of chromosomal regions and genes that, taken together, are much more likely to contain allelic variants that alter vulnerability to substance dependence than expected by chance.

Methodology/Principal Findings

We report pooled “nontemplate” genome-wide association studies of two independent samples of substance dependent vs control research volunteers (n = 1620), one European-American and the other African-American using 1 million SNP (single nucleotide polymorphism) Affymetrix genotyping arrays. We assess convergence between results from these two samples using two related methods that seek clustering of nominally-positive results and assess significance levels with Monte Carlo and permutation approaches. Both “converge then cluster” and “cluster then converge” analyses document convergence between the results obtained from these two independent datasets in ways that are virtually never found by chance. The genes identified in this fashion are also identified by individually-genotyped dbGAP data that compare allele frequencies in cocaine dependent vs control individuals.

Conclusions/Significance

These overlapping results identify small chromosomal regions that are also identified by genome wide data from studies of other relevant samples to extents much greater than chance. These chromosomal regions contain more genes related to “cell adhesion” processes than expected by chance. They also contain a number of genes that encode potential targets for anti-addiction pharmacotherapeutics. “Nontemplate” GWA approaches that seek chromosomal regions in which nominally-positive associations are found in multiple independent samples are likely to complement classical, “template” GWA approaches in which “genome wide” levels of significance are sought for SNP data from single case vs control comparisons.     

Comprehensive Characterization of Human Genome Variation by High Coverage Whole-Genome Sequencing of Forty Four Caucasians

Whole genome sequencing studies are essential to obtain a comprehensive understanding of the vast pattern of human genomic variations. Here we report the results of a high-coverage whole genome sequencing study for 44 unrelated healthy Caucasian adults, each sequenced to over 50-fold coverage (averaging 65.8×). We identified approximately 11 million single nucleotide polymorphisms (SNPs), 2.8 million short insertions and deletions, and over 500,000 block substitutions. We showed that, although previous studies, including the 1000 Genomes Project Phase 1 study, have catalogued the vast majority of common SNPs, many of the low-frequency and rare variants remain undiscovered. For instance, approximately 1.4 million SNPs and 1.3 million short indels that we found were novel to both the dbSNP and the 1000 Genomes Project Phase 1 data sets, and the majority of which (∼96%) have a minor allele frequency less than 5%. On average, each individual genome carried ∼3.3 million SNPs and ∼492,000 indels/block substitutions, including approximately 179 variants that were predicted to cause loss of function of the gene products. Moreover, each individual genome carried an average of 44 such loss-of-function variants in a homozygous state, which would completely “knock out” the corresponding genes. Across all the 44 genomes, a total of 182 genes were “knocked-out” in at least one individual genome, among which 46 genes were “knocked out” in over 30% of our samples, suggesting that a number of genes are commonly “knocked-out” in general populations. Gene ontology analysis suggested that these commonly “knocked-out” genes are enriched in biological process related to antigen processing and immune response. Our results contribute towards a comprehensive characterization of human genomic variation, especially for less-common and rare variants, and provide an invaluable resource for future genetic studies of human variation and diseases.     

Complete Genome Sequence of Francisella tularensis Subspecies holarctica FTNF002-00

Francisella tularensis subspecies holarctica FTNF002-00 strain was originally obtained from the first known clinical case of bacteremic F. tularensis pneumonia in Southern Europe isolated from an immunocompetent individual. The FTNF002-00 complete genome contains the RD₂₃ deletion and represents a type strain for a clonal population from the first epidemic tularemia outbreak in Spain between 1997–1998. Here, we present the complete sequence analysis of the FTNF002-00 genome. The complete genome sequence of FTNF002-00 revealed several large as well as small genomic differences with respect to two other published complete genome sequences of F. tularensis subsp. holarctica strains, LVS and OSU18. The FTNF002-00 genome shares >99.9% sequence similarity with LVS and OSU18, and is also ∼5 MB smaller by comparison. The overall organization of the FTNF002-00 genome is remarkably identical to those of LVS and OSU18, except for a single 3.9 kb inversion in FTNF002-00. Twelve regions of difference ranging from 0.1–1.5 kb and forty-two small insertions and deletions were identified in a comparative analysis of FTNF002-00, LVS, and OSU18 genomes. Two small deletions appear to inactivate two genes in FTNF002-00 causing them to become pseudogenes; the intact genes encode a protein of unknown function and a drug:H⁺ antiporter. In addition, we identified ninety-nine proteins in FTNF002-00 containing amino acid mutations compared to LVS and OSU18. Several non-conserved amino acid replacements were identified, one of which occurs in the virulence-associated intracellular growth locus subunit D protein. Many of these changes in FTNF002-00 are likely the consequence of direct selection that increases the fitness of this subsp. holarctica clone within its endemic population. Our complete genome sequence analyses lay the foundation for experimental testing of these possibilities.     

