Rockefeller University

About: Rockefeller University is a education organization based out in New York, New York, United States. It is known for research contribution in the topics: Population & Gene. The organization has 15867 authors who have published 32938 publications receiving 2940261 citations. The organization is also known as: Rockefeller University & Rockefeller Institute.

Isolation and characterization of postsynaptic densities from various brain regions: enrichment of different types of postsynaptic densities.

Abstract: Postsynaptic densities (PSDs) have been isolated from cerebral cortex, midbrain, cerebellum, and brain stem by the Triton X-100 method previously used in the isolation of cerebral PSDs (Cohen et al., 1977, J. Cell Biol. 74:181). These PSDs have been compared in protein composition, protein phosphorylation, and morphology. Thin-section electron microscopy revealed that cerebral cortex and midbrain PSDs were identical, being approximately 57 nm thick and composed of apparent aggregates 20-30 nm in diameter. Isolated cerebellar PSDs appeared thinner (33 nm) than cerebral cortex PSDs and lacked the apparent 20- to 30-nm aggregates, but had a latticelike structure. In unidirectional and rotary-shadowed replicas, the cerebrum and midbrain PSDs were circular in shape with a large central perforation or hole in the center of them. Cerebellum PSDs did not have a large perforation, but did have numerous smaller perforations in a lattice like structure. Filaments (6-9 nm) were observed connecting possible 20- to 30-nm aggregates in cerebrum PSDs and were also observed radiating from one side of the PSD. Both cerebral cortex and midbrain PSDs exhibited identical protein patterns on SDS gel electrophoresis. In comparison, cerebellar PSDs (a) lacked the major 51,000 Mr protein, (b) contained two times less calmodulin, and (c) contained a unique protein at 73,000 Mr. Calcium plus calmodulin stimulated the phosphorylation of the 51,000 and 62,000 Mr bands in both cerebral cortex and midbrain PSDs. In cerebellar PSDs, only the 58,000 and 62,000 Mr bands were phosphorylated. In the PSDs from all brain regions, cAMP stimulated the phosphorylation of Protein Ia (73,000 Mr), Protein Ib (68.000 Mr), and a 60,000 Mr protein, although cerebrum and midbrain PSDs contained very much higher levels of phosphorylated protein than did the cerebellum. On the basis of the morphological criteria, it is possible that PSDs isolated from cerebrum and midbrain were derived from the Gray type I, or asymmetric, synapses, whereas cerebellum PSDs were derived from the Gray type II, or symmetric, synapses. Since there is some evidence that the type I synapses are involved in excitatory mechanisms while the type II are involved in inhibitory mechanisms, the role of the PSD and of some of its proteins in these synaptic responses is discussed.

Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9

Abstract: The bacterial CRISPR/Cas9 system allows sequence-specific gene editing in many organisms and holds promise as a tool to generate models of human diseases, for example, in human pluripotent stem cells. CRISPR/Cas9 introduces targeted double-stranded breaks (DSBs) with high efficiency, which are typically repaired by non-homologous end-joining (NHEJ) resulting in nonspecific insertions, deletions or other mutations (indels). DSBs may also be repaired by homology-directed repair (HDR) using a DNA repair template, such as an introduced single-stranded oligo DNA nucleotide (ssODN), allowing knock-in of specific mutations. Although CRISPR/Cas9 is used extensively to engineer gene knockouts through NHEJ, editing by HDR remains inefficient and can be corrupted by additional indels, preventing its widespread use for modelling genetic disorders through introducing disease-associated mutations. Furthermore, targeted mutational knock-in at single alleles to model diseases caused by heterozygous mutations has not been reported. Here we describe a CRISPR/Cas9-based genome-editing framework that allows selective introduction of mono- and bi-allelic sequence changes with high efficiency and accuracy. We show that HDR accuracy is increased dramatically by incorporating silent CRISPR/Cas-blocking mutations along with pathogenic mutations, and establish a method termed 'CORRECT' for scarless genome editing. By characterizing and exploiting a stereotyped inverse relationship between a mutation's incorporation rate and its distance to the DSB, we achieve predictable control of zygosity. Homozygous introduction requires a guide RNA targeting close to the intended mutation, whereas heterozygous introduction can be accomplished by distance-dependent suboptimal mutation incorporation or by use of mixed repair templates. Using this approach, we generated human induced pluripotent stem cells with heterozygous and homozygous dominant early onset Alzheimer's disease-causing mutations in amyloid precursor protein (APP(Swe)) and presenilin 1 (PSEN1(M146V)) and derived cortical neurons, which displayed genotype-dependent disease-associated phenotypes. Our findings enable efficient introduction of specific sequence changes with CRISPR/Cas9, facilitating study of human disease.

The structural basis of the activation of Ras by Sos

Abstract: The crystal structure of human H-Ras complexed with the Ras guanine-nucleotide-exchange-factor region of the Son of sevenless (Sos) protein has been determined at 2.8 A resolution. The normally tight interaction of nucleotides with Ras is disrupted by Sos in two ways. First, the insertion into Ras of an α-helix from Sos results in the displacement of the Switch 1 region of Ras, opening up the nucleotide-binding site. Second, side chains presented by this helix and by a distorted conformation of the Switch 2 region of Ras alter the chemical environment of the binding site for the phosphate groups of the nucleotide and the associated magnesium ion, so that their binding is no longer favoured. Sos does not impede the binding sites for the base and the ribose of GTP or GDP, so the Ras–Sos complex adopts a structure that allows nucleotide release and rebinding.

A module map showing conditional activity of expression modules in cancer.

Abstract: DNA microarrays are widely used to study changes in gene expression in tumors, but such studies are typically system-specific and do not address the commonalities and variations between different types of tumor. Here we present an integrated analysis of 1,975 published microarrays spanning 22 tumor types. We describe expression profiles in different tumors in terms of the behavior of modules, sets of genes that act in concert to carry out a specific function. Using a simple unified analysis, we extract modules and characterize gene-expression profiles in tumors as a combination of activated and deactivated modules. Activation of some modules is specific to particular types of tumor; for example, a growth-inhibitory module is specifically repressed in acute lymphoblastic leukemias and may underlie the deregulated proliferation in these cancers. Other modules are shared across a diverse set of clinical conditions, suggestive of common tumor progression mechanisms. For example, the bone osteoblastic module spans a variety of tumor types and includes both secreted growth factors and their receptors. Our findings suggest that there is a single mechanism for both primary tumor proliferation and metastasis to bone. Our analysis presents multiple research directions for diagnostic, prognostic and therapeutic studies.