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

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 heterologou s 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. OVERVIEW SUMMARYThe optimization of transgene expression at the posttranscriptional level will enhance the capacity of vector delivery systems to produce therapeutic results in target tissues. In this study, the woodchuck hepatitis virus posttranscriptional regulatory elem ent (WPRE) is shown to enhance the expression of an eGFP reporter gene in both transient and stable contexts, when delivered either by transfection or by transduction with recombinant AAV. This enhancement of transgene expression is observed both in cell lines and in primary cells. Inclusion of the WPRE in transgenic messages will better the prospects for therapeutic levels of transgene expression using currently existing gene delivery technologies.

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.

The turns joining segments of secondary structure have been proposed to be key elements in dictating the folded structures of native proteins. An alternative view assumes that turns play a passive role and are merely default structures that occur as a consequence of interactions between antiparallel segments of secondary structure, with chain reversal being dictated by the context surrounding the turn and not by the sequence of the turn itself. The solvent-exposure of turns and their tolerance to evolutionary variance suggests that they may have little or no effect on the formation of native structures. Previous investigations have focused on various types of beta-turns that connect antiparallel beta-strands with comparatively little reported on the structural role of interhelical turns. Here we probe the structural importance of such a turn in an antiparallel 4-helix bundle by randomly substituting an interhelical tripeptide in cytochrome b-562 with many different amino-acid sequences. Thirty-one of the resulting substituted proteins were characterized and all of them were shown to fold into stable, native-like structures. These results suggest that this interhelical turn does not does not play a dominant role in determining the folded structure of this antiparallel 4-helix bundle.

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Monoclonal human B cell tumors are a model system for the study of somatic hypermutation of the Ig genes of humans. It was previously shown that a number of B cell lymphomas exhibited striking V region point mutation, hypothesized to result from the somatic hypermutation mechanism. In this study we have extended the analysis to chronic lymphocytic leukemia. We have cloned and sequenced the productive Vh representing five different cells from a monoclonal chronic lymphocytic leukemia. All five Vh sequences were identical. Therefore, the Vh region in this leukemia was not the subject of detectable somatic mutation. These data suggest that chronic lymphocytic leukemia might lack the mechanism for somatic hypermutation and represent a stage of normal B lymphocyte differentiation in which the somatic hypermutation mechanism is not active.