Nicholas A. Stover

The Oxytricha trifallax Macronuclear Genome: A Complex Eukaryotic Genome with 16,000 Tiny Chromosomes

With more chromosomes than any other sequenced genome, the macronuclear genome of Oxytricha trifallax has a unique and complex architecture, including alternative fragmentation and predominantly single-gene chromosomes.

Tetrahymena Genome Database (TGD): a new genomic resource for Tetrahymena thermophila research

We have developed a web-based resource (available at ) for researchers studying the model ciliate organism Tetrahymena thermophila. Employing the underlying database structure and programming of the Saccharomyces Genome Database, the Tetrahymena Genome Database (TGD) integrates the wealth of knowledge generated by the Tetrahymena research community about genome structure, genes and gene products with the newly sequenced macronuclear genome determined by The Institute for Genomic Research (TIGR). TGD provides information curated from the literature about each published gene, including a standardized gene name, a link to the genomic locus in our graphical genome browser, gene product annotations utilizing the Gene Ontology, links to published literature about the gene and more. TGD also displays automatic annotations generated for the gene models predicted by TIGR. A variety of tools are available at TGD for searching the Tetrahymena genome, its literature and information about members of the research community.

The Draft Assembly of the Radically Organized Stylonychia lemnae Macronuclear Genome

Stylonychia lemnae is a classical model single-celled eukaryote, and a quintessential ciliate typified by dimorphic nuclei: A small, germline micronucleus and a massive, vegetative macronucleus. The genome within Stylonychia’s macronucleus has a very unusual architecture, comprised variably and highly amplified “nanochromosomes,” each usually encoding a single gene with a minimal amount of surrounding noncoding DNA. As only a tiny fraction of the Stylonychia genes has been sequenced, and to promote research using this organism, we sequenced its macronuclear genome. We report the analysis of the 50.2-Mb draft S. lemnae macronuclear genome assembly, containing in excess of 16,000 complete nanochromosomes, assembled as less than 20,000 contigs. We found considerable conservation of fundamental genomic properties between S. lemnae and its close relative, Oxytricha trifallax, including nanochromosomal gene synteny, alternative fragmentation, and copy number. Protein domain searches in Stylonychia revealed two new telomere-binding protein homologs and the presence of linker histones. Among the diverse histone variants of S. lemnae and O. trifallax, we found divergent, coexpressed variants corresponding to four of the five core nucleosomal proteins (H1.2, H2A.6, H2B.4, and H3.7) suggesting that these ciliates may possess specialized nucleosomes involved in genome processing during nuclear differentiation. The assembly of the S. lemnae macronuclear genome demonstrates that largely complete, well-assembled highly fragmented genomes of similar size and complexity may be produced from one library and lane of Illumina HiSeq 2000 shotgun sequencing. The provision of the S. lemnae macronuclear genome sets the stage for future detailed experimental studies of chromatin-mediated, RNA-guided developmental genome rearrangements.

Whole Genome Studies of Tetrahymena

Within the past decade, genomic studies have emerged as essential and highly productive tools to explore the biology of Tetrahymena thermophila. The current major resources, which have been extensively mined by the research community, are the annotated macronuclear genome assembly, transcriptomic data and the databases that house this information. Efforts in progress will soon improve these data sources and expand their scope, including providing annotated micronuclear and comparative genomic sequences. Future studies of Tetrahymena cell and molecular biology, development, physiology, evolution and ecology will benefit greatly from these resources and the advanced genomic technologies they enable.

Multiple Independent Fusions of Glucose-6-Phosphate Dehydrogenase with Enzymes in the Pentose Phosphate Pathway

Fusions of the first two enzymes in the pentose phosphate pathway, glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconolactonase (6PGL), have been previously described in two distant clades, chordates and species of the malarial parasite Plasmodium. We have analyzed genome and expressed sequence data from a variety of organisms to identify the origins of these gene fusion events. Based on the orientation of the domains and range of species in which homologs can be found, the fusions appear to have occurred independently, near the base of the metazoan and apicomplexan lineages. Only one of the two metazoan paralogs of G6PD is fused, showing that the fusion occurred after a duplication event, which we have traced back to an ancestor of choanoflagellates and metazoans. The Plasmodium genes are known to contain a functionally important insertion that is not seen in the other apicomplexan fusions, highlighting this as a unique characteristic of this group. Surprisingly, our search revealed two additional fusion events, one that combined 6PGL and G6PD in an ancestor of the protozoan parasites Trichomonas and Giardia, and another fusing G6PD with phosphogluconate dehydrogenase (6PGD) in a species of diatoms. This study extends the range of species known to contain fusions in the pentose phosphate pathway to many new medically and economically important organisms.

