Extrachromosomal DNA (ecDNA) amplification promotes intratumoral genetic heterogeneity and accelerated tumor evolution
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, but its frequency and clinical impact are unclear. Here we show, using computational analysis of whole-genome sequencing data from 3,212 cancer patients, that ecDNA amplification frequently occurs in most cancer types, but not in blood or normal tissue. Oncogenes were highly enriched on amplified ecDNA and the most common recurrent oncogene amplifications arise on ecDNA. EcDNA amplifications resulted in higher levels of oncogene transcription compared to copy number matched linear DNA, coupled with enhanced chromatin accessibility and more frequently resulted in transcript fusions. Patients whose cancers carry ecDNAs have significantly shorter survival, even when controlled for tissue type, than do patients whose cancers are not driven by ecDNA-based oncogene amplification. The results presented here demonstrate that ecDNA-based oncogene amplification is common in cancer, is different from chromosomal amplification and drives poor outcome for patients across many cancer types.
Site-specific incorporation of unnatural amino acids into proteins
provides a powerful tool to study protein function. Here we report genetic code
expansion in zebrafish embryos and its application to the optogenetic control of
cell signaling. We genetically encoded four unnatural amino acids with a diverse
set of functional groups, which included a photocaged lysine that was applied to
the light-activation of luciferase and kinase activity. This approach enables
versatile manipulation of protein function in live zebrafish embryos, a
transparent and commonly used model organism to study embryonic development.
Expanding the genetic code to enable the incorporation of unnatural amino acids into proteins in biological systems provides a powerful tool for studying protein structure and function. While this technology has been mostly developed and applied in bacterial and mammalian cells, it recently expanded into animals, including worms, fruit flies, zebrafish, and mice. In this review, we highlight recent advances toward the methodology development of genetic code expansion in animal model organisms. We further illustrate the applications, including proteomic labeling in fruit flies and mice and optical control of protein function in mice and zebrafish. We summarize the challenges of unnatural amino acid mutagenesis in animals and the promising directions toward broad application of this emerging technology.
We genetically encoded three new caged tyrosine analogues with improved photochemical properties by using an engineered pyrrolysyl-tRNA synthetase/tRNA pair in bacterial and mammalian cells. We applied the new tyrosine analogues to the photoregulation of firefly luciferase by caging its key tyrosine residue, Tyr340, and observed excellent off-to-on light switching. This reporter was then used to evaluate the activation rates of the different light-removable protecting groups in live cells. We identified the nitropiperonyl caging group as an excellent compromise between incorporation efficiency and photoactivation properties. To demonstrate applicability of the new caged tyrosines, an important proteolytic enzyme, tobacco etch virus (TEV) protease, was engineered for optical control. The ability to incorporate differently caged tyrosine analogues into proteins in live cells further expands the unnatural amino acid and optogenetic toolbox.
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