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.

Promoter directionality is controlled by U1 snRNP and polyadenylation signals

Abstract: Transcription of the mammalian genome is pervasive, but productive transcription outside of protein-coding genes is limited by unknown mechanisms. In particular, although RNA polymerase II (RNAPII) initiates divergently from most active gene promoters, productive elongation occurs primarily in the sense-coding direction. Here we show in mouse embryonic stem cells that asymmetric sequence determinants flanking gene transcription start sites control promoter directionality by regulating promoter-proximal cleavage and polyadenylation. We find that upstream antisense RNAs are cleaved and polyadenylated at poly(A) sites (PASs) shortly after initiation. De novo motif analysis shows PAS signals and U1 small nuclear ribonucleoprotein (snRNP) recognition sites to be the most depleted and enriched sequences, respectively, in the sense direction relative to the upstream antisense direction. These U1 snRNP sites and PAS sites are progressively gained and lost, respectively, at the 5' end of coding genes during vertebrate evolution. Functional disruption of U1 snRNP activity results in a dramatic increase in promoter-proximal cleavage events in the sense direction with slight increases in the antisense direction. These data suggest that a U1-PAS axis characterized by low U1 snRNP recognition and a high density of PASs in the upstream antisense region reinforces promoter directionality by promoting early termination in upstream antisense regions, whereas proximal sense PAS signals are suppressed by U1 snRNP. We propose that the U1-PAS axis limits pervasive transcription throughout the genome.

Mediateurs d'interference arn specifiques de sequences arn

Abstract: L'invention concerne un systeme drosophile in vitro qui a ete utilise en vue de demontrer que l'ARN ds (double brin) est traite en segments ARN de 21-23 nucleotides (nt) de longueur. En outre, lorsque ces 21-23 nt fragments sont purifies et re-ajoutes aux extraits drosophiles, ils assurent la mediation de l'interference ARN en l'absence d'ARN ds long. Ainsi, ces 21-23 nt fragments sont les mediateurs de degradation d'ARN specifiques de la sequence. Un signal moleculaire, qui peut etre leur longueur specifique, doit etre present dans ces 21-23 nt fragments pour recruter des facteurs cellulaires impliques dans l'ARNi. L'invention concerne ces 21-23 nt fragments et leur utilisation pour l'inactivation specifique d'une fonction genique. L'utilisation de ces fragments (ou d'oligonucleotides synthetises chimiquement de meme nature ou de nature similaire) permet le ciblage de mARNs specifiques pour la degradation dans des cellules mammiferes, la ou l'utilisation d'ARNs ds longs pour obtenir l'ARNi n'est generalement pas pratique, vraisemblablement en raison des effets nuisibles de la reponse d'interferon. Ce ciblage specifique d'une fonction genique particuliere est utilise dans des applications genomiques et therapeutiques fonctionnelles.

Iron and Zinc Interactions: Does Entero-Pancreatic-Zinc Excretion Cross-Talk with Intestinal Iron Absorption?

Abstract: Iron and zinc are essential micronutrients required for growth and health. Deficiencies of these nutrients are highly prevalent among populations, but can be alleviated by supplementation. Cross-sectional studies in humans showed positive association of serum zinc levels with hemoglobin and markers of iron status. Dietary restriction of zinc or intestinal specific conditional knock out of ZIP4 (SLC39A4), an intestinal zinc transporter, in experimental animals demonstrated iron deficiency anemia and tissue iron accumulation. Similarly increased iron accumulation has been observed in cultured cells exposed to zinc deficient media. These results together suggest a potential role of zinc in modulating whole body iron metabolism. Studies in intestinal cell culture models demonstrate that zinc induces iron uptake and transcellular transport via induction of divalent metal iron transporter-1 (DMT1) and ferroportin (FPN) expression, respectively. It is interesting to note that intestinal cells are exposed to very high levels of zinc through pancreatic secretions, which is a major route of zinc excretion from the body. Therefore, zinc appears to be modulating the iron metabolism possibly via regulating the DMT1 and FPN1 levels. Herein we critically reviewed the available evidence to hypothesize novel mechanism of Zinc-DMT1/FPN axis in regulating intestinal iron absorption and tissue iron accumulation to facilitate future research aimed at understanding the yet elusive mechanisms of iron and zinc interactions.

