We have developed a new T-DNA vector, pGA2715, which can be used for promoter trapping and activation tagging of rice (Oryza sativa) genes. The binary vector contains the promoterless ␤-glucuronidase (GUS) reporter gene next to the right border. In addition, the multimerized transcriptional enhancers from the cauliflower mosaic virus 35S promoter are located next to the left border. A total of 13,450 T-DNA insertional lines have been generated using pGA2715. Histochemical GUS assays have revealed that the GUS-staining frequency from those lines is about twice as high as that from lines transformed with the binary vector pGA2707, which lacks the enhancer element. This result suggests that the enhancer sequence present in the T-DNA improves the GUS-tagging efficiency. Reverse transcriptase-PCR analysis of a subset of randomly selected pGA2715 lines shows that expression of the genes immediately adjacent to the inserted enhancer is increased significantly. Therefore, the large population of T-DNA-tagged lines transformed with pGA2715 could be used to screen for promoter activity using the gus reporter, as well as for creating gain-of-function mutants.Recent completion of the draft sequence for the rice (Oryza sativa) genome has resulted in an explosion of information on rice genes (Goff et al., 2002; Yu et al., 2002). The challenge for the post-sequencing era is to identify the biological functions for these genes. Of all the approaches used to discover gene function, the most direct is to disrupt the genes and analyze the consequences. Various methods have been developed in plants for this purpose. These include using ethyl methanesulfonate, fast-neutron treatment, or insertion of an element, such as a transposable element or T-DNA (Koornneef et al., 1982;Sundaresan, 1996;Krysan et al., 1999). Insertional mutagenesis has the advantage that the inserted element acts as a tag for gene identification. However, all gene disruption approaches also have some limitations. For example, it is difficult to identify the function of redundant genes, or of genes required in early embryogenesis or gametophyte development.To overcome those limitations, modified insertional elements have been developed. One of these modified designs is the gene trap system that involves creating fusions between the tagged genes and a reporter gene, such as ␤-glucuronidase (gus) or green fluorescent protein (gfp; Sundaresan et al., 1995;Springer, 2000). This system provides a way of identifying novel genes based on their expression patterns. Insertion of the promoterless reporter not only destroys normal gene function but also activates expression of the reporter gene. Because expression levels can be monitored in heterozygote plants, the gene trap system is useful for studying the patterns of most plant genes, including essential genes that cause lethal mutations. This system is convenient for observing mutant phenotypes because reporter activation indicates the location, condition, and time of expression for the disrupted gene. In Arabidopsis, ...

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SummaryA late-¯owering mutant was isolated from rice T-DNA-tagging lines. T-DNA had been integrated into the K-box region of Oryza sativa MADS50 (OsMADS50), which shares 50.6% amino acid identity with the Arabidopsis MADS-box gene SUPPRESSOR OF OVEREXPRESSION OF CO 1/AGAMOUS-LIKE 20 (SOC1/ AGL20). While overexpression of OsMADS50 caused extremely early¯owering at the callus stage, OsMADS50 RNAi plants exhibited phenotypes of late¯owering and an increase in the number of elongated internodes. This con®rmed that the phenotypes observed in the knockout (KO) plants are because of the mutation in OsMADS50. RT-PCR analyses of the OsMADS50 KO and ubiquitin (ubi):OsMADS50 plants showed that OsMADS50 is an upstream regulator of OsMADS1, OsMADS14, OsMADS15, OsMADS18, and Hd (Heading date)3a, but works either parallel with or downstream of Hd1 and O. sativa GIGANTEA (OsGI). These results suggest that OsMADS50 is an important¯owering activator that controls various¯oral regulators in rice.

With methotrexate, joint damage progresses even at low and moderate disease activity levels, whereas methotrexate plus infliximab inhibits radiographic progression across all disease activity states.

