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.

The major late transcription factor binds to and activates the mouse metallothionein I promoter.

Abstract: Human (HeLa) cells contain a protein, MLTF, which specifically binds to a DNA sequence in the adenovirus 2 major late promoter and activates transcription of that promoter. The presence of MLTF in uninfected cells suggests that this factor contributes to the transcription of some cellular genes. We find that MLTF binds in a sequence-specific manner to the 5'-flanking region of the mouse metallothionein I (mMTI) gene. Binding was localized between -101 and -94 (relative to the initiation site at +1) by DNA-binding gel electrophoresis assay and DNA methylation interference analysis. As in adenovirus, binding occurred in a region containing the sequence CPuCGTGAC. Deletion of this sequence both eliminated the binding of MLTF and produced a fourfold reduction in transcriptional efficiency in vitro. In contrast to the intact promoter, transcription from the deletion mutant promoter was not stimulated by addition of purified MLTF to an in vitro reconstituted reaction. These results suggest that MLTF contributes to the transcription of cellular genes.

Topoisomerase I enhances TFIID-TFIIA complex assembly during activation of transcription.

Abstract: The mechanism of coactivation by DNA topoisomerase I (topo I) was examined in a highly defined in vitro transcription system containing Pol II and purified factors. Both stimulation of the basal reaction and coactivation occurred dependent on TAF(II)s. Activation was first observed at the TFIID-TFIIA stage of initiation and maximal activation required the concomitant presence of TFIID, TFIIA, topo I, and activator. Electrophoretic mobility shift assay demonstrated a dramatic enhancement in the formation of the TFIID-TFIIA complex by topo I and activator, dependent on the TAF(II)s. DNase I footprinting confirmed this recruitment. A catalytically inactive topo I, which coactivated transcription, similarly stimulated the rapid formation of the TFIID-TFIIA complex in the presence of activator. A camptothecin-mediated DNA cleavage assay demonstrated the recruitment of topo I to the template by TFIID. Topo I likely functions during activation by enhancing the formation of an active TFIID-TFIIA complex on the promoter.

Splicing of adenovirus RNA in a cell-free transcription system

Abstract: A soluble whole-cell extract prepared accurately from HeLa cells splices 2-3% of the RNA transcribed from a DNA template containing the first and second leader exons of late adenovirus RNA. The spliced RNA was detected by a sensitive technique using hybridization to a single-stranded phage M13 cDNA clone, followed by binding to nitrocellulose filters. The identity of the spliced RNA was established by RNase T1 and pancreatic RNase two-dimensional peptide mapping. The bond formed during the in vitro splicing reaction appears to be a typical 3',5'-phosphodiester bond as judged by its sensitivity to RNase T1. The splicing reaction is specifically inhibited by KCl at concentrations greater than 50 mM and by the addition of cellular RNA. Three features of this system may account for the detection of splicing in a soluble extract: (i) the sensitive and unambiguous hybridization assay, (ii) the high transcriptional activity of the major late promoter of adenovirus, and (iii) the use of the first and second leader exon splice of adenovirus, which may be unusually rapid.

The mammalian TFIID protein is present in two functionally distinct complexes.

Abstract: The TFIID activity recognizes a TATA-box element and supports formation of an initiation complex containing RNA polymerase II. Antisera specific for the 38-kD human TFIID protein were used to determine whether this protein cofractionated with the TFIID activity. Surprisingly, the TFIID activity in HeLa whole-cell extracts was resolved into two different size complexes, one of 300 kD and one of greater than 700 kD. Cofractionation studies suggest that both complexes contain the 38-kD protein; thus, this component of the large complexes is probably responsible for recognition of the TATA sequence and interaction with the other general transcription factors in formation of the initiation complex. Interestingly, in contrast to the TFIID activity characterized previously, the 300-kD form of TFIID activity, B-TFIID, does not support stimulation of transcription by factors containing acidic or glutamine-rich activating motifs. We propose that the functional and physical differences between these two forms of TFIID activity are caused by differences in the protein composition of the TFIID complexes of which the 38-kD hTFIID protein is an integral part.

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.