What transposable elements tell us about genome organization and evolution: the case of Drosophila

Transposable elements (TEs) have been identified in every organism in which they have been looked for. The sequencing of large genomes, such as the human genome and those of Drosophila, Arabidopsis, Caenorhabditis, has also shown that they are a major constituent of these genomes, accounting for 15% of the genome of Drosophila, 45% of the human genome, and more than 70% in some plants and amphibians. Compared with the 1% of genomic DNA dedicated to protein-coding sequences in the human genome, this has prompted various researchers to suggest that the TEs and the other repetitive sequences that constitute the so-called "noncoding DNA", are where the most stimulating discoveries will be made in the future (Bromham, 2002). We are therefore getting further and further from the original idea that this DNA was simply "junk DNA", that owed its presence in the genome entirely to its capacity for selfish transposition. Our understanding of the structures of TEs, their distribution along the genomes, their sequence and insertion polymorphisms within genomes, and within and between populations and species, their impact on genes and on the regulatory mechanisms of genetic expression, their effects on exon shuffling and other phenomena that reshape the genome, and their impact on genome size has increased dramatically in recent years. This leads to a more general picture of the impact of TEs on genomes, though many copies are still mainly selfish or junk DNA. In this review we focus mainly on discoveries made in Drosophila, but we also use information about other genomes when this helps to elucidate the general processes involved in the organization, plasticity, and evolution of genomes.     

Zebrafish as a genomics model for human neurological and polygenic disorders

Whole exome sequencing and, to a lesser extent, genome-wide association studies, have provided unprecedented advances in identifying genes and candidate genomic regions involved in the development of human disease. Further progress will come from sequencing the entire genome of multiple patients and normal controls to evaluate overall mutational burden and disease risk. A major challenge will be the interpretation of the resulting data and distinguishing true pathogenic mutations from rare benign variants.While in model organisms such as the zebrafish,mutants are sought that disrupt the function of individual genes, human mutations that cause, or are associated with, the development of disease, are often not acting in a Mendelian fashion, are frequently of small effect size, are late onset, and may reside in noncoding parts of the genome. The zebrafish model is uniquely poised for understanding human coding- and noncoding variants because of its sequenced genome, a large body of knowledge on gene expression and function, rapid generation time, and easy access to embryos. A critical advantage is the ease of zebrafish transgenesis, both for the testing of human regulatory DNA driving expression of fluorescent reporter proteins, and the expression of mutated disease-associated human proteins in specific neurons to rapidly model aspects of neurological disorders. The zebrafish affords progress both through its model genome and it is rapidly developing transparent model vertebrate embryo.     

Caenorhabditis elegans functional genomics: omic resonance.

The nematode Caenorhabditis elegans is widely used as a model organism for studying many fundamental aspects of development and cell biology, including processes underlying human disease. The genome of C. elegans encodes over 19,000 protein-coding genes and hundreds of non-coding RNAs. The availability of whole genome sequence has facilitated the development of high throughput techniques for elucidating the function of individual genes and gene products. Furthermore, attempts can now be made to integrate these substantial functional genomics data collections and to understand at a global level how the flow of genomic information that is at the core of the central dogma leads to the development of a multicellular organism.     

DNA sequence alterations affect nucleosome array formation of the chicken ovalbumin gene

The role of the large amount (more than half of the genome) of noncoding DNA in higher organisms is not well understood. DNA evolved to function in the context of chromatin, and the possibility exists that some of the noncoding DNA serves to influence chromatin structure and function. In this age of genomics and bioinformatics, genomic DNA sequences are being searched for informational content beyond the known genetic code. The discovery that period-10 non-T, A/T, G (VWG) triplets are among the most abundant motifs in human genomic DNA suggests that they may serve some function in higher organisms. In this paper, we provide direct evidence that the regular oscillation of period-10 VWG that occurs in the chicken ovalbumin gene sequence with a dinucleosome-like period facilitates nucleosome array formation. Using a linker histone-dependent in vitro chromatin assembly system that spontaneously aligns nucleosomes into a physiological array, we show that nucleosomes tend to avoid DNA regions with low period-10 VWG counts. This avoidance leads to the formation of an array with a nucleosome repeat equal to half the period value of the oscillation in period-10 VWG, as determined by Fourier analysis. Two different half-period deletions in the wild-type DNA sequence altered the nucleosome array, as predicted computationally. In contrast, a full-period deletion had an insignificant effect on the nucleosome array formed, also consistent with the prediction. An inversion mutation, with no DNA sequences deleted, again altered the nucleosome array formed, as predicted computationally. Hence, a VWG dinucleosome signal is plausible.     