Conserved linkage of two genes on the same macronuclear chromosome in spirotrichous ciliates

Macronuclear chromosomes of spirotrichous ciliates are mainly “nanochromosomes” containing only a single gene. We identified a two-gene chromosome in the spirotrich Sterkiella histriomuscorum (formerly Oxytricha trifallax) which, unlike other characterized two-gene molecules, contains reading frames oriented tail to tail. These are homologs of ribosomal protein L29 (RPL29) and cyclophilin. We found that both genes are transcribed, with their polyadenylation sites on opposite strands separated by only 135 bp. Furthermore, both genes in S. histriomuscorum are present only on one macronuclear chromosome and do not occur alone or linked to other genes. The corresponding micronuclear locus is fragmented into three nonscrambled gene segments (MDSs), separated by two noncoding segments (IESs). We also found that these two genes are linked on a macronuclear chromosome, similarly arranged tail to tail, in the three spirotrichs Stylonychia lemnae, Uroleptus sp., and Holosticha sp.. In addition, single-gene macronuclear chromosomes containing only the RPL29 gene were detected in the earlier diverged Holosticha and Uroleptus. These observations suggest a possible evolutionary trend towards loss of chromosomal breakage between these two genes. This study is the first to examine gene linkage in the macronucleus of several spirotrichs and may provide insight into the evolution of multi-gene macronuclear chromosomes and chromosomal fragmentation in spirotrichs. Electronic Supplementary Material Supplementary material is available for this article at     

Internal sequences are eliminated from genes during macronuclear development in the ciliated protozoan Oxytricha nova

During the life cycle of the hypotrichous ciliate Oxytricha nova, a macronucleus containing short, gene-sized DNA molecules is produced from a copy of the chromosomal micronuclear genome. In order to characterize the process of macronuclear development, we have isolated and determined the DNA sequence of a particular macronuclear gene and its micronuclear precursor. The results of this analysis indicate that macronuclear telomeric sequences (5'C4A4(3') repeats) are not present at the ends of the gene in its micronuclear chromosomal location and must be added during development. In addition, the micronuclear copy of the gene contains three short blocks of sequence that must be removed during development, implying the involvement of a nucleic acid-splicing process in generating mature macronuclear genes.     

The Micronuclear Gene Encoding β-Telomere Binding Protein in Oxytricha nova

ABSTRACT The micronuclear version of the gene encoding β-telomere binding protein (β-TBP) in Oxytricha nova has been sequenced and compared to the macronuclear β-TBP gene, previously described. The micronuclear gene contains three AT-rich internal eliminated sequences (IES) of 37, 40, and 43 bp and four macronuclear destined sequences (MDS). The IES interrupt the gene once near the 5′ end of the coding region and twice in the 3′ trailer downstream from the TGA stop codon. The sequences of the micronuclear and macronuclear genes are colinear. Thus, the micronuclear β-TBP gene is not scrambled, which contrasts with the highly scrambled state among the 14 MDS in the micronuclear α;-TBP gene.     

Deletion of the Tetrahymena thermophila rDNA replication fork barrier region disrupts macronuclear rDNA excision and creates a fragile site in the micronuclear genome

During macronuclear development the Tetrahymena thermophila ribosomal RNA gene is excised from micronuclear chromosome 1 by site-specific cleavage at chromosome breakage sequence (Cbs) elements, rearranged into a ‘palindromic’ 21 kb minichromosome and extensively amplified. Gene amplification initiates from origins in the 5′ non-transcribed spacer, and forks moving toward the center of the palindrome arrest at a developmentally regulated replication fork barrier (RFB). The RFB is inactive during vegetative cell divisions, suggesting a role in the formation or amplification of macronuclear rDNA. Using micronuclear (germline) transformation, we show that the RFB region facilitates Cbs-mediated excision. Deletion of the RFB inhibits chromosome breakage in a sub-population of developing macronuclei and promotes alternative processing by a Cbs-independent mechanism. Remarkably, the RFB region prevents spontaneous breakage of chromosome 1 in the diploid micronucleus. Strains heterozygous for ΔRFB and wild-type rDNA lose the ΔRFB allele and distal left arm of chromosome 1 during vegetative propagation. The wild-type chromosome is subsequently fragmented near the rDNA locus, and both homologs are progressively eroded, suggesting that broken micronuclear chromosomes are not ‘healed’ by telomerase. Deletion of this 363 bp segment effectively creates a fragile site in the micronuclear genome, providing the first evidence for a non-telomere cis-acting determinant that functions to maintain the structural integrity of a mitotic eukaryotic chromosome.