The tuatara genome reveals ancient features of amniote evolution.

The tuatara (Sphenodon punctatus)-the only living member of the reptilian order Rhynchocephalia (Sphenodontia), once widespread across Gondwana1,2-is an iconic species that is endemic to New Zealand2,3. A key link to the now-extinct stem reptiles (from which dinosaurs, modern reptiles, birds and mammals evolved), the tuatara provides key insights into the ancestral amniotes2,4. Here we analyse the genome of the tuatara, which-at approximately 5 Gb-is among the largest of the vertebrate genomes yet assembled. Our analyses of this genome, along with comparisons with other vertebrate genomes, reinforce the uniqueness of the tuatara. Phylogenetic analyses indicate that the tuatara lineage diverged from that of snakes and lizards around 250 million years ago. This lineage also shows moderate rates of molecular evolution, with instances of punctuated evolution. Our genome sequence analysis identifies expansions of proteins, non-protein-coding RNA families and repeat elements, the latter of which show an amalgam of reptilian and mammalian features. The sequencing of the tuatara genome provides a valuable resource for deep comparative analyses of tetrapods, as well as for tuatara biology and conservation. Our study also provides important insights into both the technical challenges and the cultural obligations that are associated with genome sequencing.

Publisher Correction: The tuatara genome reveals ancient features of amniote evolution.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The effect of compound kushen injection on cancer cells: Integrated identification of candidate molecular mechanisms.

Traditional Chinese Medicine (TCM) preparations are often extracts of single or multiple herbs containing hundreds of compounds, and hence it has been difficult to study their mechanisms of action. Compound Kushen Injection (CKI) is a complex mixture of compounds extracted from two medicinal plants and has been used in Chinese hospitals to treat cancer for over twenty years. To demonstrate that a systematic analysis of molecular changes resulting from complex mixtures of bioactives from TCM can identify a core set of differentially expressed (DE) genes and a reproducible set of candidate pathways. We used in vitro cancer models to measure the effect of CKI on cell cycle phases and apoptosis, and correlated those phenotypes with CKI induced changes in gene expression. We treated two cancer cell lines with or without CKI and assessed the resulting phenotypes by employing cell viability and proliferation assays. Based on these results, we carried out high-throughput transcriptome data analysis to identify genes and candidate pathways perturbed by CKI. We integrated these differential gene expression results with previously reported results and carried out validation of selected differentially expressed genes. CKI induced cell-cycle arrest and apoptosis in the cancer cell lines tested. In these cells CKI also altered the expression of 363 core candidate genes associated with cell cycle, apoptosis, DNA replication/repair, and various cancer pathways. Of these, 7 are clinically relevant to cancer diagnosis or therapy, 14 are cell cycle regulators, and most of these 21 candidates are downregulated by CKI. Comparison of our core candidate genes to a database of plant medicinal compounds and their effects on gene expression identified one-to-one, one-to-many and many-to-many regulatory relationships between compounds in CKI and DE genes. By identifying genes and promising candidate pathways associated with CKI treatment based on our transcriptome-based analysis, we have shown that this approach is useful for the systematic analysis of molecular changes resulting from complex mixtures of bioactives.

A New Strategy for Identifying Mechanisms of Drug-drug Interaction Using Transcriptome Analysis: Compound Kushen Injection as a Proof of Principle.

Drug-drug interactions (DDIs), especially with herbal medicines, are complex, making it difficult to identify potential molecular mechanisms and targets. We introduce a workflow to carry out DDI research using transcriptome analysis and interactions of a complex herbal mixture, Compound Kushen Injection (CKI), with cancer chemotherapy drugs, as a proof of principle. Using CKI combined with doxorubicin or 5-Fu on cancer cells as a model, we found that CKI enhanced the cytotoxic effects of doxorubicin on A431 cells while protecting MDA-MB-231 cells treated with 5-Fu. We generated and analysed transcriptome data from cells treated with single treatments or combined treatments and our analysis showed that opposite directions of regulation for pathways related to DNA synthesis and metabolism which appeared to be the main reason for different effects of CKI when used in combination with chemotherapy drugs. We also found that pathways related to organic biosynthetic and metabolic processes might be potential targets for CKI when interacting with doxorubicin and 5-Fu. Through co-expression analysis correlated with phenotype results, we selected the MYD88 gene as a candidate major regulator for validation as a proof of concept for our approach. Inhibition of MYD88 reduced antagonistic cytotoxic effects between CKI and 5-Fu, indicating that MYD88 is an important gene in the DDI mechanism between CKI and chemotherapy drugs. These findings demonstrate that our pipeline is effective for the application of transcriptome analysis to the study of DDIs in order to identify candidate mechanisms and potential targets.