How can we deliver the large plant genomes? Strategies and perspectives

The first sequenced plant genome, from the small mustard plant Arabidopsis thaliana, was published at the end of 2000. The sequencing of the rice genome is well under way. The sizes of plant genomes vary by a factor of up to 1000, and many important crop plants have genomes that are several times larger than the human genome. To gain insight into the gene toolbox of plant species, numerous large-scale EST sequencing projects have been launched successfully, and analysis procedures are constantly being refined to add maximum value to the sequence data. In addition, an alternative approach to exclude repetitive noncoding DNA and to enrich sequence libraries for gene-containing genomic regions has been developed. This strategy has the potential to deliver information about both genes and regulatory regions outside the transcribed regions.     

Genomic organization of selected genes in the small monogonont rotifer, Brachionus koreanus

Information of genome structure with its size variation may provide important clues for evolutionary processes at lower taxon level in eukaryotes. Here, we analyzed the compact genome structure of the monogonont rotifer, Brachionus koreanus in the light of transphyletic genome comparison and economic genome usage. To confirm the genome compactness of B. koreanus, we compared the genomic structure of several selected genes with those of human and pufferfish. For example, one of the large genes, DNA-dependent protein kinase (DNA-PK) with dimeric protein Ku70 and Ku80, showed high similarity, even though genomic DNA lengths were quite different. The replication protein As (RPAs) as a heterotrimeric protein also showed a compact genomic structure including all the essential domains and motifs in B. koreanus. Regarding transmembrane protein-containing genes, the B. koreanus P-glycoprotein (P-gp) showed exactly the same topology of the TM domain compared to those of human and pufferfish, even though it had a compact genome structure. In addition, the gene structure of an inducible repair enzyme O(6)-methylguanine DNA methyltransferase (O(6)-MGMT) of B. koreanus showed the highest compactness among the genes tested. The objective of this report is to evaluate the potential for whole genome sequencing and functional genomic research using the monogonont rotifer B. koreanus as a non-model organism that plays important roles in aquatic food-webs. Subsequently, we discussed possible reasons for compact genome structures as well as small and fewer introns from several perspectives. We conclude that the small size genome of B. koreanus would make this species potentially useful for comparative genome structure analysis of non-model species through whole genome sequencing and genetic mapping.     

Methods for integration site distribution analyses in animal cell genomes

The question of where retroviral DNA becomes integrated in chromosomes is important for understanding (i) the mechanisms of viral growth, (ii) devising new anti-retroviral therapy, (iii) understanding how genomes evolve, and (iv) developing safer methods for gene therapy. With the completion of genome sequences for many organisms, it has become possible to study integration targeting by cloning and sequencing large numbers of host–virus DNA junctions, then mapping the host DNA segments back onto the genomic sequence. This allows statistical analysis of the distribution of integration sites relative to the myriad types of genomic features that are also being mapped onto the sequence scaffold. Here we present methods for recovering and analyzing integration site sequences.     

