The process of apoptosis (programmed cell death) has become the subject of intensive and extensive research over the past few years. Various approaches are being used to identify and study genes which function as positive mediators of apoptosis. Here, we address a novel approach of gene cloning aimed at isolating intracellular death promoting genes by utilizing a functional screen. This method, called TKO, was based on transfection of cells with an anti-sense cDNA library, followed by the selection of transfectants which survived in the continuous presence of a killing cytokine-interferon-gamma. It led to the identification of five novel apoptotic genes and to the finding that a known protease-cathepsin D, is actively recruited to the death process. The five novel apoptotic genes (named DAP genes for: Death Associated Proteins) code for proteins which display a diverse spectrum of biochemical activities. The list comprises a novel type of calcium/calmodulin-regulated kinase which carries ankyrin repeats and a death domain (DAP-kinase), a nucleotide-binding protein (DAP-3), a small proline-rich cytoplasmic protein (DAP-1), and a novel homolog of the eIF4G translation initiation factor (DAP-5). Extensive studies proved that these genes are critical for mediating cell death initiated by interferon-gamma, and in some of the tested cases also cell death induced by Fas/APO-1, TNF-alpha, and a detachment from extracellular matrix. Moreover, one of these genes, DAP-kinase, was recently found to display strong tumor suppressive activities, coupling the control of apoptosis to metastasis. The advantage of functional approaches of gene cloning is that they select the relevant rate limiting genes along the death pathways in a complete unbiased manner. As a consequence, novel targets and unpredicted mechanisms emerged. A few examples illustrating this important point will be discussed. One relates to the calcium/calmodulin-dependent DAP-kinase, which is localized to the actin microfilaments. It was found that the correct localization of DAP-kinase to the microfilament network was critical for the execution of the apoptotic process, and more specifically for the disruption of the stress fibers--a typical hallmark of apoptosis. Another important breakthrough step in our understanding of apoptotic processes relates to the identification and analysis of the DAP-5 gene. The structure/ function features of this novel translation regulator resemble the proteolytically cleaved eIF4G which appears in cells upon infection with some RNA viruses and which directs cap-independent translation. Thus, the rescue of DAP-5 highlighted the importance of regulation of protein translation in certain apoptotic systems. Finally, the isolation of cathespin D by our method suggests that lysosomal proteases are recruited during apoptosis, in addition to the well known caspase family of proteases, and that a unique pattern of regulation affecting the processing of this protease takes place. The major challenge now is to analyse how these diverse DAP gene activities constitute biochemical pathway(s) leading to programmed cell death, and what is their functional position with respect to other known positive mediators and suppressors of apoptosis such as the Bcl2 and caspase family members.