Understanding the Mechanistic Contribution of Herbal Extracts in Compound Kushen Injection With Transcriptome Analysis.

Herbal compatibility is the knowledge of which herbs to combine in traditional Chinese medicine (TCM) formulations. The lack of understanding of herbal compatibility is one of the key problems for the application and popularization of TCM in western society. Because of the chemical complexity of herbal medicines, it is simpler to begin to conduct compatibility research based on herbs rather than component plant secondary metabolites. We have used transcriptome analysis to explore the effects and interactions of two plant extracts (Kushen and Baituling) combined in Compound Kushen Injection (CKI). Based on shared chemical compounds and in vitro cytotoxicity comparisons, we found that both the major compounds in CKI, and the cytotoxicity effects of CKI were mainly derived from the extract of Kushen (Sophorae flavescentis). We generated and analyzed transcriptome data from MDA-MB-231 cells treated with single-herb extracts or CKI and results showed that Kushen contributed to the perturbation of the majority of cytotoxicity/cancer related pathways in CKI such as cell cycle and DNA replication. We also found that Baituling (Heterosmilax yunnanensis Gagnep) could not only enhance the cytotoxic effects of Kushen in CKI, but also activate immune-related pathways. Our analyses predicted that IL-1ß gene expression was upregulated by Baituling in CKI and we confirmed that IL-1ß protein expression was increased using an ELISA assay. Altogether, these findings help to explain the rationale for combining Kushen and Baituling in CKI, and show that transcriptome analysis using single herb extracts is an effective method for understanding herbal compatibility in TCM.

Horizontal transfer of BovB and L1 retrotransposons in eukaryotes.

BACKGROUND: Transposable elements (TEs) are mobile DNA sequences, colloquially known as jumping genes because of their ability to replicate to new genomic locations. TEs can jump between organisms or species when given a vector of transfer, such as a tick or virus, in a process known as horizontal transfer. Here, we propose that LINE-1 (L1) and Bovine-B (BovB), the two most abundant TE families in mammals, were initially introduced as foreign DNA via ancient horizontal transfer events. RESULTS: Using analyses of 759 plant, fungal and animal genomes, we identify multiple possible L1 horizontal transfer events in eukaryotic species, primarily involving Tx-like L1s in marine eukaryotes. We also extend the BovB paradigm by increasing the number of estimated transfer events compared to previous studies, finding new parasite vectors of transfer such as bed bug, leech and locust, and BovB occurrences in new lineages such as bat and frog. Given that these transposable elements have colonised more than half of the genome sequence in today's mammals, our results support a role for horizontal transfer in causing long-term genomic change in new host organisms. CONCLUSIONS: We describe extensive horizontal transfer of BovB retrotransposons and provide the first evidence that L1 elements can also undergo horizontal transfer. With the advancement of genome sequencing technologies and bioinformatics tools, we anticipate our study to be a valuable resource for inferring horizontal transfer from large-scale genomic data.

Transposable elements and gene expression during the evolution of amniotes.