Macronuclear Genome Sequence of the Ciliate Tetrahymena thermophila, a Model Eukaryote

The ciliate Tetrahymena thermophila is a model organism for molecular and cellular biology. Like other ciliates, this species has separate germline and soma functions that are embodied by distinct nuclei within a single cell. The germline-like micronucleus (MIC) has its genome held in reserve for sexual reproduction. The soma-like macronucleus (MAC), which possesses a genome processed from that of the MIC, is the center of gene expression and does not directly contribute DNA to sexual progeny. We report here the shotgun sequencing, assembly, and analysis of the MAC genome of T. thermophila, which is approximately 104 Mb in length and composed of approximately 225 chromosomes. Overall, the gene set is robust, with more than 27,000 predicted protein-coding genes, 15,000 of which have strong matches to genes in other organisms. The functional diversity encoded by these genes is substantial and reflects the complexity of processes required for a free-living, predatory, single-celled organism. This is highlighted by the abundance of lineage-specific duplications of genes with predicted roles in sensing and responding to environmental conditions (e.g., kinases), using diverse resources (e.g., proteases and transporters), and generating structural complexity (e.g., kinesins and dyneins). In contrast to the other lineages of alveolates (apicomplexans and dinoflagellates), no compelling evidence could be found for plastid-derived genes in the genome. UGA, the only T. thermophila stop codon, is used in some genes to encode selenocysteine, thus making this organism the first known with the potential to translate all 64 codons in nuclear genes into amino acids. We present genomic evidence supporting the hypothesis that the excision of DNA from the MIC to generate the MAC specifically targets foreign DNA as a form of genome self-defense. The combination of the genome sequence, the functional diversity encoded therein, and the presence of some pathways missing from other model organisms makes T. thermophila an ideal model for functional genomic studies to address biological, biomedical, and biotechnological questions of fundamental importance.     

Macronuclear genome sequence of the ciliate Tetrahymena thermophila, a model eukaryote

The ciliate Tetrahymena thermophila is a model organism for molecular and cellular biology. Like other ciliates, this species has separate germline and soma functions that are embodied by distinct nuclei within a single cell. The germline-like micronucleus (MIC) has its genome held in reserve for sexual reproduction. The soma-like macronucleus (MAC), which possesses a genome processed from that of the MIC, is the center of gene expression and does not directly contribute DNA to sexual progeny. We report here the shotgun sequencing, assembly, and analysis of the MAC genome of T. thermophila, which is approximately 104 Mb in length and composed of approximately 225 chromosomes. Overall, the gene set is robust, with more than 27,000 predicted protein-coding genes, 15,000 of which have strong matches to genes in other organisms. The functional diversity encoded by these genes is substantial and reflects the complexity of processes required for a free-living, predatory, single-celled organism. This is highlighted by the abundance of lineage-specific duplications of genes with predicted roles in sensing and responding to environmental conditions (e.g., kinases), using diverse resources (e.g., proteases and transporters), and generating structural complexity (e.g., kinesins and dyneins). In contrast to the other lineages of alveolates (apicomplexans and dinoflagellates), no compelling evidence could be found for plastid-derived genes in the genome. UGA, the only T. thermophila stop codon, is used in some genes to encode selenocysteine, thus making this organism the first known with the potential to translate all 64 codons in nuclear genes into amino acids. We present genomic evidence supporting the hypothesis that the excision of DNA from the MIC to generate the MAC specifically targets foreign DNA as a form of genome self-defense. The combination of the genome sequence, the functional diversity encoded therein, and the presence of some pathways missing from other model organisms makes T. thermophila an ideal model for functional genomic studies to address biological, biomedical, and biotechnological questions of fundamental importance.     

Chromatin structure analysis enables detection of DNA insertions into the mammalian nuclear genome

BackgroundGenetically modified organisms (GMOs) have numerous biomedical, agricultural and environmental applications. Development of accurate methods for the detection of GMOs is a prerequisite for the identification and control of authorized and unauthorized release of these engineered organisms into the environment and into the food chain. Current detection methods are unable to detect uncharacterized GMOs, since either the DNA sequence of the transgene or the amino acid sequence of the protein must be known for DNA-based or immunological-based detection, respectively.MethodsHere we describe the application of an epigenetics-based approach for the detection of mammalian GMOs via analysis of chromatin structural changes occurring in the host nucleus upon the insertion of foreign or endogenous DNA.ResultsImmunological methods combined with DNA next generation sequencing enabled direct interrogation of chromatin structure and identification of insertions of various size foreign (human or viral) DNA sequences, DNA sequences often used as genome modification tools (e.g. viral sequences, transposon elements), or endogenous DNA sequences into the nuclear genome of a model animal organism.ConclusionsThe results provide a proof-of-concept that epigenetic approaches can be used to detect the insertion of endogenous and exogenous sequences into the genome of higher organisms where the method of genetic modification, the sequence of inserted DNA, and the exact genomic insertion site(s) are unknown.General significanceMeasurement of chromatin dynamics as a sensor for detection of genomic manipulation and, more broadly, organism exposure to environmental or other factors affecting the epigenomic landscape are discussed.     