Targeting TRAIL receptors with either recombinant TRAIL or agonistic DR4-or DR5-specific antibodies has been considered a promising treatment for cancer, particularly due to the preferential apoptotic susceptibility of tumor cells over normal cells to TRAIL. However, the realization that many tumors are unresponsive to TRAIL treatment has stimulated interest in identifying apoptotic agents that when used in combination with TRAIL can sensitize tumor cells to TRAIL-mediated apoptosis. Our studies suggest that various apoptosis defects that block TRAIL-mediated cell death at different points along the apoptotic signaling pathway shift the signaling cascade from default apoptosis toward cytoprotective autophagy. We also obtained evidence that inhibition of such a TRAIL-mediated autophagic response by specific knockdown of autophagic genes initiates an effective mitochondrial apoptotic response that is caspase-8-dependent. Currently, the molecular mechanisms linking disabled autophagy to mitochondrial apoptosis are not known. Our analysis of the molecular mechanisms involved in the shift from protective autophagy to apoptosis in response to TRAIL sheds new light on the negative regulation of apoptosis by the autophagic process and by some of its individual components.Accumulating evidence suggests that autophagy functions as an adaptive cell response, allowing the cell to survive bioenergetic stress via a mechanism associated with clearance of damaged organelles and the degradation of mutant or misfolded proteins (1). Certain therapeutic approaches to cancer, including radiation and cytotoxic drugs that have been known to activate apoptosis, were observed to induce autophagy in certain human cancer cell lines (2). The functional relationship between apoptosis and autophagy and the potential cross-regulation between these two processes are complex and remain to be resolved. The complexity stems partly from the findings that in certain cellular scenarios, autophagy constitutes a stress adaptation response that avoids and suppresses cell death, whereas in other cellular settings, autophagy constitutes an alternative pathway to cellular demise that is called autophagic cell death (type II cell death) (3-5). Thus, the autophagy genes beclin-1 and atg7 are required to induce nonapoptotic cell death in murine fibroblast L929 cells treated with the caspase inhibitor Z-VAD 3 (6). In addition, Atg5 and Beclin-1 are required for etoposide-and staurosporin-induced cell death in apoptosis-resistant Bax/Bak double knock-out mouse embryonic fibroblasts (7). Current evidence suggests that the removal or functional inhibition of proteins essential for the apoptotic machinery can switch a cellular stress response from apoptotic default to massive autophagy (4, 6 -8). In this regard, dogmaaltering studies were reported by Craig Thompson's group, who discovered that when apoptosis-resistant cells are exposed to stress mediated by the decreased availability of growth factor, the ensuing autophagy actually protects cells from death (8). ...

The current study demonstrates a novel cross-talk mechanism between the TRAIL receptor death signaling pathway and the mitochondria. This newly identified pathway is regulated at the mito

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Apoptotic defects endow tumor cells with survival advantages. Such defects allow the cellular stress response to take the path of cytoprotective autophagy, which either precedes or effectively blocks an apoptotic cascade. Inhibition of the cytoprotective autophagic response shifts the cells toward apoptosis, by interfering with an underlying molecular mechanism of cytoprotection. The current study has identified such a mechanism that is centered on the regulation of caspase-8 activity. The study took advantage of Bax(-/-) Hct116 cells that are TRAIL-resistant despite significant DISC processing of caspase-8, and of the availability of a caspase-8-specific antibody that exclusively detects the caspase-8 large subunit or its processed precursor. Utilizing these biological tools, we investigated the expression pattern and subcellular localization of active caspase-8 in TRAIL-mediated autophagy and in the autophagy-to-apoptosis shift upon autophagy inhibition. Our results suggest that the TRAIL-mediated autophagic response counter-balances the TRAIL-mediated apoptotic response by the continuous sequestration of the large caspase-8 subunit in autophagosomes and its subsequent elimination in lysosomes. The current findings are the first to provide evidence for regulation of caspase activity by autophagy and thus broaden the molecular basis for the observed polarization between autophagy and apoptosis.