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.

Expression of chimeric genes in the early region of SV40.

Abstract: Chimeric genes have been constructed by inserting foreign gene sequences in the early region of SV40. The genes contained the first exon of the SV40 large T gene with 180 bp of its intron and either the third exon of the rat preproinsulin gene II with 488 bp of its large intron or the third exon of the mouse beta globin gene with 63 bp of its intron. The chimeric genes contained a 5' splicing site (SS) from SV40 and a 3' SS from the inserted gene. Both the preproinsulin and the globin insertions contained a polyadenylation signal. The SV40 early poly(A) addition signal was also retained. High-titer virus stocks were obtained when the recombinants, which contained SV40 origin of replication and the entire late region, were used to transfect a cloned line of COS cells (COS-M6). These stocks typically contained no detectable wild-type virus. RNA mapping demonstrated the following: (a) The SV40-rat preproinsulin chimeric RNA was initiated at the SV40 early promoter, spliced from the SV40 5' SS to the rat preproinsulin 3' SS, and polyadenylated solely at the SV40 poly(A) addition signal. (b) The SV40-mouse beta globin chimeric RNA was initiated at the SV40 early promoter, spliced from the 5' SS to the mouse beta globin 3' SS, and polyadenylated at the mouse beta globin poly(A) site. The chimeric RNAs were overproduced, owing to low levels of T antigen in the COS-M6 cells, which did not completely repress transcription from the early region. Fusion proteins of 15,500 molecular weight resulted from expression in vivo of the SV40-rat preproinsulin chimeric gene and of 11,500 molecular weight for the SV40-mouse beta globin chimeric gene. The molecular weights of the proteins suggested that they were initiated at the early SV40 AUG and that translation continued across the chimeric splice sites. The chimeric proteins were also overproduced.

1918 flu and responsible science.

Abstract: T he influenza pandemic of 1918 is estimated to have caused 50 million deaths worldwide; 675,000 in the United States. The reconstruction of the 1918 virus by the synthesis of all eight subunits and the generation of infectious virus are described on p. 77 of this issue,[*][1] and the sequences of the final three gene segments of the virus are described in a concurrent Nature paper.[†][2] Predictably, but alarmingly, this virus is more lethal to mice than are other influenza strains, suggesting that this property of the 1918 virus has been recovered in the published sequence. The good news is that we now have the sequence of this virus, perhaps permitting the development of new therapies and vaccines to protect against another such pandemic. The concern is that a terrorist group or a careless investigator could convert this new knowledge into another pandemic. Should the sequence of the 1918 virus have been published, given its potential use by terrorists? The dual-use nature of biological information has been debated widely since September 11, 2001. In 2003, a committee of the U.S. National Academies chaired by Gerald Fink considered this issue, weighing the benefits against the risks of restricting the publication of such biological information. They outlined the tradeoff between erring on the side of prudence, thus potentially hindering the progress of critical science, and erring on the side of disclosure, thus potentially aiding terrorists. The U.S. National Science Advisory Board for Biosecurity (NSABB) was established to advise governmental agencies and the scientific community on policies relative to public disclosure. This board has begun to deliberate, but the questions are complex, as typified by these papers on the 1918 virus. It is reassuring that the NSABB was asked to consider these papers before publication and concluded that the scientific benefit of the future use of this information far outweighs the potential risk of misuse. People may be reassured that the system is working, because agencies representing the public, the scientific community, and the publishing journals were involved in the decision. ![Figure][3] CREDIT: COURTESY OF THE NATIONAL MUSEUM OF HEALTH AND MEDICINE, ARMED FORCES INSTITUTE OF PATHOLOGY, WASHINGTON, D.C. (NCP 1603) I firmly believe that allowing the publication of this information was the correct decision in terms of both national security and public health. It is impossible to forecast how scientific observations might stimulate others to create new treatments or procedures to control future pandemics. For example, in the Nature article, sequence comparisons suggest that the 1918 virus was generated not by incremental changes in the polymerase genes, but by the movement of these genes, in total, from an avian source into a human influenza virus. The availability of these sequences will permit identification of their avian origin and should show why this particular set of genes was selected. Similarly, the results in the Science article suggest that the cleavage of a protein on the surface of the 1918 virus, a step critical for virulent infection, may occur by a previously unknown mechanism—a hint that could lead to new drugs for inhibiting this step and thus preventing future pandemic eruptions. Influenza is highly infectious, and a new strain could spread around the world in a matter of months, if not weeks. The public needs confidence that the 1918 virus will not escape from research labs. All of the described experiments were done in a Biosafety Level 3 laboratory, a high-containment environment recommended by the U.S. Centers for Disease Control and Prevention and the National Institutes of Health on an interim basis, whose use should become a permanent requirement for such experiments. Current evidence suggests that some available drugs and possible future vaccines could suppress infections by the 1918 virus. Given the prospect of another natural influenza pandemic, the recent decision by the U.S. administration to stockpile antivirals for influenza treatment seems wise. Finally, although a sequence of the 1918 virus has been determined and is highly virulent in mice, this may not be the specific form of the virus that caused the pandemic of 1918. An article in the same issue of Nature [‡][4] reports the existence of sequence variation in a natural population of influenza virus; yet we have only one sequence for the 1918 pandemic strain, and the reconstructed virus described in the Science article was built into the backbone of a laboratory strain. Because a pandemic infection is dependent on many unknown properties, there is no certainty that the reconstructed 1918 virus is capable of causing a pandemic. [1]: #fn-1 [2]: #fn-2 [3]: pending:yes [4]: #fn-3

Viral DNA in Transformed Cells. III. The Amounts of Different Regions of the SV40 Genome Present in a Line of Transformed Mouse Cells

Abstract: 32P-Labeled SV40 DNA was treated sequentially with restricting endonucleases EcoRI and Hpa I, and the resulting four fragments of DNA were separated by gel electrophoresis. The kinetics of renaturation of each of the fragments and of complete SV40 DNA were measured in the presence of DNA extracted from the SVT2 line of SV40-transformed mouse cells. It was found that these cells contain about six copies of a segment of DNA which includes the early region of the SV40 genome, and about one copy of the late viral sequences. To map the region of the viral genome which is transcribed in SVT2 cells, separated strands of each of the four fragments were prepared and hybridized to total transformed cell RNA. Part of the E strands of the two DNA fragments (A and C) which span the early region of the SV40 genome were found to enter the hybrid.

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.