Small‐ and large‐scale heterogeneity in genetic variation across the collard flycatcher genome: implications for estimating genetic diversity in nonmodel organisms

Population genetic studies in nonmodel organisms are often hampered by a lack of reference genomes that are essential for whole‐genome resequencing. In the light of this, genotyping methods have been developed to effectively eliminate the need for a reference genome, such as genotyping by sequencing or restriction site‐associated DNA sequencing (RAD‐seq). However, what remains relatively poorly studied is how accurately these methods capture both average and variation in genetic diversity across an organism's genome. In this issue of Molecular Ecology Resources, Dutoit et al. (2016) use whole‐genome resequencing data from the collard flycatcher to assess what factors drive heterogeneity in nucleotide diversity across the genome. Using these data, they then simulate how well different sequencing designs, including RAD sequencing, could capture most of the variation in genetic diversity. They conclude that for evolutionary and conservation‐related studies focused on the estimating genomic diversity, researchers should emphasize the number of loci analysed over the number of individuals sequenced.     

Unfolding large-scale maps.

This is an account of the development and use of genetic maps, from humble beginnings at the hands of Thomas Hunt Morgan, to the sophistication of genome sequencing. The review charters the emergence of molecular marker maps exploiting DNA polymorphism, the renaissance of cytogenetics through the use of fluorescence in situ hybridisation, and the discovery and isolation of genes by map-based cloning. The historical significance of sequencing of DNA prefaces a section describing the sequencing of genomes, the ascendancy of particular model organisms, and the utility and limitations of comparative genomic and functional genomic approaches to further our understanding of the control of biological processes. Emphasis is given throughout the treatise as to how the structure and biological behaviour of the DNA molecule underpin the technological development and biological applications of maps.     

A case for evolutionary genomics and the comprehensive examination of sequence biodiversity

Comparative analysis is one of the most powerful methods available for understanding the diverse and complex systems found in biology, but it is often limited by a lack of comprehensive taxonomic sampling. Despite the recent development of powerful genome technologies capable of producing sequence data in large quantities (witness the recently completed first draft of the human genome), there has been relatively little change in how evolutionary studies are conducted. The application of genomic methods to evolutionary biology is a challenge, in part because gene segments from different organisms are manipulated separately, requiring individual purification, cloning, and sequencing. We suggest that a feasible approach to collecting genome-scale data sets for evolutionary biology (i.e., evolutionary genomics) may consist of combination of DNA samples prior to cloning and sequencing, followed by computational reconstruction of the original sequences. This approach will allow the full benefit of automated protocols developed by genome projects to be realized; taxon sampling levels can easily increase to thousands for targeted genomes and genomic regions. Sequence diversity at this level will dramatically improve the quality and accuracy of phylogenetic inference, as well as the accuracy and resolution of comparative evolutionary studies. In particular, it will be possible to make accurate estimates of normal evolution in the context of constant structural and functional constraints (i.e., site-specific substitution probabilities), along with accurate estimates of changes in evolutionary patterns, including pairwise coevolution between sites, adaptive bursts, and changes in selective constraints. These estimates can then be used to understand and predict the effects of protein structure and function on sequence evolution and to predict unknown details of protein structure, function, and functional divergence. In order to demonstrate the practicality of these ideas and the potential benefit for functional genomic analysis, we describe a pilot project we are conducting to simultaneously sequence large numbers of vertebrate mitochondrial genomes.     

