Since the advent of recombinant DNA technology, maximization of exogenous gene expression has been an important issue for molecular biologists. Efforts at enhancing transgene expression have mostly been directed at improving the efficiency of delivery and increasing levels of transcription and translation. Less progress has been made in the application of post-transcriptional methods for improving gene expression. Here is described the use of an element derived from the woodchuck hepatitis virus (WHV) that possesses the ability to enhance the expression of heterologous genes post-transcriptionally. Green fluorescent protein (GFP) is frequently employed as a fusion protein to enable the detection, visualization, and quantification of molecules under study. Problems of expression are often encountered when using this strategy, making the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) a useful addition to vectors designed to express fusion proteins. This chapter discusses general issues of cloning and placement of the WPRE when designing such vectors.KeywordsGreen Fluorescent ProteinGreen Fluorescent Protein ExpressionEnhance Green Fluorescent Protein ExpressionGreen Fluorescent Protein Fusion ProteinHEPES Buffer SalineThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

heard. At the same time, the progress that these communities have made is truly remarkable. The stories are inspiring in terms of the work that has gone into the creation of a vision and the job of putting it into practice, and they provide a sense of the long-term commitment and belief in a project required to sustain it. The Land Tllat Could Bebegins with the premise that an environmental perspective leads to democracy. Bythe end of the book, the author seems to be arguing the opposite: True democracy and the active participation of its citizenry lead to environmentalism. Either way, Shutkin's basic idea that environmental projects must be rooted in communities is strong. Although he points out the weaknesses of the top-down environmental strategy, he does not deny its role; he simply argues for the strengthening of the bottom-up perspective as well.

The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) evolved to stimulate the expression of intronless viral messages. To determine whether this ability to enhance expression could be useful in nonviral and heterologous viral gene delivery systems, we analyzed the ability of the WPRE to elevate the expression of a cDNA encoding the green fluorescent protein (GFP) in these contexts. We find that the WPRE can stimulate the expression of GFP when the gene is delivered by transfection or transduction with recombinant adeno-associated virus (AAV). Enhancement occurred both during transient expression and when the gene is stably incorporated into the genome of target cells. This enhancement required that the WPRE be located in cis within the GFP message, and was observed in both transformed cell lines and primary human fibroblasts. These results demonstrate that the WPRE will be an effective tool for increasing the long-term expression of transgenes in gene therapy.

The ubiquitously expressed nonreceptor tyrosine kinase c-Abl contains three nuclear localization signals, however, it is found in both the nucleus and the cytoplasm of proliferating fibroblasts. A rapid and transient loss of c-Abl from the nucleus is observed upon the initial adhesion of fibroblasts onto a fibronectin matrix, suggesting the possibility of nuclear export [Lewis, J,, Baskaran, R,, Taagepera, S., Schwartz, M, & Wang, J, (1996) Proc. Natl. Acad. Sci. USA 93, 15174-15179]. Here we show that the C terminus of c- Abl does indeed contain a functional nuclear export signal (NES) with the characteristic leucine-rich motif. The c-Abl NES can functionally complement an NES-defective HIV Rev protein (Rev Delta 3NI) and can mediate the nuclear export of glutathione-S-transferase. The c-Abl NES function is sensitive to the nuclear export inhibitor leptomycin B, Mutation of a single leucine (L1064A) in the c-Abl NES abrogates export function. The NES-mutated c-Abl, termed c-Abl NES(-), is localized exclusively to the nucleus. Treatment of cells with leptomycin B also leads to the nuclear accumulation of wild-type c-Abl protein. The c-Abl NES(-) is not lost from the nucleus when detached fibroblasts are replated onto fibronectin matrix, Taken together, these results demonstrate that c-Abl shuttles continuously between the nucleus and the cytoplasm and that the rate of nuclear import and export can be modulated by the adherence status of fibroblastic cells.

ABSTRACT The hepatitis B virus posttranscriptional regulatory element (HBVPRE) is a cis -acting RNA element that partially overlaps with enhancer I and is required for the cytoplasmic accumulation of HBV surface RNAs. We find that the closely related woodchuck hepatitis virus (WHV), which has been shown to lack a functional enhancer I, also contains a posttranscriptional regulatory element (WPRE). Deletion analysis suggests that the WPRE consists of three independent subelements. Comparison of the bipartite HBVPRE and tripartite WPRE activities reveals that the tripartite WPRE is two to three times more active than the bipartite HBVPRE. Mutation of a single WPRE subelement decreases WPRE activity to the level of the HBVPRE. Bipartite and tripartite chimeras of the WPRE and HBVPRE possess activities which suggest that elements containing three subelements are posttranscriptionally stronger than those containing two. These data demonstrate that the posttranscriptional regulatory element is conserved within the mammalian hepadnaviruses and that its strength is determined by the number of subelements within the RNA.