Statistical tests for intra-tumour clonal co-occurrence and exclusivity

Tumour progression is an evolutionary process in which different clones evolve over time, leading to intra-tumour heterogeneity. Interactions between clones can affect tumour evolution and hence disease progression and treatment outcome. Intra-tumoural pairs of mutations that are overrepresented in a co-occurring or clonally exclusive fashion over a cohort of patient samples may be suggestive of a synergistic effect between the different clones carrying these mutations. We therefore developed a novel statistical testing framework, called GeneAccord, to identify such gene pairs that are altered in distinct subclones of the same tumour. We analysed our framework for calibration and power. By comparing its performance to baseline methods, we demonstrate that to control type I errors, it is essential to account for the evolutionary dependencies among clones. In applying GeneAccord to the single-cell sequencing of a cohort of 123 acute myeloid leukaemia patients, we find 1 clonally co-occurring and 8 clonally exclusive gene pairs. The clonally exclusive pairs mostly involve genes of the key signalling pathways

Population Structure and Mating Dynamics in the Social Amoeba Dictyostelium discoideum

Successfully investigating the evolution and maintenance of sex and mating systems can often have as much to do with choosing the right study system as it has to do with asking the right questions. Dictyostelium discoideum has long been the focus of researchers interested in understanding a number of biological processes, such as motility, chemotaxis and development. More recently, attentions have shifted to include questions about the evolution of social and sexual interactions both within and between species. The D. discoideum life cycles, both asexual and sexual, are uniquely social, each requiring a costly sacrificial act. This offers an ideal system for exploring questions about kin recognition, conflict, and the evolution of multicellularity, as well as the evolution of differential sexual investment and mating types. This dissertation focused on understanding the phylogenetic and geographical relationships between clones in D. discoideum and identifying the social and selective pressures that shape its mating system. I introduce this mating system in Chapter 1. In Chapter 2, I investigated genetic variation and population structure in D. discoideum to identify possible factors that could affect interactions between clones. I used DNA sequence data and phylogenetic techniques to show that though D. discoideum clones form a monophyletic group, there is evidence of genetic differentiation among locations (FST = 0.242, P = 0.011), suggesting geographic or other barriers limit gene flow between populations. In chapter 3, I again looked for population structure, this time concentrating on gamete size and sex ratio, to understand selective pressures maintaining multiple mating types in D. discoideum. Evidence suggests that both balancing selection and drift are likely acting on the D. discoideum mating system. I found no differences in gamete size across the three mating types and also no genetic differentiation across three wild populations at the mating type locus. However, I found that mating type frequency varied across these populations, likely due to drift. Chapter 4 focused on understanding the social dynamics of mating in D. discoideum. During macrocyst formation, two cells of complementary mating types fuse to form a zygote. This zygote then consumes hundreds of surrounding amoebae, likely clones of the original two cells, for use as protection and food. I varied the frequencies at which two clones of differing mating types interacted to investigate the possibility that one mating type cheats another by differentially contributing to the cannibalized cells. Contrary to previous claims that mating type I induces mating type II, coercing it to contribute disproportionately more of these cannibalized cells during macrocyst production, I found that these cells are likely contributed relative to their frequency in the population, regardless of mating type. However, I did find evidence for differential contribution to macrocyst production between some pairs of clones, suggesting that cheating can happen between partners during sex, but is rare and clone-specific. Overall, these studies looked for evidence of underlying population structure in D. discoideum that could impact our understanding of social and sexual interactions in this species. I also applied questions about the maintenance of sex usually only asked in two-sex systems to the unique social sexual interactions within D. discoideum in order to expand the understanding of how mating systems evolve and are maintained in nature. I developed and used new tools and techniques for observing the processes important to understanding this unique system and identified genetic and social factors that could impact how individuals interact during both the asexual and sexual life cycles

CRISPR-Cas9 Editing of Human Histone Deubiquitinase Gene USP16 in Human Monocytic Leukemia Cell Line THP-1

USP16 is a histone deubiquitinase which facilitates G2/M transition during the cell cycle, regulates DNA damage repair and contributes to inducible gene expression. We mutated the USP16 gene in a high differentiation clone of the acute monocytic leukemia cell line THP-1 using the CRISPR-Cas9 system and generated four homozygous knockout clones. All were able to proliferate and to differentiate in response to phorbol ester (PMA) treatment. One line was highly proliferative prior to PMA treatment and shut down proliferation upon differentiation, like wild type. Three clones showed sustained expression of the progenitor cell marker MYB, indicating that differentiation had not completely blocked proliferation in these clones. Network analysis of transcriptomic differences among wild type, heterozygotes and homozygotes showed clusters of genes that were up- or down-regulated after differentiation in all cell lines. Prior to PMA treatment, the homozygous clones had lower levels than wild type of genes relating to metabolism and mitochondria, including SRPRB, encoding an interaction partner of USP16. There was also apparent loss of interferon signaling. In contrast, a number of genes were up-regulated in the homozygous cells compared to wild type at baseline, including other deubiquitinases (USP12, BAP1, and MYSM1). However, three homozygotes failed to fully induce USP3 during differentiation. Other network clusters showed effects prior to or after differentiation in the homozygous clones. Thus the removal of USP16 affected the transcriptome of the cells, although all these lines were able to survive, which suggests that the functions attributed to USP16 may be redundant. Our analysis indicates that the leukemic line can adapt to the extreme selection pressure applied by the loss of USP16, and the harsh conditions of the gene editing and selection protocol, through different compensatory pathways. Similar selection pressures occur during the evolution of a cancer in vivo, and our results can be seen as a case study in leukemic cell adaptation. USP16 has been considered a target for cancer chemotherapy, but our results suggest that treatment would select for escape mutants that are resistant to USP16 inhibitors

Assessment of Genetic Relationship and Hybrid Evaluation Studies in Tea (Camellia sp.) by RAPD.