Bacillus species proteins involved in spore formation and degradation: from identification in the genome,to sequence analysis,and determination of function and structure

The members of Bacillus species are Gram-positive, ubiquitous spore-forming bacilli. Several genomic sequences have been made available during recent years, including Bacillus subtilis, a model organism among this genus, Bacillus anthracis, and their analyses provided a wealth of information about spore-forming bacteria. Some members of this species can cause serious diseases in livestock and humans. An important pathogen in this group of organisms is B. anthracis, which is the causative agent of anthrax. A summary of the B. subtilis genome information, based on the publicly released sequence, that allowed for the identification and characterization of new and novel proteins of this organism as well as similar proteins from other members of Bacillus species is provided. The primary goal for this work is to present a review of the genome sequence-identified genes that encode proteins involved in the sporulation, germination, and outgrowth processes. These three processes are essential for spore development and later its transformation into a vegetative cell. Additionally, for a few selected examples of the protein products of the identified genes, the application of bioinformatics and modeling tools is illustrated in order to determine their likely structure and function. Two three-dimensional models of the structures of such proteins, PrfA endonuclease and phosphatase PhoE, are presented together with the structure-based functional conclusions. The review of such studies provides an example of how the genomic sequence can be utilized in order to elucidate the structure and function of proteins, in particular proteins of the Bacillus species. Because only a limited number of proteins of Bacillus species organisms are involved in the synthesis and degradation of spores and have been characterized to date, this genome-based analysis may provide new insights into the developmental processes of bacterial organism.     

Status of genome projects for nonpathogenic bacteria and archaea

Since the first microbial genome was sequenced in 1995, 30 others have been completed and an additional 99 are known to be in progress. Although the early emphasis of microbial genomics was on human pathogens for obvious reasons, a significant number of sequencing projects have focused on nonpathogenic organisms, beginning with the release of the complete genome sequence of the archaeon Methanococcus jannaschii in 1996. The past 18 months have seen the completion of the genomes of several unusual organisms, including Thermotoga maritima, whose genome reveals extensive potential lateral transfer with archaea; Deinococcus radiodurans, the most radiation-resistant microorganism known; and Aeropyrum pernix, the first Crenarchaeota to be completely sequenced. Although the functional characterization of genomic data is still in its initial stages, it is likely that microbial genomics will have a significant impact on environmental, food, and industrial biotechnology as well as on genomic medicine.     

Bacillus Species Proteins Involved in Spore Formation and Degradation: From Identification in the Genome,to Sequence Analysis,and Determination of Function and Structure

The members of Bacillus species are Gram-positive, ubiquitous spore-forming bacilli. Several genomic sequences have been made available during recent years, including Bacillus subtilis, a model organism among this genus, Bacillus anthracis, and their analyses provided a wealth of information about spore-forming bacteria. Some members of this species can cause serious diseases in livestock and humans. An important pathogen in this group of organisms is B. anthracis, which is the causative agent of anthrax. A summary of the B. subtilis genome information, based on the publicly released sequence, that allowed for the identification and characterization of new and novel proteins of this organism as well as similar proteins from other members of Bacillus species is provided. The primary goal for this work is to present a review of the genome sequence-identified genes that encode proteins involved in the sporulation, germination, and outgrowth processes. These three processes are essential for spore development and later its transformation into a vegetative cell. Additionally, for a few selected examples of the protein products of the identified genes, the application of bioinformatics and modeling tools is illustrated in order to determine their likely structure and function. Two three-dimensional models of the structures of such proteins, PrfA endonuclease and phosphatase PhoE, are presented together with the structure-based functional conclusions. The review of such studies provides an example of how the genomic sequence can be utilized in order to elucidate the structure and function of proteins, in particular proteins of the Bacillus species. Because only a limited number of proteins of Bacillus species organisms are involved in the synthesis and degradation of spores and have been characterized to date, this genome-based analysis may provide new insights into the developmental processes of bacterial organism.