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Full-Text Articles in Molecular Biology

Nmecas9 Is An Intrinsically High-Fidelity Genome Editing Platform, Nadia Amrani, Xin D. Gao, Pengpeng Liu, Alireza Edraki, Aamir Mir, Raed Ibraheim, Ankit Gupta, Kanae E. Sasaki, Tong Wu, Thomas G. Fazzio, Lihua Julie Zhu, Scot A. Wolfe, Erik J. Sontheimer May 2018

Nmecas9 Is An Intrinsically High-Fidelity Genome Editing Platform, Nadia Amrani, Xin D. Gao, Pengpeng Liu, Alireza Edraki, Aamir Mir, Raed Ibraheim, Ankit Gupta, Kanae E. Sasaki, Tong Wu, Thomas G. Fazzio, Lihua Julie Zhu, Scot A. Wolfe, Erik J. Sontheimer

University of Massachusetts Medical School Faculty Publications

Background: The development of CRISPR genome editing has transformed biomedical research. Most applications reported thus far rely upon the Cas9 protein from Streptococcus pyogenes SF370 (SpyCas9). With many RNA guides, wild-type SpyCas9 can induce significant levels of unintended mutations at near-cognate sites, necessitating substantial efforts toward the development of strategies to minimize off-target activity. Although the genome-editing potential of thousands of other Cas9 orthologs remains largely untapped, it is not known how many will require similarly extensive engineering to achieve single-site accuracy within large (e.g. mammalian) genomes. In addition to its off-targeting propensity, SpyCas9 is encoded by a relatively ...


Small Rnas Gained During Epididymal Transit Of Sperm Are Essential For Embryonic Development In Mice, Colin C. Conine, Fengyun Sun, Lina Song, Jaime A. Rivera-Perez, Oliver J. Rando Apr 2018

Small Rnas Gained During Epididymal Transit Of Sperm Are Essential For Embryonic Development In Mice, Colin C. Conine, Fengyun Sun, Lina Song, Jaime A. Rivera-Perez, Oliver J. Rando

University of Massachusetts Medical School Faculty Publications

The small RNA payload of mammalian sperm undergoes dramatic remodeling during development, as several waves of microRNAs and tRNA fragments are shipped to sperm during post-testicular maturation in the epididymis. Here, we take advantage of this developmental process to probe the function of the sperm RNA payload in preimplantation development. We generated zygotes via intracytoplasmic sperm injection (ICSI) using sperm obtained from the proximal (caput) vs. distal (cauda) epididymis, then characterized development of the resulting embryos. Embryos generated using caput sperm significantly overexpress multiple regulatory factors throughout preimplantation development, and subsequently implant inefficiently and fail soon after implantation. Remarkably, microinjection ...


Primate Immunodeficiency Virus Vpx And Vpr Counteract Transcriptional Repression Of Proviruses By The Hush Complex, Leonid Yurkovetskiy, Mehmet Hakan Guney, Kyusik Kim, Shih Lin Goh, Sean M. Mccauley, Ann Dauphin, William E. Diehl, Jeremy Luban Apr 2018

Primate Immunodeficiency Virus Vpx And Vpr Counteract Transcriptional Repression Of Proviruses By The Hush Complex, Leonid Yurkovetskiy, Mehmet Hakan Guney, Kyusik Kim, Shih Lin Goh, Sean M. Mccauley, Ann Dauphin, William E. Diehl, Jeremy Luban

University of Massachusetts Medical School Faculty Publications

Drugs that inhibit HIV-1 replication and prevent progression to AIDS do not eliminate HIV-1 proviruses from the chromosomes of long-lived CD4+ memory T cells. To escape eradication by these antiviral drugs, or by the host immune system, HIV-1 exploits poorly defined host factors that silence provirus transcription. These same factors, though, must be overcome by all retroviruses, including HIV-1 and other primate immunodeficiency viruses, in order to activate provirus transcription and produce new virus. Here we show that Vpx and Vpr, proteins from a wide range of primate immunodeficiency viruses, activate provirus transcription in human CD4+ T cells. Provirus activation ...


Fluorescence Polarization Control For On-Off Switching Of Single Molecules At Cryogenic Temperatures, Christiaan Hulleman, Maximiliaan Huisman, Robert Moerland, David Grünwald, Sjoerd Stallinga, Bernd Rieger Oct 2017

Fluorescence Polarization Control For On-Off Switching Of Single Molecules At Cryogenic Temperatures, Christiaan Hulleman, Maximiliaan Huisman, Robert Moerland, David Grünwald, Sjoerd Stallinga, Bernd Rieger

University of Massachusetts Medical School Faculty Publications

Light microscopy allowing sub-diffraction limited resolution has been among the fastest developing techniques at the interface of biology, chemistry and physics. Intriguingly no theoretical limit exists on how far the underlying measurement uncertainty can be lowered. In particular data fusion of large amounts of images can reduce the measurement error to match the resolution of structural methods like cryo-electron microscopy. Fluorescence, although reliant on a reporter molecule and therefore not the first choice to obtain ultra resolution structures, brings highly specific labeling of molecules in a large assemble to the table and inherently allows the detection of multiple colors, which ...


