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.

Transcription by RNA Polymerase II

Abstract: Gene expression in eukaryotic cells is often regulated at the level of transcription of the gene (see Darnell, 1982). In order to understand viral growth, the response of cells to external stimuli, and the processes of differentiation and development, it is of importance to understand what controls the initiation of transcription at a gene. The enzyme of major interest in this regard is the eukaryotic DNA-dependent RNA polymerase (pol II), which is responsible for all cellular messenger RNA synthesis as well as messenger RNA synthesis from many DNA viruses and proviral forms of RNA viruses.

Measurement of hepcidin isoforms in human serum by liquid chromatography with high resolution mass spectrometry

Abstract: Aim: Hepcidin-25 is the master regulator of iron homeostasis. N-truncated isoforms of hepcidin-25 have been identified (hepcidin-20, -22, -24), although data on the concentrations of these isoforms are sparse. Materials & methods: Serum was mixed with aqueous formic acid, and the supernatant loaded onto a 96-well-SPE-plate. Eluted analytes were analyzed using LC–HR-MS. Forty-seven paired dipotassium-EDTA human plasma and serum samples were analyzed. Results: The LLOQ was 1 μg/l (all analytes). Accuracy and precision were acceptable. There was a good correlation (R2 >0.90, all analytes) between matrices. The median (range) serum hepcidin-20, -22, -24 and -25 concentrations measured were 4 (1–40), 8 (2–20), 8 (1–50) and 39 (1–334) μg/l, respectively. Conclusion: LC–HR-MS is widely applicable to the measurement of hepcidin-25, and truncated isoforms.

DNA methylation of hepatic iron sensing genes and the regulation of hepcidin expression

Abstract: Production of the iron regulatory peptide hepcidin is tightly controlled by a network of proteins in hepatocytes that sense levels of iron in the circulation (as diferric-transferrin) and in tissues (in ferritin). Human studies show high variability in the normal range of serum hepcidin levels. We have postulated that this may, in part, be related to inter-individual variability in the expression of genes in the iron sensing pathway, potentially governed by epigenetic factors. Here, we have investigated whether genes encoding hepatic iron sensing proteins and hepcidin are regulated by DNA methylation. Experiments were performed on two human hepatoma cell lines, HepG2 cells and Huh7 cells. Basal expression of TFR2 and HAMP was significantly lower in Huh7 cells compared with HepG2 cells. Analysis of bisulphite-converted DNA from Huh7 cells revealed partial methylation of TFR2 (alpha transcript), which could result in gene silencing. Demethylation using 5-aza-2’-deoxycitidine (AZA) increased TFR2 mRNA expression in Huh7. PCR analysis of bisulphite-converted HAMP promoter DNA, using methylation-specific primers, revealed no differences between cell lines. However, HAMP mRNA expression in Huh7 was increased by AZA treatment, suggesting that methylation of one or more iron sensing genes may indirectly influence HAMP expression. Our study provides evidence that DNA methylation might control expression of HAMP and other hepatic iron sensing genes, and indicates that epigenetic influences on iron homeostasis warrant further investigation.

Deconvolution of seed and RNA-binding protein crosstalk in RNAi-based functional genomics

Abstract: RNA interference (RNAi) is a major, powerful platform for gene perturbations, but is restricted by off-target mechanisms. Communication between RNAs, small RNAs, and RNA-binding proteins (RBPs) is a pervasive feature of cellular RNA networks. We present a crosstalk scenario, designated as ‘crosstalk with endogenous RBPs’ (ceRBP), in which small interfering RNAs or microRNAs with seed sequences that overlap RBP motifs have extended biological effects by perturbing endogenous RBP activity. Systematic analysis of small interfering RNA (siRNA) off-target data and genome-wide RNAi cancer lethality screens using 501 human cancer cell lines, a cancer dependency map, identified that seed-to-RBP crosstalk is widespread, contributes to off-target activity, and affects RNAi performance. Specifically, deconvolution of the interactions between gene knockdown and seed-mediated silencing effects in the cancer dependency map showed widespread contributions of seed-to-RBP crosstalk to growth-phenotype modulation. These findings suggest a novel aspect of microRNA biology and offer a basis for improvement of RNAi agents and RNAi-based functional genomics. This study presents evidence that siRNAs or miRNAs with seed sequences that overlap RBP motifs have extended biological effects by perturbing RBP activity. Seed-to-RBP crosstalk contributes to off-target activity and growth phenotype modulation.

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.