The genetic relationships among 12 tea accessions representing three species in the genus Camellia were studied using random amplified polymorphic DNA (RAPD) markers. The genetic distance matrix based on Euclidian Distances showed a minimum genetic distance of 2.24 between ‘UPASI-2’ and ‘UPASI-3’ clones and the maximum was 4.47 between ‘TRF-1’ and ‘TRI-2025’. The dendrogram based on Ward’s method of cluster analysis clearly characterized all 12 tea varieties into three clusters based on their types namely China, Assam and Cambod. Pair-wise genetic similarity index between parent and hybrid clones generated showed a highest mean of 0.59 between ‘TRI-2025’ and ‘BSS-1’ and a lowest of 0.34 between ‘UPASI-10’ and ‘BSS-1’. This study revealed that all the varieties analysed fall the present taxonomic framework of Camellia species and that the hybrid is of Cambod type. RAPD markers can thus be successfully applied in this taxon for the study of relationships and to confirm hybrid origin. The study offers a sound platform for future tea bre eding programmes in tea as well as evolution of hybrids in the commercially important tea varieties

Evolution of retrovirus-infected premalignant T-cell clones prior to Adult T-cell leukemia/lymphoma diagnosis

Adult T cell leukemia/lymphoma (ATL) is an aggressive hematological malignancy caused by Human T-cell leukemia virus type-1 (HTLV-1). ATL is preceded by decades of chronic HTLV-1 infection, and the tumors carry both somatic mutations and proviral DNA integrated into the tumor genome. In order to gain insight into the oncogenic process, we used targeted sequencing to track the evolution of the malignant clone in six individuals, 2-10 years before the diagnosis of ATL. Clones of premalignant HTLV-1-infected cells bearing known driver mutations were detected in the blood up to 10 years before individuals developed acute and lymphoma subtype ATL. Six months before diagnosis, the total number and variant allele fraction of mutations increased in the blood. Peripheral blood mononuclear cells from premalignant cases (1 year pre-diagnosis) had significantly higher mutational burden in genes frequently mutated in ATL than did high risk, age-matched HTLV-1-carriers who remained ATL-free after a median of 10 years of follow up. These data show that HTLV-1-infected T cell clones carrying key oncogenic driver mutations can be detected in cases of ATL years before the onset of symptoms. Early detection of such mutations may enable earlier and more effective intervention to prevent the development of ATL

Mutant spectrum of dengue type 1 virus in the plasma of patients from the 2006 epidemic in South China

SummaryThe aim of the present study was to explore the mutant spectrum of dengue type 1 virus (DENV-1) within individuals during the 2006 dengue epidemic in South China. A 513-bp fragment including most of domain III of the envelope (E) gene was amplified directly from clinical samples, then cloned and sequenced. A total of 89 clones from six patients (range 11–17 clones per patient) were sequenced. Genetic diversity was calculated using MEGA 4 package. The total number of nucleotide mutations was 113 (3.7%) within the sequenced 513-bp E gene, with a range of 15 (3%) to 24 (4.7%) within individual viral populations, harboring more non-synonymous than synonymous mutations. The extent of sequence diversity varied among patients, with the mean diversity ranging from 0.19% to 0.32%, and the mean pairwise p-distance ranging from 0.34% to 0.65%. No genome-defective virus was detected in any clone in this study. Purifying selection may be the main driving force for the intrahost evolution: the mean dN/dS ratio was 0.532. Our findings contribute to the understanding of the genetic variation of DENV-1 in South China

Parallel compensatory evolution stabilizes plasmids across the parasitism-mutualism continuum

Plasmids drive genomic diversity in bacteria via horizontal gene transfer [1 and 2]; nevertheless, explaining their survival in bacterial populations is challenging [3]. Theory predicts that irrespective of their net fitness effects, plasmids should be lost: when parasitic (costs outweigh benefits), plasmids should decline due to purifying selection [4, 5 and 6], yet under mutualism (benefits outweigh costs), selection favors the capture of beneficial accessory genes by the chromosome and loss of the costly plasmid backbone [4]. While compensatory evolution can enhance plasmid stability within populations [7, 8, 9, 10, 11, 12, 13, 14 and 15], the propensity for this to occur across the parasitism-mutualism continuum is unknown. We experimentally evolved Pseudomonas fluorescens and its mercury resistance mega-plasmid, pQBR103 [ 16], across an environment-mediated parasitism-mutualism continuum. Compensatory evolution stabilized plasmids by rapidly ameliorating the cost of plasmid carriage in all environments. Genomic analysis revealed that, in both parasitic and mutualistic treatments, evolution repeatedly targeted the gacA/gacS bacterial two-component global regulatory system while leaving the plasmid sequence intact. Deletion of either gacA or gacS was sufficient to completely ameliorate the cost of plasmid carriage. Mutation of gacA/gacS downregulated the expression of ∼17% of chromosomal and plasmid genes and appears to have relieved the translational demand imposed by the plasmid. Chromosomal capture of mercury resistance accompanied by plasmid loss occurred throughout the experiment but very rarely invaded to high frequency, suggesting that rapid compensatory evolution can limit this process. Compensatory evolution can explain the widespread occurrence of plasmids and allows bacteria to retain horizontally acquired plasmids even in environments where their accessory genes are not immediately useful