Phillip A. Sharp

Bio: Phillip A. Sharp is an academic researcher from Massachusetts Institute of Technology. The author has contributed to research in topic(s): RNA & RNA splicing. The author has an hindex of 172, co-authored 614 publication(s) receiving 117126 citation(s). Previous affiliations of Phillip A. Sharp include McGovern Institute for Brain Research & Medical Research Council.

Histone H3K27ac separates active from poised enhancers and predicts developmental state

Abstract: Developmental programs are controlled by transcription factors and chromatin regulators, which maintain specific gene expression programs through epigenetic modification of the genome. These regulatory events at enhancers contribute to the specific gene expression programs that determine cell state and the potential for differentiation into new cell types. Although enhancer elements are known to be associated with certain histone modifications and transcription factors, the relationship of these modifications to gene expression and developmental state has not been clearly defined. Here we interrogate the epigenetic landscape of enhancer elements in embryonic stem cells and several adult tissues in the mouse. We find that histone H3K27ac distinguishes active enhancers from inactive/poised enhancer elements containing H3K4me1 alone. This indicates that the amount of actively used enhancers is lower than previously anticipated. Furthermore, poised enhancer networks provide clues to unrealized developmental programs. Finally, we show that enhancers are reset during nuclear reprogramming.

RNAi: Double-Stranded RNA Directs the ATP-Dependent Cleavage of mRNA at 21 to 23 Nucleotide Intervals

Abstract: Double-stranded RNA (dsRNA) directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). Using a recently developed Drosophila in vitro system, we examined the molecular mechanism underlying RNAi. We find that RNAi is ATP dependent yet uncoupled from mRNA translation. During the RNAi reaction, both strands of the dsRNA are processed to RNA segments 21-23 nucleotides in length. Processing of the dsRNA to the small RNA fragments does not require the targeted mRNA. The mRNA is cleaved only within the region of identity with the dsRNA. Cleavage occurs at sites 21-23 nucleotides apart, the same interval observed for the dsRNA itself, suggesting that the 21-23 nucleotide fragments from the dsRNA are guiding mRNA cleavage.

Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids

Abstract: We have developed a simple and sensitive method for detecting, sizing and mapping RNA transcripts from viral or cloned DNAs This technique has been used to examine the cytoplasmic transcripts produced during the early phase of adenovirus 2 (Ad2) infection of HeLa cells Unlabeled total cytoplasmic or oligo (dT)-selected cytoplasmic RNA is hybridized to restriction fragments of 32 P-labeled viral DNA in 80% formamide under conditions above the Tm of the DNA duplex, but below the Tm of the RNA-DNA hybrid duplex (Casey and Davidson, 1977) DNA complements precisely the length of the hybridized RNA are generated by treating with single-strand-specific S1 endonuclease under conditions which do not introduce strand breaks into hybrid duplex The sizes of the S1-resistant single-stranded DNAs are then determined by alkaline agarose gel electrophoresis (McDonnell, Simon and Studier, 1977) A restriction fragment which terminates within a region coding for an mRNA yields a band equal in size to the portion of the mRNA transcribed from that restriction fragment This allows unique mapping of coding regions relative to restriction endonuclease cleavage sites All early Ad2 transcripts are clustered in four regions of the viral DNA At least five early stable cytoplasmic colinear transcripts are transcribed to the right from the left end of the viral genome, the region of the genome coding functions necessary for transformation of mammalian cells Transcripts of 650, 350 and 1750 nucleotides map from 17 ± 05 to 36 ± 05, 36 ± 02 to 46 ± 04, and 47 ± 03 to 95 ± 05 units, respectively, and there are two 450 nucleotide transcripts tentatively mapped in the region of 30 and 110 units Two overlapping transcripts of 1600 and 1700 nucleotides having the same 3′ terminus and 5′ termini displaced by 100 nucleotides are transcribed to the left from 662 ± 03 to 616 ± 02 and 665 ± 03 to 616 ± 02 units, respectively Seven other distinct early stable cytoplasmic transcripts have also been positioned on the genome

MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells

Abstract: MicroRNAs are predicted to regulate thousands of mammalian genes, but relatively few targets have been experimentally validated and few microRNA loss-of-function phenotypes have been assigned. As an alternative to chemically modified antisense oligonucleotides, we developed microRNA inhibitors that can be expressed in cells, as RNAs produced from transgenes. Termed 'microRNA sponges', these competitive inhibitors are transcripts expressed from strong promoters, containing multiple, tandem binding sites to a microRNA of interest. When vectors encoding these sponges are transiently transfected into cultured cells, sponges derepress microRNA targets at least as strongly as chemically modified antisense oligonucleotides. They specifically inhibit microRNAs with a complementary heptameric seed, such that a single sponge can be used to block an entire microRNA seed family. RNA polymerase II promoter (Pol II)-driven sponges contain a fluorescence reporter gene for identification and sorting of sponge-treated cells. We envision the use of stably expressed sponges in animal models of disease and development.

In vivo genome editing using Staphylococcus aureus Cas9

Abstract: The RNA-guided endonuclease Cas9 has emerged as a versatile genome-editing platform. However, the size of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for basic research and therapeutic applications that use the highly versatile adeno-associated virus (AAV) delivery vehicle. Here, we characterize six smaller Cas9 orthologues and show that Cas9 from Staphylococcus aureus (SaCas9) can edit the genome with efficiencies similar to those of SpCas9, while being more than 1 kilobase shorter. We packaged SaCas9 and its single guide RNA expression cassette into a single AAV vector and targeted the cholesterol regulatory gene Pcsk9 in the mouse liver. Within one week of injection, we observed >40% gene modification, accompanied by significant reductions in serum Pcsk9 and total cholesterol levels. We further assess the genome-wide targeting specificity of SaCas9 and SpCas9 using BLESS, and demonstrate that SaCas9-mediated in vivo genome editing has the potential to be efficient and specific.

MicroRNAs: Genomics, Biogenesis, Mechanism, and Function

Abstract: MicroRNAs (miRNAs) are endogenous ∼22 nt RNAs that can play important regulatory roles in animals and plants by targeting mRNAs for cleavage or translational repression. Although they escaped notice until relatively recently, miRNAs comprise one of the more abundant classes of gene regulatory molecules in multicellular organisms and likely influence the output of many protein-coding genes.

Initial sequencing and analysis of the human genome.

Abstract: The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

Abstract: 抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

MicroRNAs: Target Recognition and Regulatory Functions

Abstract: MicroRNAs (miRNAs) are endogenous ∼23 nt RNAs that play important gene-regulatory roles in animals and plants by pairing to the mRNAs of protein-coding genes to direct their posttranscriptional repression. This review outlines the current understanding of miRNA target recognition in animals and discusses the widespread impact of miRNAs on both the expression and evolution of protein-coding genes.

A rapid alkaline extraction procedure for screening recombinant plasmid DNA

Abstract: A procedure for extracting plasmid DNA from bacterial cells is described. The method is simple enough to permit the analysis by gel electrophoresis of 100 or more clones per day yet yields plasmid DNA which is pure enough to be digestible by restriction enzymes. The principle of the method is selective alkaline denaturation of high molecular weight chromosomal DNA while covalently closed circular DNA remains double-stranded. Adequate pH control is accomplished without using a pH meter. Upon neutralization, chromosomal DNA renatures to form an insoluble clot, leaving plasmid DNA in the supernatant. Large and small plasmid DNAs have been extracted by this method.