A Synthetic Biology Approach To Probing Nucleosome Symmetry, Yuichi Ichikawa, Yuanyuan Chen, Vineeta Bajaj, Caitlin M. Connolly, Hsin-Jung Chou, Upasna Sharma, Hsiuyi V. Chen, Daniel N. Bolon, Oliver J. Rando, Paul D. Kaufman Sep 2017

A Synthetic Biology Approach To Probing Nucleosome Symmetry, Yuichi Ichikawa, Yuanyuan Chen, Vineeta Bajaj, Caitlin M. Connolly, Hsin-Jung Chou, Upasna Sharma, Hsiuyi V. Chen, Daniel N. Bolon, Oliver J. Rando, Paul D. Kaufman

University of Massachusetts Medical School Faculty Publications

The repeating subunit of chromatin, the nucleosome, includes two copies of each of the four core histones, and several recent studies have reported that asymmetrically modified nucleosomes occur at regulatory elements in vivo. To probe the mechanisms by which histone modifications are read out, we designed an obligate pair of H3 heterodimers, termed H3X and H3Y, which we validated genetically and biochemically. Comparing the effects of asymmetric histone tail point mutants with those of symmetric double mutants revealed that a single methylated H3K36 per nucleosome was sufficient to silence cryptic transcription in vivo. We also demonstrate the utility of this ...


Fundamental Limits On Dynamic Inference From Single Cell Snapshots, Caleb Weinreb, Samuel Wolock, Betsabeh K. Tusi, Merav Socolovsky, Allon M. Klein Aug 2017

Fundamental Limits On Dynamic Inference From Single Cell Snapshots, Caleb Weinreb, Samuel Wolock, Betsabeh K. Tusi, Merav Socolovsky, Allon M. Klein

University of Massachusetts Medical School Faculty Publications

Single cell expression profiling reveals the molecular states of individual cells with unprecedented detail. However, because these methods destroy cells in the process of analysis, they cannot measure how gene expression changes over time. But some information on dynamics is present in the data: the continuum of molecular states in the population can reflect the trajectory of a typical cell. Many methods for extracting single cell dynamics from population data have been proposed. However, all such attempts face a common limitation: for any measured distribution of cell states, there are multiple dynamics that could give rise to it, and by ...


Ki-67 Contributes To Normal Cell Cycle Progression And Inactive X Heterochromatin In P21 Checkpoint-Proficient Human Cells, Xiaoming Sun, Aizhan Bizhanova, Timothy D. Matheson, Jun Yu, Lihua Julie Zhu, Paul D. Kaufman May 2017

Ki-67 Contributes To Normal Cell Cycle Progression And Inactive X Heterochromatin In P21 Checkpoint-Proficient Human Cells, Xiaoming Sun, Aizhan Bizhanova, Timothy D. Matheson, Jun Yu, Lihua Julie Zhu, Paul D. Kaufman

University of Massachusetts Medical School Faculty Publications

Ki-67 protein is widely used as a tumor proliferation marker. However, whether Ki-67 affects cell cycle progression has been controversial. Here, we demonstrate that depletion of Ki-67 in human hTERT-RPE1, WI-38, IMR90, hTERT-BJ cell lines and primary fibroblast cells slowed entry into S phase and coordinately downregulated genes related to DNA replication. Some gene expression changes were partially relieved in Ki-67-depleted hTERT-RPE1 cells by co-depletion of the Rb checkpoint protein, but more thorough suppression of the transcriptional and cell cycle defects was observed upon depletion of cell cycle inhibitor p21. Notably, induction of p21 upon depletion of Ki-67 was a ...


Hnrnp A1 And Secondary Structure Coordinate Alternative Splicing Of Mag, Nancy R. Zearfoss, Emily S. Johnson, Sean P. Ryder Jul 2013

Hnrnp A1 And Secondary Structure Coordinate Alternative Splicing Of Mag, Nancy R. Zearfoss, Emily S. Johnson, Sean P. Ryder

University of Massachusetts Medical School Faculty Publications

Myelin-associated glycoprotein (MAG) is a major component of myelin in the vertebrate central nervous system. MAG is present in the periaxonal region of the myelin structure, where it interacts with neuronal proteins to inhibit axon outgrowth and protect neurons from degeneration. Two alternatively spliced isoforms of Mag mRNA have been identified. The mRNA encoding the shorter isoform, known as S-MAG, contains a termination codon in exon 12, while the mRNA encoding the longer isoform, known as L-MAG, skips exon 12 and produces a protein with a longer C-terminal region. L-MAG is required in the central nervous system. How inclusion of ...