RESUMO
Retrons are bacterial immune systems that use reverse transcribed DNA as a detector of phage infection. They are also increasingly deployed as a component of biotechnology. For genome editing, for instance, retrons are modified so that the reverse transcribed DNA (RT-DNA) encodes an editing donor. Retrons are commonly found in bacterial genomes; thousands of unique retrons have now been predicted bioinformatically. However, only a small number have been characterized experimentally. Here, we add substantially to the corpus of experimentally studied retrons. We synthesized >100 previously untested retrons to identify the natural sequence of RT-DNA they produce, quantify their RT-DNA production, and test the relative efficacy of editing using retron-derived donors to edit bacterial genomes, phage genomes, and human genomes. We add 62 new empirically determined, natural RT-DNAs, which are not predictable from the retron sequence alone. We report a large diversity in RT-DNA production and editing rates across retrons, finding that top performing editors outperform those used in previous studies, and are drawn from a subset of the retron phylogeny.
RESUMO
Cooperativity is a central feature of protein folding, but the thermodynamic and structural origins of cooperativity remain poorly understood. To quantify cooperativity, we measured guanidine-induced unfolding transitions of single helix-hairpin-helix (HhH)2 repeats and tandem pairs from a seven-repeat segment of Methanopyrus kandleri Topoisomerase V (Topo V) to determine intrinsic repeat stability and interfacial free energies between repeats. Most single-repeat constructs are folded and stable; moreover, several pairs have unfolding midpoints that exceed midpoints of the single repeats they comprise, demonstrating favorable coupling between repeats. Analyzing unfolding transitions with a modified Ising model, we find a broad range of intrinsic and interfacial free energies. Surprisingly, the G repeat, which lacks density in the crystal structure of Topo V without DNA, is the most stable repeat in the array. Using nuclear magnetic resonance spectroscopy, we demonstrate that the isolated G repeat adopts a canonical (HhH)2 fold and forms an ordered interface with the F-repeat but not with the H repeat. Using parameters from our paired Ising fit, we built a partition function for the seven-repeat array. The multistate unfolding transition predicted from this partition function is in excellent agreement with the experimental unfolding transition, providing strong justification for the nearest-neighbor model. The seven-repeat partition function predicts a native state in which three independent segments ("stability islands") of interacting repeats are separated by two unstable interfaces. We confirm this segmented architecture by measuring the unfolding transition of an equimolar mixture of these three separate polypeptides. This segmented structural organization may facilitate wrapping around DNA.
Assuntos
DNA Topoisomerases Tipo I , Dobramento de Proteína , Ilhas , Termodinâmica , DNARESUMO
Altered mitochondrial quality control and dynamics may contribute to neurodegenerative diseases, including Parkinson's disease, but we understand little about these processes in neurons. We combined time-lapse microscopy and correlative light and electron microscopy to track individual mitochondria in neurons lacking the fission-promoting protein dynamin-related protein 1 (Drp1) and delineate the kinetics of PINK1-dependent pathways of mitochondrial quality control. Depolarized mitochondria recruit Parkin to the outer mitochondrial membrane, triggering autophagosome formation, rapid lysosomal fusion, and Parkin redistribution. Unexpectedly, these mitolysosomes are dynamic and persist for hours. Some are engulfed by healthy mitochondria, and others are deacidified before bursting. In other cases, Parkin is directly recruited to the matrix of polarized mitochondria. Loss of PINK1 blocks Parkin recruitment, causes LC3 accumulation within mitochondria, and exacerbates Drp1KO toxicity to dopamine neurons. These results define a distinct neuronal mitochondrial life cycle, revealing potential mechanisms of mitochondrial recycling and signaling relevant to neurodegeneration.
RESUMO
Decades of research suggest that species richness depends on spatial characteristics of habitat patches, especially their size and isolation. In contrast, the habitat amount hypothesis predicts that (1) species richness in plots of fixed size (species density) is more strongly and positively related to the amount of habitat around the plot than to patch size or isolation; (2) habitat amount better predicts species density than patch size and isolation combined, (3) there is no effect of habitat fragmentation per se on species density and (4) patch size and isolation effects do not become stronger with declining habitat amount. Data on eight taxonomic groups from 35 studies around the world support these predictions. Conserving species density requires minimising habitat loss, irrespective of the configuration of the patches in which that habitat is contained.
Assuntos
EcossistemaRESUMO
OBJECTIVE: To identify causes of the autosomal-recessive malformation, diencephalic-mesencephalic junction dysplasia (DMJD) syndrome. METHODS: Eight families with DMJD were studied by whole-exome or targeted sequencing, with detailed clinical and radiological characterization. Patient-derived induced pluripotent stem cells were derived into neural precursor and endothelial cells to study gene expression. RESULTS: All patients showed biallelic mutations in the nonclustered protocadherin-12 (PCDH12) gene. The characteristic clinical presentation included progressive microcephaly, craniofacial dysmorphism, psychomotor disability, epilepsy, and axial hypotonia with variable appendicular spasticity. Brain imaging showed brainstem malformations and with frequent thinned corpus callosum with punctate brain calcifications, reflecting expression of PCDH12 in neural and endothelial cells. These cells showed lack of PCDH12 expression and impaired neurite outgrowth. INTERPRETATION: DMJD patients have biallelic mutations in PCDH12 and lack of protein expression. These patients present with characteristic microcephaly and abnormalities of white matter tracts. Such pathogenic variants predict a poor outcome as a result of brainstem malformation and evidence of white matter tract defects, and should be added to the phenotypic spectrum associated with PCDH12-related conditions. Ann Neurol 2018;84:646-655.