Iron uptake by rat proximal colon is increased in animals fed an iron deficient diet

Abstract: The typical western diet provides 10 mg of iron each day. However only 10% of this is absorbed in the duodenum (i.e. 1mg/day), meaning that 90% of our daily intake reaches the distal small intestine and colon. It is presumed that this excess iron is simply excreted in the faeces. However, a recent report has suggested that the proximal colon might have some iron transport capacity (Bougle et al. 2002). In the present study we have investigated this possibility by measuring iron flux across the proximal colonic mucosa in animals fed either an iron replete (control) or an iron deficient (FeD) diet. In parallel studies the effect of dietary iron on the expression of the intestinal iron transporters DMT1 and IREG1 has also been determined. All studies were carried out on male Wistar rats (250g). Rats were fed diets containing 44mg Fe/Kg (control) or <0.5mg/Kg (FeD) for 14 days prior to experimentation. For in vivo iron uptake studies, animals were anaesthetised with intraperitoneal pentobarbitone sodium (60 mg/Kg body weight) and 0.2mM 59Fe2+ (complexed with 4mM ascorbate) was instilled into a tied-off segment of proximal colon. Blood samples were removed after 20, 40 and 60 min via a femoral artery cannula to determine iron transfer into the blood. Tissue iron uptake was measured at the end of each experiment by gamma counting of the colonic segments. In a separate group of animals the mucosa was isolated and used as a source of membrane protein and total RNA for analysis of iron transporter expression by Western blotting and RT-PCR respectively. Data are mean ± SEM. Statistical analysis was performed using Student’s unpaired t-test. Iron uptake was significantly increased in FeD group (162.4 ± 36.1 pmoles/mg dry wt tissue n=6) compared with control (71.4 ± 18.3 pmoles/mg dry wt tissue, p<0.05 n=6). This corresponded with a significant increase in DMT1 mRNA and protein in the FeD group. Interestingly, there was no difference in either iron efflux into the blood or in IREG1 expression in the two animal groups. These data indicate that the proximal colon has some capacity to absorb iron from the intestinal lumen.However, rather than transferring the iron into the blood for physiological utilisation, it is retained within the colonic mucosa. The physiological rationale for such a mechanism requires further investigation.

Flavonoid-rich berry-extract treatment influences expression of genes in the copper-uptake pathway in human intestinal Caco-2 cells

Abstract: Berries are a rich dietary source of bioactive polyphenols, including flavonoids, such as anthocyanins. Dietary flavonoids are known to chelate Cu + and are known to alter the uptake of metal ions in human intestinal Caco-2 cells. However, little is known about the effects of dietary polyphenols on the expression of genes involved in the Cu-uptake pathway in the human intestine. The present study investigated the influence of a flavonoid-rich berry-extract on the expression of the following genes which co-ordinate the intestinal uptake of Cu: the cell surface metalloreductase (DCYTB); the Cu importers, divalent metal ion transporter (DMT1) and Cu transporter 1 (CTR1); the intracellular Cu chaperone (HAH1) and metallothionein (MT); the Cu transporting ATPases (ATP7A and ATP7B). Human intestinal Caco-2 cells, cultured for 19 d, were treated for 16 h with a flavonoid-rich berry-extract (OptiBerry; InterHealth Nutraceuticals, Benicia, CA, USA) at a final concentration of 0.125% (w/v). RNA was isolated for quantitative RT–PCR. All gene expression data were normalised to 18S and GAPDH as housekeeping genes and presented as mean normalised expression ratios SEM. Statistical significance was determined by Student’s t test with significance indicated at P £ 0.05 (n 12). Following treatment with the berry extract there were significant decreases in DMT1 (0.73 0.08, P<0.04), CTR1 (0.67 0.06, P<0.01), HAH1 (0.82 0.06, P<0.03) and ATP7B (0.72 0.05, P<0.001) mRNA expression (Fig. 1). The mRNA expression of the other genes did not change significantly in response to the berry-extract treatment. These results indicate that berry flavonoids influence the expression of components of the Cu-uptake pathway. Studies are in progress to investigate the biological relevance of the observed effects in relation to berry consumption and the bioavailability of dietary Cu.

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.