Phillip A. Sharp

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

Identification and characterization of a HeLa nuclear protein that specifically binds to the trans-activation-response (TAR) element of human immunodeficiency virus

Abstract: Human immunodeficiency virus type 1 RNAs contain a sequence, trans-activation-response (TAR) element, which is required for tat protein-mediated trans-activation of viral gene expression. We have identified a nuclear protein from extracts of HeLa cells that binds to the TAR element RNA in a sequence-specific manner. The binding of this 68-kDa polypeptide was detected by UV cross-linking proteins to TAR element RNA transcribed in vitro. Competition experiments were performed by using a partially purified preparation of the protein to quantify the relative binding affinities of TAR element RNA mutants. The binding affinity of the TAR mutants paralleled the reported ability of those mutants to support tat trans-activation in vivo. We propose that this cellular protein moderates TAR activity in vivo.

Transcriptional silencing of a transgene by RNAi in the soma of C. elegans

Abstract: The silencing of transgene expression at the level of transcription in the soma of Caenorhabditis elegans through an RNAi-dependent pathway has not been previously characterized. Most gene silencing due to RNAi in C. elegans occurs at the post-transcriptional level. We observed transcriptional silencing when worms containing the elt-2::gfp/LacZ transgene were fed RNA produced from the commonly used L4440 vector. The transgene and the vector share plasmid backbone sequences. This transgene silencing depends on multiple RNAi pathway genes, including dcr-1, rde-1, rde-4, and rrf-1. Unlike post-transcriptional gene silencing in worms, elt-2::gfp/LacZ silencing is dependent on the PAZ-PIWI protein Alg-1 and on the HP1 homolog Hpl-2. The latter is a chromatin silencing factor, and expression of the transgene is inhibited at the level of intron-containing precursor mRNA. This inhibition is accompanied by a decrease in the acetylation of histones associated with the transgene. This transcriptional silencing in the soma can be distinguished from transgene silencing in the germline by its inability to be transmitted across generations and its dependence on the rde-1 gene. We therefore define this type of silencing as RNAi-induced Transcriptional Gene Silencing (RNAi-TGS). Additional chromatin-modifying components affecting RNAi-TGS were identified in a candidate RNAi screen.

Mapping of adenovirus 2 RNA sequences in lytically infected cells and transformed cell lines.

Abstract: The strands of the six EcoRI fragments and the HpaI fragments E and C of Ad2 DNA were separated by electrophoresis in agarose gels. Using 32P-labeled fragment strands in solution hybridization experiments, the fraction of each strand complementary to RNA extracted from infected or transformed cells was assayed by chromatography on hydroxylapatite. In this manner, a tentative map of the cytoplasmic RNA sequences has been constructed for viral RNA extracted from cells both early and late during infection (see Fig. 16; in the map shown, the two strands of Ad2 are named the r and l strands following the bacteriophage convention). Since early cytoplasmic RNA anneals to four distinct regions of the genome, Ad2 probably codes for at least four early gene functions. Summation experiments have shown that all RNA sequences found in the cytoplasm of cells early during infection are also present in the cells' cytoplasm at late times. Viral RNA sequences in five independently isolated and cloned transformed rat cell lines were also mapped on the Ad2 genome. One class of Ad2-transformed rat cells contains RNA sequences complementary to only the segment of Ad2 DNA from 0.03-0.10 on the physical map, and this corresponds to one of the four regions of the genome expressed early during infection. If a viral gene product is necessary to maintain the transformed phenotype of the cell or codes for the virus-specific tumor (T) antigen, this genetic information must be at the left end of the genome (see Fig. 16). The two other classes of Ad2-transformed rat cells contain viral RNA sequences complementary to two or three of the regions of the genome transcribed into early cytoplasmic RNA. At both early and late times during the lytic cycle, the nucleus of the infected cell contains viral RNA sequences that are not transported to the cell's cytoplasm, suggesting that RNA processing and selection may play a role in the regulation of viral mRNA production.

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。

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.