BACKGROUND: Transposable elements (TEs) are primarily responsible for the DNA losses and gains in genome sequences that occur over time within and between species. TEs themselves evolve, with clade specific LTR/ERV, LINEs and SINEs responsible for the bulk of species-specific genomic features. Because TEs can contain regulatory motifs, they can be exapted as regulators of gene expression. While TE insertions can provide evolutionary novelty for the regulation of gene expression, their overall impact on the evolution of gene expression is unclear. Previous investigators have shown that tissue specific gene expression in amniotes is more similar across species than within species, supporting the existence of conserved developmental gene regulation. In order to understand how species-specific TE insertions might affect the evolution/conservation of gene expression, we have looked at the association of gene expression in six tissues with TE insertions in six representative amniote genomes. RESULTS: A novel bootstrapping approach has been used to minimise the conflation of effects of repeat types on gene expression. We compared the expression of orthologs containing recent TE insertions to orthologs that contained older TE insertions, and the expression of non-orthologs containing recent TE insertions to non-orthologs with older TE insertions. Both orthologs and non-orthologs showed significant differences in gene expression associated with TE insertions. TEs were found associated with species-specific changes in gene expression, and the magnitude and direction of expression changes were noteworthy. Overall, orthologs containing species-specific TEs were associated with lower gene expression, while in non-orthologs, non-species specific TEs were associated with higher gene expression. Exceptions were SINE elements in human and chicken, which had an opposite association with gene expression compared to other species. CONCLUSIONS: Our observed species-specific associations of TEs with gene expression support a role for TEs in speciation/response to selection by species. TEs do not exhibit consistent associations with gene expression and observed associations can vary depending on the age of TE insertions. Based on these observations, it would be prudent to refrain from extrapolating these and previously reported associations to distantly related species.

Divergent genome evolution caused by regional variation in DNA gain and loss between human and mouse.

The forces driving the accumulation and removal of non-coding DNA and ultimately the evolution of genome size in complex organisms are intimately linked to genome structure and organisation. Our analysis provides a novel method for capturing the regional variation of lineage-specific DNA gain and loss events in their respective genomic contexts. To further understand this connection we used comparative genomics to identify genome-wide individual DNA gain and loss events in the human and mouse genomes. Focusing on the distribution of DNA gains and losses, relationships to important structural features and potential impact on biological processes, we found that in autosomes, DNA gains and losses both followed separate lineage-specific accumulation patterns. However, in both species chromosome X was particularly enriched for DNA gain, consistent with its high L1 retrotransposon content required for X inactivation. We found that DNA loss was associated with gene-rich open chromatin regions and DNA gain events with gene-poor closed chromatin regions. Additionally, we found that DNA loss events tended to be smaller than DNA gain events suggesting that they were able to accumulate in gene-rich open chromatin regions due to their reduced capacity to interrupt gene regulatory architecture. GO term enrichment showed that mouse loss hotspots were strongly enriched for terms related to developmental processes. However, these genes were also located in regions with a high density of conserved elements, suggesting that despite high levels of DNA loss, gene regulatory architecture remained conserved. This is consistent with a model in which DNA gain and loss results in turnover or "churning" in regulatory element dense regions of open chromatin, where interruption of regulatory elements is selected against.

Superior ab initio identification, annotation and characterisation of TEs and segmental duplications from genome assemblies.

Transposable Elements (TEs) are mobile DNA sequences that make up significant fractions of amniote genomes. However, they are difficult to detect and annotate ab initio because of their variable features, lengths and clade-specific variants. We have addressed this problem by refining and developing a Comprehensive ab initio Repeat Pipeline (CARP) to identify and cluster TEs and other repetitive sequences in genome assemblies. The pipeline begins with a pairwise alignment using krishna, a custom aligner. Single linkage clustering is then carried out to produce families of repetitive elements. Consensus sequences are then filtered for protein coding genes and then annotated using Repbase and a custom library of retrovirus and reverse transcriptase sequences. This process yields three types of family: fully annotated, partially annotated and unannotated. Fully annotated families reflect recently diverged/young known TEs present in Repbase. The remaining two types of families contain a mixture of novel TEs and segmental duplications. These can be resolved by aligning these consensus sequences back to the genome to assess copy number vs. length distribution. Our pipeline has three significant advantages compared to other methods for ab initio repeat identification: 1) we generate not only consensus sequences, but keep the genomic intervals for the original aligned sequences, allowing straightforward analysis of evolutionary dynamics, 2) consensus sequences represent low-divergence, recently/currently active TE families, 3) segmental duplications are annotated as a useful by-product. We have compared our ab initio repeat annotations for 7 genome assemblies to other methods and demonstrate that CARP compares favourably with RepeatModeler, the most widely used repeat annotation package.

