RNA interference (RNAi) is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene. The mediators of sequence-specific messenger RNA degradation are 21- and 22-nucleotide small interfering RNAs (siRNAs) generated by ribonuclease III cleavage from longer dsRNAs. Here we show that 21-nucleotide siRNA duplexes specifically suppress expression of endogenous and heterologous genes in different mammalian cell lines, including human embryonic kidney (293) and HeLa cells. Therefore, 21-nucleotide siRNA duplexes provide a new tool for studying gene function in mammalian cells and may eventually be used as gene-specific therapeutics.

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RNA interference (RNAi) is a highly conserved gene silencing mechanism that uses double-stranded RNA (dsRNA) as a signal to trigger the degradation of homologous mRNA. The mediators of sequence-specific mRNA degradation are 21- to 23-nt small interfering RNAs (siRNAs) generated by ribonuclease III cleavage from longer dsRNAs. Twenty-one-nucleotide siRNA duplexes trigger specific gene silencing in mammalian somatic cells without activation of the unspecific interferon response. Here we provide a collection of protocols for siRNA-mediated knockdown of mammalian gene expression. Because of the robustness of the siRNA knockdown technology, genomewide analysis of human gene function in cultured cells has now become possible.

Long protrusions that contain a microtubular axoneme, the beating of which can drive a cell through liquid media. Note that bacterial flagella are constructed very differently from eukaryotic flagella. PROTISTS Single-celled eukaryotic organisms that are either free living or parasitic.

The major protein of intermediate-sized fila-

Abstract. mAbs specific for titin or nebulin were characterized by immunoblotting and fluorescence microscopy. Immunoelectron microscopy on relaxed chicken breast muscle revealed unique transverse striping patterns. Each of the 10 distinct titin antibodies provided a pair of delicate decoration lines per sarcomere. The position of these pairs was centrally symmetric to the M line and was antibody dependent. The results provided a linear epitope map, which starts at the Z line (antibody T20), covers five distinct positions along the I band (T21, T12, T4, T1, Tll), the A-I junction (T3), and three distinct positions within the A band (T10, T22, T23). The epitope of T23 locates 0.2 txm before the M line. In immunoblots, the two antibodies decorating at or just before the Z line (T20, T21) specifically recognized the insoluble titin TI component but did not recognize TII, a proteolytic derivative. All other titin antibodies recognized TI and TII. Thus titin molecules appear as polar structures lacking over large regions repetitive epitopes. One physical end seems related to Z line anchorage, while the other may bind close to the M line. Titin epitopes influenced by the contractional state of the sarcomere locate between the N1 line and the A-I junction (T4, T1, Tll). We discuss the results in relation to titin molecules having half-sarcomere lengths. The three nebulin antibodies so far characterized again give rise to distinct pairs of stripes. These locate close to the N2 line.T HE pioneering work of the laboratories of Maruyama and Wang has suggested that sarcomeric structure relies not only on the well-known thick and thin filaments with their various associated proteins but also involves an elastic component (for recent reviews see 19,35). While the nature of the elastic filaments is still poorly understood, it seems clear that they are built from very high molecular weight polypeptides (20,22,38). One major component of the system is titin. Its polypeptide molecular weight is variously given as 1 or 2.8 million (21,31,32,39). Titin is usually present as a doublet on low porosity gels and only the second species TII can be purified under native conditions (10,11,31,40). TII is thought to arise by limited proteolysis from the nonextractable TI species (19,35). Although electron micrographs of purified TII indicate morphological heterogeneity, they clearly document very thin and rather long threads. While the exact biophysical properties are still in dispute, length measurements range from ~0.6 to 1.2 Ixm (21,31,40). In agreement with the proposal that it plays a pivotal role in sarcomere integrity, titin is found both in skeletal and cardiac muscle, although possibly not as identical molecules (7,9,17). Much less is known about nebulin from skeletal muscle. Its molecular weight is ~0. 5-0.8 million (21, 35, 38). As it has not been isolated in native form, its function and shape are not known. In addition, there are reports that nebulin is not expressed in cardiac muscle (9,17).There is distinct disagreement about...

Caenorhabditis elegans has a single lamin gene, designated lmn-1 (previously termed CeLam-1). Antibodies raised against the lmn-1 product (Ce-lamin) detected a 64-kDa nuclear envelope protein. Ce-lamin was detected in the nuclear periphery of all cells except sperm and was found in the nuclear interior in embryonic cells and in a fraction of adult cells. Reductions in the amount of Ce-lamin protein produce embryonic lethality. Although the majority of affected embryos survive to produce several hundred nuclei, defects can be detected as early as the first nuclear divisions. Abnormalities include rapid changes in nuclear morphology during interphase, loss of chromosomes, unequal separation of chromosomes into daughter nuclei, abnormal condensation of chromatin, an increase in DNA content, and abnormal distribution of nuclear pore complexes (NPCs). Under conditions of incomplete RNA interference, a fraction of embryos escaped embryonic arrest and continue to develop through larval life. These animals exhibit additional phenotypes including sterility and defective segregation of chromosomes in germ cells. Our observations show that lmn-1 is an essential gene in C. elegans, and that the nuclear lamins are involved in chromatin organization, cell cycle progression, chromosome segregation, and correct spacing of NPCs. INTRODUCTIONThe nuclear lamina is a filamentous meshwork that is present between the inner nuclear membrane and the peripheral chromatin. The inner nuclear membrane and the nuclear lamina are involved in organizing nuclear structure and regulating nuclear events. These include the organization of the higher order structure of chromatin and regulation of nuclear assembly and disassembly. The nuclear lamina is a primary target for caspases in apoptosis (reviewed in Goldberg et al., 1999b). Lamins are the major proteins of the nuclear lamina. They are classified as type-V intermediate filaments and are composed of an ␣-helical rod domain flanked by a short amino (head) and a long carboxy (tail) domains. The rod domain of lamins is 52-nm long and contains four ␣-helices, each composed of heptad repeats. Coiled-coil interactions and head-to-tail associations between lamin monomers form 10-to 200-nm thick lamin filaments (reviewed in Stuurman et al., 1998) In vivo, lamin filaments are closely associated with the chromatin fibers (Belmont et al., 1993). In vitro, lamins can bind interphase chromatin (Hoger et al., 1991;Yuan et al., 1991;Taniura et al., 1995;Ulitzur et al., 1997;Goldberg et al., 1999a), mitotic chromosomes (Glass and Gerace, 1990;Glass et al., 1993), or specific DNA sequences (Shoeman and Traub, 1990;Luderus et al., 1992;Luderus et al., 1994;Baricheva et al., 1996;Zhao et al., 1996). The binding site of vertebrate lamins to chromatin is localized to specific sequences in the tail domain and can be displaced with the core histones H2A and H2B (Taniura et al., 1995;Goldberg et al., 1999a).The composition of the nuclear lamina varies in different cell types and is under developmental regulation ...