Showing papers by "Phillip A. Sharp published in 2001"

Rna sequence-specific mediators of rna interference

Abstract: The present invention relates to a Drosophila in vitro system which was used to demonstrate that dsRNA is processed to RNA segments 21-23 nucleotides (nt) in length. Furthermore, when these 21-23 nt fragments are purified and added back to Drosophila extracts, they mediate RNA interference in the absence of long dsRNA. Thus, these 21-23 nt fragments are the sequence-specific mediators of RNA degradation. A molecular signal, which may be their specific length, must be present in these 21-23 nt fragments to recruit cellular factors involved in RNAi. This present invention encompasses these 21-23 nt fragments and their use for specifically inactivating gene function. The use of these fragments (or chemically synthesized oligonucleotides of the same or similar nature) enables the targeting of specific mRNAs for degradation in mammalian cells, where the use of long dsRNAs to elicit RNAi is usually not practical, presumably because of the deleterious effects of the interferon response. This specific targeting of a particular gene function is useful in functional genomic and therapeutic applications.

RNA interference—2001

Abstract: In the few years since the discovery of RNA interference (RNAi; Fire et al. 1998), it has become clear that this process is ancient. RNAi, the oldest and most ubiquitous antiviral system, appeared before the divergence of plants and animals. Because aspects of RNAi, known as cosuppression, also control the expression of transposable elements and repetitive sequences (Ketting et al. 1999; Tabara et al. 1999), the interplay of RNAi and transposon activities have almost certainly shaped the structure of the genome of most organisms. Surprisingly, we are only now beginning to explore the molecular processes responsible for RNAi and to appreciate the breadth of its function in biology. Practical applications of this knowledge have allowed rapid surveys of gene functions (see Fraser et al. 2000 and Gönczey et al. 2000 for RNAi analysis of genes on chromosome I and III of Caenorhabditis elegans) and will possibly result in new therapeutic interventions. Genetic studies have expanded the biology of RNAi to cosuppression, transposon silencing, and the first hints of relationships to regulation of translation and development. The possible roles of RNA-dependent RNA polymerase (RdRp) in RNAi have been expanded. Many experiments indicate that dsRNA directs gene-specific methylation of DNA and, thus, regulation at the stage of transcription in plants. Cosuppression may involve regulation by polycomb complexes at the level of transcription in C. elegans and Drosophila. This article will review these topics and primarily summarize advances in the study of RNAi over the past year.

Engineering of a mammalian cell line for reduction of Lactate formation and high monoclonal antibody production

Abstract: Lactate and ammonia are the two major waste products formed during mammalian cell growth. Accumulation of these side products can have a negative effect on cell growth, and has drawn recent attention because of their inhibitory effects on the specific product synthesis rate. Our aim is to reduce lactate formation in the cell culture by genetically manipulating of the pathway of lactate synthesis with an aim to achieve high monoclonal antibody production. We have partially disrupted the LDH-A gene by homologous recombination in hybridoma cells (ATCC-CRL-1606). The cells that received the newly introduced DNA were selected by G418, and an LDH-deficient cell was identified by a screening method based on medium color changing in 96-well plates. A variant cell, LDH-neo21, was identified through this screening method and was characterized. The specific productivity of lactate by LDH-neo21 cells was 50% lower than that of parental cells. Intracellular LDH enzyme activity was significantly reduced. The cell growth was improved both in terms of cell density and cell viability. Total cell density potentially reached 5 x 10(6) cells/mL while the parental hybridoma cells had a cell density of 3.5 x 10(6) cells/mL, which represented a 30% increase. The antibody production of LDH-neo21 cells was threefold greater than that of parental cells during 5-day batch culture. Polymerase chain reaction (PCR) results showed that at least one copy of the LDH-A gene was disrupted in the LDH-neo21 cells. The variant of the hybridoma cell exhibited a significant advantage of reduced lactate formation in the cell culture with a high concentration of glucose, which led to a higher production of monoclonal antibody. 2001 John Wiley & Sons, Inc.

A ribozyme selected from variants of U6 snRNA promotes 2′,5′-branch formation.

Abstract: In vitro selection was used to sample SnRNA-related sequences for ribozyme activities, and several 2',5'-branch-forming ribozymes were isolated One such ribozyme is highly dependent upon an 11-nt motif that contains a conserved U6 snRNA sequence (ACAGAGA-box) known to be important for pre-mRNA splicing The ribozyme reaction is similar to the first step of splicing in that an internal 2'-hydroxyl of an unpaired adenosine attacks at the 5'-phosphate of a guanosine It differs in that the leaving group is diphosphate rather than a 5' exon The finding that lariat formation can be accomplished by a small RNA with sequences related to U6 snRNA indicates that the RNA available in the spliceosome may be involved in RNA-catalyzed branch formation

Nobel laureates' letter to President Bush.

Abstract: We the undersigned urge you to support Federal funding for research using human pluripotent stem cells. We join with other research institutions and patient groups in our belief that the current National Institutes of Health (NIH) guidelines, which enable scientists to conduct stem cell research within the rigorous constraints of federal oversight and standards, should be permitted to remain in effect. The discovery of human pluripotent stem cells is a significant milestone in medical research. Federal support for the enormous creativity of the US biomedical community is essential to translate this discovery into novel therapies for a range of serious and currently intractable diseases.

Methods of producing knockdown cells or organisms by means of RNA sequence-specific mediators of RNA interference and uses thereof.

Abstract: The present invention relates to a Drosophila in vitro system which was used to demonstrate that dsRNA is processed to RNA segments 21-23 nucleotides (nt) in length. Furthermore, when these 21-23 nt fragments are purified and added back to Drosophila extracts, they mediate RNA interference in the absence of long dsRNA. Thus, these 21-23 nt fragments are the sequence-specific mediators of RNA degradation. A molecular signal, which may be their specific length, must be present in these 21-23 nt fragments to recruit cellular factors involved in RNAi. This present invention encompasses these 21-23 nt fragments and their use for specifically inactivating gene function.; The use of these fragments (or chemically synthesized oligonucleotides of the same or similar nature) enables the targeting of specific mRNAs for degradation in mammalian cells, where the use of long dsRNAs to elicit RNAi is usually not practical, presumably because of the deleterious effects of the interferon response. This specific targeting of a particular gene function is useful in functional genomic and therapeutic applications.

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.