A comprehensive genomic history of extinct and living elephants.

Elephantids are the world's most iconic megafaunal family, yet there is no comprehensive genomic assessment of their relationships. We report a total of 14 genomes, including 2 from the American mastodon, which is an extinct elephantid relative, and 12 spanning all three extant and three extinct elephantid species including an ∼120,000-y-old straight-tusked elephant, a Columbian mammoth, and woolly mammoths. Earlier genetic studies modeled elephantid evolution via simple bifurcating trees, but here we show that interspecies hybridization has been a recurrent feature of elephantid evolution. We found that the genetic makeup of the straight-tusked elephant, previously placed as a sister group to African forest elephants based on lower coverage data, in fact comprises three major components. Most of the straight-tusked elephant's ancestry derives from a lineage related to the ancestor of African elephants while its remaining ancestry consists of a large contribution from a lineage related to forest elephants and another related to mammoths. Columbian and woolly mammoths also showed evidence of interbreeding, likely following a latitudinal cline across North America. While hybridization events have shaped elephantid history in profound ways, isolation also appears to have played an important role. Our data reveal nearly complete isolation between the ancestors of the African forest and savanna elephants for ∼500,000 y, providing compelling justification for the conservation of forest and savanna elephants as separate species.

Similar Evolutionary Trajectories for Retrotransposon Accumulation in Mammals.

The factors guiding retrotransposon insertion site preference are not well understood. Different types of retrotransposons share common replication machinery and yet occupy distinct genomic domains. Autonomous long interspersed elements accumulate in gene-poor domains and their nonautonomous short interspersed elements accumulate in gene-rich domains. To determine genomic factors that contribute to this discrepancy we analyzed the distribution of retrotransposons within the framework of chromosomal domains and regulatory elements. Using comparative genomics, we identified large-scale conserved patterns of retrotransposon accumulation across several mammalian genomes. Importantly, retrotransposons that were active after our sample-species diverged accumulated in orthologous regions. This suggested a similar evolutionary interaction between retrotransposon activity and conserved genome architecture across our species. In addition, we found that retrotransposons accumulated at regulatory element boundaries in open chromatin, where accumulation of particular retrotransposon types depended on insertion size and local regulatory element density. From our results, we propose a model where density and distribution of genes and regulatory elements canalize retrotransposon accumulation. Through conservation of synteny, gene regulation and nuclear organization, mammalian genomes with dissimilar retrotransposons follow similar evolutionary trajectories.

Understanding the Effectiveness of Natural Compound Mixtures in Cancer through Their Molecular Mode of Action.

Many approaches to cancer management are often ineffective due to adverse reactions, drug resistance, or inadequate target specificity of single anti-cancer agents. In contrast, a combinatorial approach with the application of two or more anti-cancer agents at their respective effective dosages can achieve a synergistic effect that boosts cytotoxicity to cancer cells. In cancer, aberrant apoptotic pathways allow cells that should be killed to survive with genetic abnormalities, leading to cancer progression. Mutations in apoptotic mechanism arising during the treatment of cancer through cancer progression can consequently lead to chemoresistance. Natural compound mixtures that are believed to have multiple specific targets with minimal acceptable side-effects are now of interest to many researchers due to their cytotoxic and chemosensitizing activities. Synergistic interactions within a drug mixture enhance the search for potential molecular targets in cancer cells. Nonetheless, biased/flawed scientific evidence from natural products can suggest false positive therapeutic benefits during drug screening. In this review, we have taken these factors into consideration when discussing the evidence for these compounds and their synergistic therapeutic benefits in cancer. While there is limited evidence for clinical efficacy for these mixtures, in vitro data suggest that these preparations merit further investigation, both in vitro and in vivo.

