All normal somatic cells are thought to acquire mutations. However, characterisation of the patterns and consequences of somatic mutation in normal tissues is limited. Uterine endometrium is a dynamic tissue that undergoes cyclical shedding and reconstitution and is lined by a gland-forming epithelium. Whole genome sequencing of normal endometrial glands showed that most are clonal cell populations derived from a recent common ancestor with mutation burdens differing from other normal cell types and manyfold lower than endometrial cancers. Mutational signatures found ubiquitously account for most mutations.Many, in some women potentially all, endometrial glands are colonised by cell clones carrying driver mutations in cancer genes, often with multiple drivers. Total and driver mutation burdens increase with age but are also influenced by other factors including body mass index and parity. Clones with drivers often originate during early decades of life. The somatic mutational landscapes of normal cells differ between cell types and are revealing the procession of neoplastic change leading to cancer.
Focal chromosomal amplification is an important route to generating cancer through mediating over-expression of oncogenes 1 – 3 or to developing cancer therapy resistance by increasing expression of a gene whose action diminishes efficacy of an anti-cancer drug. Here we used whole-genome sequencing of clonal isolates developing chemotherapeutic resistance to identify chromothripsis as a major driver of extrachromosomal DNA (ecDNA) amplification into circular double minutes (DMs) through PARP- and DNA-PKcs-dependent mechanisms. Longitudinal analyses revealed that DMs undergo continuing structural evolution to promote increased drug tolerance through additional chromothriptic events. In-situ Hi-C sequencing is used to demonstrate that DMs preferentially tether near chromosome ends where they re-integrate when DNA damage is present. Intrachromosomal amplifications formed initially under low-level drug selection undergo continuing breakage-fusion-bridge cycles, generating >100 megabase-long amplicons that we show become trapped within interphase bridges and then shattered, producing micronuclei that mediate DM formation. Similar genome rearrangement profiles linked to localized gene amplification are identified in human cancers with acquired drug resistance or with oncogene amplifications. We propose that chromothripsis is a primary mechanism accelerating genomic DNA amplification and which enables rapid acquisition of tolerance to altered growth conditions.
The commonest causes of chronic liver disease are excess alcohol intake, viral hepatitis or nonalcoholic fatty liver disease, with the clinical spectrum ranging in severity from hepatic inflammation through cirrhosis to liver failure or hepatocellular carcinoma. The hepatocellular Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
Cancer genomes are frequently characterized by numerical and structural chromosomal abnormalities. Here we integrated a centromere-specific inactivation approach with selection for a conditionally essential gene, a strategy termed ‘CEN-SELECT’, to systematically interrogate the structural landscape of missegregated chromosomes. We show that single-chromosome missegregation into a micronucleus can directly trigger a broad spectrum of genomic rearrangement types. Cytogenetic profiling revealed that missegregated chromosomes exhibit 120-fold higher susceptibility to developing seven major categories of structural aberrations, including translocations, insertions, deletions, and complex reassembly through chromothripsis coupled to classical non-homologous end joining. Whole-genome sequencing of clonally propagated rearrangements identified random patterns of clustered breakpoints with copy-number alterations resulting in interspersed gene deletions and extrachromosomal DNA amplification events. We conclude that individual chromosome segregation errors during mitotic cell division are sufficient to drive extensive structural variations that recapitulate genomic features commonly associated with human disease.
Synthetic recoding of genomes, to remove targeted sense codons, may facilitate the encoded cellular synthesis of unnatural polymers by orthogonal translation systems. However, our limited understanding of allowed synonymous codon substitutions and the absence of methods that enable the stepwise replacement of the E. coli genome with long synthetic DNA, and provide feedback on allowed and disallowed design features in synthetic genomes, have restricted progress on this goal. Here we endow E. coli with a system for efficient, programmable replacement of genomic DNA with long (~100 kb) synthetic DNA, through the in vivo excision of double stranded DNA from an episomal replicon by CRISPR/Cas9, coupled to lambda red mediated recombination and simultaneous positive and negative selection. We iterate the approach, providing a basis for stepwise whole-genome replacement. We attempt systematic recoding in an essential operon using eight synonymous recoding schemes. Each scheme systematically replaces target codons with defined synonyms and is compatible with codon reassignment. Our results define allowed and disallowed synonymous recoding schemes, and enable the identification and repair of recoding at idiosyncratic positions in the genome.The design and synthesis of genomes provides a powerful approach for understanding and engineering biology1-6. Genome synthesis has the potential to elucidate synonymous codon function7, accelerate metabolic engineering8, and facilitate genetically encoded unnatural polymer synthesis9,10.Methods that i) replace the genome in sections6, ii) provide feedback on precisely where a given design fails and on how to repair it, and that iii) can be rapidly iterated for whole
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