RNA interference (RNAi) was first recognized inCaenorhabditis elegans as a biological response to exogenous double-stranded RNA (dsRNA), which induces sequence-specific gene silencing. RNAi represents a conserved regulatory motif, which is present in a wide range of eukaryotic organisms. Recently, we and others have shown that endogenously encoded triggers of gene silencing act through elements of the RNAi machinery to regulate the expression of protein-coding genes. These small temporal RNAs (stRNAs) are transcribed as short hairpin precursors (∼70 nt), processed into active, 21-nt RNAs by Dicer, and recognize target mRNAs via base-pairing interactions. Here, we show that short hairpin RNAs (shRNAs) can be engineered to suppress the expression of desired genes in cultured Drosophila and mammalian cells. shRNAs can be synthesized exogenously or can be transcribed from RNA polymerase III promoters in vivo, thus permitting the construction of continuous cell lines or transgenic animals in which RNAi enforces stable and heritable gene silencing.

The cdc25 phosphatases play key roles in cell cycle progression by activating cyclin-dependent kinases. Two members of the 14-3-3 protein family have been isolated in a yeast two-hybrid screen designed to identify proteins that interact with the human cdc25A and cdc25B phosphatases. Genes encoding the human homolog of the 14-3-3 epsilon protein and the previously described 14-3-3 beta protein have been isolated in this screening. 14-3-3 proteins constitute a family of well-conserved eukaryotic proteins that were originally isolated in mammalian brain preparations and that possess diverse biochemical activities related to signal transduction. We present evidence that indicates that cdc25 and 14-3-3 proteins physically interact both in vitro and in vivo. 14-3-3 protein does not, however, affect the phosphatase activity of cdc25A. Raf-1, which is known to bind 14-3-3 proteins, has recently been shown to associate with cdc25A and to stimulate its phosphatase activity. 14-3-3 protein, however, has no effect on the cdc25A-kinase activity of Raf-1. Instead, 14-3-3 may facilitate the association of cdc25 with Raf-1 in vivo, participating in the linkage between mitogenic signaling and the cell cycle machinery.

The COT1 gene of Saccharomyces cerevisiae has been isolated as a dosage-dependent suppressor of cobalt toxicity. Overexpression of the COT1 gene confers increased tolerance to cobalt and rhodium ions but not other divalent cations. Strains containing null alleles of COT1 are viable yet more sensitive to cobalt than are wild-type strains. Transcription of COT1 responds minimally to the extracellular cobalt concentration. Addition of cobalt ions to growth media results in a twofold increase in COT1 mRNA abundance. The gene encodes a 48-kDa protein which is found in mitochondrial membrane fractions of cells. The protein contains six possible membrane-spanning domains and several potential metal-binding amino acid residues. The COT1 protein shares 60% identity with the ZRC1 gene product, which confers resistance to zinc and cadmium ions. Cobalt transport studies indicate that the COT1 product is involved in the uptake of cobalt ions yet is not solely responsible for it. The increased tolerance of strains containing multiple copies of the COT1 gene is probably due to increased compartmentalization or sequestration of the ion within mitochondria.