Tal Kafri

Suppression of TNF-α-Induced Apoptosis by NF-κB

Tumor necrosis factor α (TNF-α) signaling gives rise to a number of events, including activation of transcription factor NF-κB and programmed cell death (apoptosis). Previous studies of TNF-α signaling have suggested that these two events occur independently. The sensitivity and kinetics of TNF-α-induced apoptosis are shown to be enhanced in a number of cell types expressing a dominant-negative IκBα (IκBαM). These findings suggest that a negative feedback mechanism results from TNF-α signaling in which NF-κB activation suppresses the signals for cell death.

Integration complexes derived from HIV vectors for rapid assays in vitro

Of three enzymes encoded by HIV–reverse transcriptase, protease, and integrase—only the first two have been exploited clinically as inhibitor targets. Efforts to develop inhibitors of purified integrase protein have yielded many compounds, but none with clinical utility. A different source of integration activity for studies in vitro is provided by replication intermediates isolated from HIV-infected cells. These preintegration complexes (PICs) can direct integration of the endogenously synthesized viral cDNA into an added target DNA in vitro. Despite their authentic activities, assays of PICs have not been widely used due to technical obstacles, particularly the requirement for handling large amounts of infectious HIV. Here, we describe greatly improved methods for producing PICs using HIV-based vectors that are capable of establishing an integrated provirus but not a spreading infection. We also report the development of a PIC integration assay using DNA-coated microtiter plates, which speeds assays of PIC integration in vitro. We used this method to screen a library of chemicals related to known integrase inhibitors and found a new compound, quinalizarin sulfate, that displayed enhanced activity against PICs.

Piecing together more efficient gene expression

The eukaryotic cell has evolved exquisite control of gene expression at many different levels. One of the more important control points is at the level of transcription of a gene into an RNA message. Luckily for the genetic engineer, the components that control this process are modular in nature, which facilitates both their analysis and their manipulation (Fig. 1A). These features are particularly important for the biotechnologist, as the ability to control gene expression specifically and meticulously is crucial for many applications. It is critical not only for the production of biological drugs and therapeutic proteins in bacteria, yeast, and mammalian cells, but also for the field of gene therapy, where the goal is to bypass completely such “middle-organisms” and deliver the gene and its product directly to the patient1. In this issue, Robert Schwartz2 and colleagues describe an approach to evolve more effective muscle-specific transcription promoters by mixing and matching operator elements from four distinct muscle-specific promoters (Fig. 1B). They succeeded in creating synthetic promoters that are eightfold more effective than the corresponding natural promoters, both in vitro and in vivo. This approach to novel promoter construction should prove to be a useful new tool to enhance tissue-specific expression for both therapeutic and transgenic applications. Early gene therapy vectors made use of viral promoters to drive gene expression because complex viral genomes had been studied intensely and their compact nature made them easier to manipulate than most eukaryotic cellular promoters. Experience with ex vivo gene therapy approaches, however, has shown that viral promoters are often inactivated by unknown mechanisms3. Promoters of endogenous cellular genes often fare no better. Some constitutive “housekeeping” promoters have shown some activity4, although in these cases expression of the gene was often weak. These problems, inherent to the use of natural promoters, have spurred