LINEs between Species: Evolutionary Dynamics of LINE-1 Retrotransposons across the Eukaryotic Tree of Life.

LINE-1 (L1) retrotransposons are dynamic elements. They have the potential to cause great genomic change because of their ability to 'jump' around the genome and amplify themselves, resulting in the duplication and rearrangement of regulatory DNA. Active L1, in particular, are often thought of as tightly constrained, homologous and ubiquitous elements with well-characterized domain organization. For the past 30 years, model organisms have been used to define L1s as 6-8 kb sequences containing a 5'-UTR, two open reading frames working harmoniously in cis, and a 3'-UTR with a polyA tail. In this study, we demonstrate the remarkable and overlooked diversity of L1s via a comprehensive phylogenetic analysis of elements from over 500 species from widely divergent branches of the tree of life. The rapid and recent growth of L1 elements in mammalian species is juxtaposed against the diverse lineages found in other metazoans and plants. In fact, some of these previously unexplored mammalian species (e.g. snub-nosed monkey, minke whale) exhibit L1 retrotranspositional 'hyperactivity' far surpassing that of human or mouse. In contrast, non-mammalian L1s have become so varied that the current classification system seems to inadequately capture their structural characteristics. Our findings illustrate how both long-term inherited evolutionary patterns and random bursts of activity in individual species can significantly alter genomes, highlighting the importance of L1 dynamics in eukaryotes.

HENMT1 and piRNA Stability Are Required for Adult Male Germ Cell Transposon Repression and to Define the Spermatogenic Program in the Mouse.

piRNAs are critical for transposable element (TE) repression and germ cell survival during the early phases of spermatogenesis, however, their role in adult germ cells and the relative importance of piRNA methylation is poorly defined in mammals. Using a mouse model of HEN methyltransferase 1 (HENMT1) loss-of-function, RNA-Seq and a range of RNA assays we show that HENMT1 is required for the 2' O-methylation of mammalian piRNAs. HENMT1 loss leads to piRNA instability, reduced piRNA bulk and length, and ultimately male sterility characterized by a germ cell arrest at the elongating germ cell phase of spermatogenesis. HENMT1 loss-of-function, and the concomitant loss of piRNAs, resulted in TE de-repression in adult meiotic and haploid germ cells, and the precocious, and selective, expression of many haploid-transcripts in meiotic cells. Precocious expression was associated with a more active chromatin state in meiotic cells, elevated levels of DNA damage and a catastrophic deregulation of the haploid germ cell gene expression. Collectively these results define a critical role for HENMT1 and piRNAs in the maintenance of TE repression in adult germ cells and setting the spermatogenic program.

Discovery of a novel long terminal repeat (LTR2i_SS) in Sus Scrofa.

Long terminal repeat (LTR) retrotransposons are transposable elements flanked by 5'/3' LTRs. They have a structure similar to endogenous retroviruses, but they lack the envelope (env) gene making them non-infectious. Long terminal repeats are motif-rich sequences and can act as bidirectional promoters or enhancers to regulate or inactivate genes by insertion. In this study, we identified a new chimeric LTR subfamily, LTR2i_SS, in the pig genome. This chimeric LTR family appears to be the ancestral form of the previously described LTR2_SS family. LTR2_SS appears to have deleted ~300 bp of un-annotated, ancestral sequence from LTR2i_SS. We identified no functional provirus sequences for either of these LTR types. LTR2i_SS sequences have been exapted into the untranslated regions of two protein-coding gene mRNAs. Both of these genes lie within previously mapped pig quantitative trait loci.

Conservation and expression of PIWI-interacting RNA pathway genes in male and female adult gonad of amniotes.

