We developed a novel simple cDNA normalization method [termed duplex-specific nuclease (DSN) normalization] that may be effectively used for samples enriched with full-length cDNA sequences. DSN normalization involves the denaturation-reassociation of cDNA, degradation of the double-stranded (ds) fraction formed by abundant transcripts and PCR amplification of the equalized single-stranded (ss) DNA fraction. The key element of this method is the degradation of the ds fraction formed during reassociation of cDNA using the kamchatka crab DSN, as described recently. This thermostable enzyme displays a strong preference for cleaving ds DNA and DNA in DNA-RNA hybrid duplexes compared with ss DNA and RNA, irrespective of sequence length. We developed normalization protocols for both first-strand cDNA [when poly(A)+ RNA is available] and amplified cDNA (when only total RNA can be obtained). Both protocols were evaluated in model experiments using human skeletal muscle cDNA. We also employed DSN normalization to normalize cDNA from nervous tissues of the marine mollusc Aplysia californica (a popular model organism in neuroscience) to illustrate further the efficiency of the normalization technique.

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Interaction of certain cytokines with their corresponding cell-surface receptors induces programmed cell death. Interferon-gamma induces in HeLa cells a type of cell death with features characteristic of programmed cell death. Here, we report the isolation of a novel gene, DAP3 (death-associated protein-3), involved in mediating interferon-gamma-induced cell death. The rescue of this gene was performed by a functional selection approach of gene cloning that is based on transfection with an antisense cDNA expression library. The antisense RNA-mediated inactivation of the DAP3 gene protected the cells from interferon-gamma-induced cell death. This property endowed the cells expressing it with a growth advantage in an environment restrictive due to the continuous presence of interferon-gamma and thus provided the basis of its selection. The gene is transcribed into a single 1.7-kilobase mRNA, which is ubiquitously expressed in different tissues and codes for a 46-kDa protein carrying a potential P-loop motif. Ectopic expression of DAP3 in HeLa cells was not compatible with cell growth, resulting in a 16-fold reduction in the number of drug-resistant stable clones. The data presented suggest that DAP3 is a positive mediator of cell death induced by interferon-gamma.

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