Detection of Fused Genes in Eukaryotic Genomes using Gene deFuser: Analysis of the Tetrahymena thermophila genome

BackgroundFused genes are important sources of data for studies of evolution and protein function. To date no service has been made available online to aid in the large-scale identification of fused genes in sequenced genomes. We have developed a program, Gene deFuser, that analyzes uploaded protein sequence files for characteristics of gene fusion events and presents the results in a convenient web interface.ResultsTo test the ability of this software to detect fusions on a genome-wide scale, we analyzed the 24,725 gene models predicted for the ciliated protozoan Tetrahymena thermophila. Gene deFuser detected members of eight of the nine families of gene fusions known or predicted in this species and identified nineteen new families of fused genes, each containing between one and twelve members. In addition to these genuine fusions, Gene deFuser also detected a particular type of gene misannotation, in which two independent genes were predicted as a single transcript by gene annotation tools. Twenty-nine of the artifacts detected by Gene deFuser in the initial annotation have been corrected in subsequent versions, with a total of 25 annotation artifacts (about 1/3 of the total fusions identified) remaining in the most recent annotation.ConclusionsThe newly identified Tetrahymena fusions belong to classes of genes involved in processes such as phospholipid synthesis, nuclear export, and surface antigen generation. These results highlight the potential of Gene deFuser to reveal a large number of novel fused genes in evolutionarily isolated organisms. Gene deFuser may also prove useful as an ancillary tool for detecting fusion artifacts during gene model annotation.