The PIWI-interacting RNA (piRNA) pathway is essential for germline development and transposable element repression. Key elements of this pathway are members of the piRNA-binding PIWI/Argonaute protein family and associated factors (e.g., VASA, MAELSTROM, and TUDOR domain proteins). PIWI-interacting RNAs have been identified in mouse testis and oocytes, but information about the expression of the different piRNA pathway genes, in particular in the mammalian ovary, remains incomplete. We investigated the evolution and expression of piRNA pathway genes in gonads of amniote species (chicken, platypus, and mouse). Database searches confirm a high level of conservation and revealed lineage-specific gain and loss of Piwi genes in vertebrates. Expression analysis in mammals shows that orthologs of Piwi-like (Piwil) genes, Mael (Maelstrom), Mvh (mouse vasa homolog), and Tdrd1 (Tudor domain-containing protein 1) are expressed in platypus adult testis. In contrast to mouse, Piwil4 is expressed in platypus and human adult testis. We found evidence for Mael and Piwil2 expression in mouse Sertoli cells. Importantly, we show mRNA expression of Piwil2, Piwil4, and Mael in oocytes and supporting cells of human, mouse, and platypus ovary. We found no Piwil1 expression in mouse and chicken ovary. The conservation of gene expression in somatic parts of the gonad and germ cells of species that diverged over 800 million yr ago indicates an important role in adult male and female gonad.

Jumping the fine LINE between species: horizontal transfer of transposable elements in animals catalyses genome evolution.

Horizontal transfer (HT) is the transmission of genetic material between non-mating species, a phenomenon thought to occur rarely in multicellular eukaryotes. However, many transposable elements (TEs) are not only capable of HT, but have frequently jumped between widely divergent species. Here we review and integrate reported cases of HT in retrotransposons of the BovB family, and DNA transposons, over a broad range of animals spanning all continents. Our conclusions challenge the paradigm that HT in vertebrates is restricted to infective long terminal repeat (LTR) retrotransposons or retroviruses. This raises the possibility that other non-LTR retrotransposons, such as L1 or CR1 elements, believed to be only vertically transmitted, can horizontally transfer between species. Growing evidence indicates that the process of HT is much more general across different TEs and species than previously believed, and that it likely shapes eukaryotic genomes and catalyses genome evolution.

A comparative analysis of algorithms for somatic SNV detection in cancer.

MOTIVATION: With the advent of relatively affordable high-throughput technologies, DNA sequencing of cancers is now common practice in cancer research projects and will be increasingly used in clinical practice to inform diagnosis and treatment. Somatic (cancer-only) single nucleotide variants (SNVs) are the simplest class of mutation, yet their identification in DNA sequencing data is confounded by germline polymorphisms, tumour heterogeneity and sequencing and analysis errors. Four recently published algorithms for the detection of somatic SNV sites in matched cancer-normal sequencing datasets are VarScan, SomaticSniper, JointSNVMix and Strelka. In this analysis, we apply these four SNV calling algorithms to cancer-normal Illumina exome sequencing of a chronic myeloid leukaemia (CML) patient. The candidate SNV sites returned by each algorithm are filtered to remove likely false positives, then characterized and compared to investigate the strengths and weaknesses of each SNV calling algorithm. RESULTS: Comparing the candidate SNV sets returned by VarScan, SomaticSniper, JointSNVMix2 and Strelka revealed substantial differences with respect to the number and character of sites returned; the somatic probability scores assigned to the same sites; their susceptibility to various sources of noise; and their sensitivities to low-allelic-fraction candidates. AVAILABILITY: Data accession number SRA081939, code at http://code.google.com/p/snv-caller-review/ CONTACT: david.adelson@adelaide.edu.au SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Contextual models and the non-Newtonian paradigm.

Biological systems exhibit a wide range of contextual effects, and this often makes it difficult to construct valid mathematical models of their behaviour. In particular, mathematical paradigms built upon the successes of Newtonian physics make assumptions about the nature of biological systems that are unlikely to hold true. After discussing two of the key assumptions underlying the Newtonian paradigm, we discuss two key aspects of the formalism that extended it, Quantum Theory (QT). We draw attention to the similarities between biological and quantum systems, motivating the development of a similar formalism that can be applied to the modelling of biological processes.