Spliced leader trans-splicing

What is spliced leader (SL) trans-splicing? It is an mRNA maturation process, similar to intron splicing, which has been shown to occur in a limited number of eukaryotes. In SL trans-splicing, the cell replaces nucleotides at the 5′ end of some pre-mRNAs with those of a special class of small nuclear RNAs, called SL RNAs. These are short molecules with two functionally distinct halves: the 5′ half consists of the leader sequence that is transferred to a pre-mRNA, along with the SL RNAs methylguanosine cap; the 3′ half contains a binding site for the Sm protein complex, which binds many of the RNAs involved in intron splicing. These two halves are separated by a splice donor site, a GT dinucleotide. Nuclear machinery trans-splices the leader sequence to splice acceptor sites (AG dinucleotides) in the 5′ region of target pre-mRNAs. As a result, many mRNAs in SL trans-splicing species have a common sequence at the 5′ end.How is SL trans-splicing related to intron splicing? The mechanism of SL trans-splicing is very similar to cis- (intron) splicing (Figure 1Figure 1). In both cases, the 2′ hydroxyl group of a nucleotide (usually adenosine) severs the pre-mRNA backbone at the splice donor site, freeing the upstream exon to displace the intron sequence at the splice acceptor. In intron splicing, the splice donor and acceptor sites lie on the same strand of RNA, separated by the intron sequence, which contains the branch point adenosine. For SL trans-splicing, the splice donor site of the SL RNA is attacked by an adenosine between the 5′ end of the pre-mRNA and its SL addition site. The region of the pre-mRNA upstream of the SL addition site, which is removed when the leader sequence is attached, is known as the ‘outron’ — it is ‘outside’ the gene, whereas introns are ‘inside’.Figure 1The leader sequence (yellow box) of an SL RNA is attached to the first exon (orange box) of a pre-mRNA by a trans-splicing reaction.The outron of the pre-mRNA and the intron-like portion of the SL RNA form a ‘Y’ branched byproduct, similar to the lariat structure formed during intron splicing. The 2,2,7-trimethylguanosine cap structure (or ‘Cap 4’ in trypanosomes) found on SL RNAs and trans-spliced mRNAs (blue circles) differs from the 7-methylguanosine caps typically found on mRNAs (red circles). These caps impart novel properties to trans-spliced messages.View Large Image | View Hi-Res Image | Download PowerPoint SlideStrikingly, experiments in the nematode Caenorhabditis elegans demonstrated that the relative location of the donor and acceptor sites is sufficient to determine whether a splice acceptor is cis- or trans-spliced. Cis-spliced acceptors can be converted to trans-splicing acceptors if the donor site upstream is mutated, and similarly an SL addition site will be cis-spliced if a donor site is inserted upstream.What is the function of SL trans-splicing? SL addition provides the cell with an alternative way of capping mRNAs, a modification required for mRNA stability, transport and translation. The standard capping machinery is typically recruited by RNA polymerase II at the beginning of transcription to cap the growing RNA. In trypanosomes, SL addition is used to cap a subset of pre-mRNAs transcribed by RNA polymerase I, which does not recruit the capping machinery. In a wider range of eukaryotes, SL addition allows the formation of operons — adjacent genes that are transcribed as a single primary transcript. The cleavage reaction that precedes polyadenylation of each gene effectively chops the transcript into smaller pieces. Since only the message from the 5′ end retains the transcripts original cap, SL trans-splicing is needed to cap the remaining fragments.Addition of an SL has also been shown to affect the translation rate of some genes, add missing start codons and trim off outron sequences. But while the role of SL addition in these processes is well established, the benefit of transcription by polymerase I, inclusion in an operon, translational regulation, substitution of a start codon and outron removal is not immediately obvious for most trans-spliced genes.How is it phylogenetically distributed? The complete phylogenetic distribution of SL trans-splicing is not currently known. To date, SL trans-splicing has been found in six diverse groups of eukaryotes: nematodes, flatworms, cnidarians, ascidians, rotifers and euglenozoans. But it has not been detected in other well-studied eukaryotic taxa, such as fungi, plants, vertebrates and arthropods.How did it evolve? There are two competing hypotheses describing the origin of SL trans-splicing. The ‘SL trans-splicing early’ hypothesis proposes that SL trans-splicing was present in the ancestral eukaryote and subsequently lost in most phyla. This hypothesis is supported in part by the continuing discovery of SL trans-splicing in an expanding range of eukaryotes. The ‘SL trans-splicing late’ hypothesis proposes that the process has emerged several times independently, and that features unique to SL addition — the SL RNA, trans-spliceosome-specific proteins, SL-specific translation enhancer proteins, operons, and so on — are also independently derived in these lineages. This hypothesis is supported by the observation that the few unique components shown to be involved in SL addition are not obviously conserved across different trans-splicing phyla. Further studies into the mechanism and effects of SL trans-splicing in different phyla will help to clarify its origin.Why should I care? Many of the organisms that perform SL trans-splicing, such as trypanosomes, flatworms and nematodes are pathogenic to humans. Drugs that target components of the trans-spliceosome or disrupt the downstream effects of SL trans-splicing may be highly effective against these parasitic species, while causing little harm to patients.

Genome Evolution: A Double Take for Paramecium

The surprising discovery of a whole-genome duplication in the otherwise compact genome of Paramecium tetraurelia displays the early forces driving gene retention and loss.

On the paucity of duplicated genes in Caenorhabditis elegans operons.

Spliced leader trans-splicing is an mRNA maturation process used by a small set of eukaryotes, including the nematode C. elegans, to cap the downstream genes of operons. We analyzed the frequency of duplication of operonic genes in C. elegans and confirmed that they are duplicated less often in the genome than monocistronic genes. Because operons account for about 15% of the genes in C. elegans, this lower duplication frequency might place a large constraint on the plasticity of the genome. Further analyses suggest that this paucity of duplicated genes results from operon organization hindering specific types of gene duplication.

Immediate Dissemination of Student Discoveries to a Model Organism Database Enhances Classroom-Based Research Experiences

A model for integrating course-based research with community genome annotation efforts at model organism databases is presented. Disseminating gene function discoveries directly to an interested audience increased student motivation to more deeply engage all aspects of an authentic research experience.