Loss Of Function Phenotype Research Articles

Ubc9 Is Essential for Viability of Higher Eukaryotic Cells

Ubc9 is an enzyme involved in the conjugation of SUMO-1 (small ubiquitin related modifier 1) to target proteins. The SUMO-1 conjugation system is well conserved from yeasts to higher eukaryotes, but many SUMO-1 target proteins reported recently in higher eukaryotic cells, including IκBα, MDM2, p53, and PML, are not present in yeasts. To determine the physiological roles of SUMO-1 conjugation in higher eukaryotic cells, we constructed a conditional UBC9 mutant of chicken DT40 cells containing the UBC9 transgene under control of a tetracycline-repressible promoter and characterized their loss of function phenotypes. Ubc9 disappeared 3 days after the addition of tetracycline and the increase in viable cell number stopped 4 days after the addition of drug. In contrast to the cases of ubc9 mutants of budding and fission yeasts, which show defects in progression of G2 or early M phase and in chromosome segregation, respectively, we did not observe accumulation of cells in G2/M phase or a considerable increase in the frequency of chromosome missegregation upon depletion of Ubc9 but we did observe an increase in the number of cells containing multiple nuclei, indicating defects in cytokinesis. A considerable portion of the Ubc9-depleted cell population was committed to apoptosis without accumulating in a specific phase of the cell cycle, suggesting that chromosome damages are accumulated in Ubc9-depleted cells, and apoptosis is triggered without activating checkpoint mechanisms under conditions of SUMO-1 conjugation system impairment.

Drosophila nemo is an essential gene involved in the regulation of programmed cell death

Nemo-like kinases define a novel family of serine/threonine kinases that are involved in integrating multiple signaling pathways. They are conserved regulators of Wnt/Wingless pathways, which may coordinate Wnt with TGFβ-mediated signaling. Drosophila nemo was identified through its involvement in epithelial planar polarity, a process regulated by a non-canonical Wnt pathway. We have previously found that ectopic expression of Nemo using the Gal4-UAS system resulted in embryonic lethality associated with defects in patterning and head development. In this study we present our analyses of the phenotypes of germline clone-derived embryos. We observe lethality associated with head defects and reduction of programmed cell death and conclude that nmo is an essential gene. We also present data showing that nmo is involved in regulating apoptosis during eye development, based on both loss of function phenotypes and on genetic interactions with the pro-apoptotic gene reaper. Finally, we present genetic data from the adult wing that suggest the activity of ectopically expressed Nemo can be modulated by Jun N-terminal kinase (JNK) signaling. Such an observation supports the model that there is cross-talk between Wnt, TGFβ and JNK signaling at multiple stages of development.

HCP-4, a CENP-C-like protein in Caenorhabditis elegans, is required for resolution of sister centromeres.

The centromere plays a critical role in the segregation of chromosomes during mitosis. In mammals, sister centromeres are resolved from one another in the G2 phase of the cell cycle. During prophase, chromosomes condense with sister centromeres oriented in a back to back configuration enabling only one chromatid to be captured by each half spindle. To study this process, we identified a centromere protein (CENP)-C-like protein, holocentric protein (HCP)-4, in Caenorhabditis elegans based on sequence identity, loss of function phenotype, and centromeric localization. HCP-4 is found in the cytoplasm during interphase, but is nuclear localized in mitosis, where it localizes specifically to the centromere. The localization of HCP-4 to the centromere is dependent on the centromeric histone HCP-3; in addition, HCP-3 and HCP-4 are both required for localization of a CENP-F-like protein, HCP-1, indicating an ordered assembly pathway. Loss of HCP-4 expression by RNA-mediated interference resulted in a failure to generate resolution of sister centromeres on chromosomes, suggesting that HCP-4 is required for sister centromere resolution. These chromosomes also failed to form a functional kinetochore. Thus, the CENP-C-like protein HCP-4 is essential for both resolution sister centromeres and attachment to the mitotic spindle.

Neural hyperplasia induced by RNA interference with m4/mα gene activity

The E(spl) complex ( E(spl)-C) contains three different classes of genes that are downstream of Notch signaling. The bHLH genes mediate the Notch signal by repressing proneural gene activity, for example during the singularization of mechanosensory organ precursor cells (SOPs). Genes of the second class, the E(spl) m4/mα family, antagonize this process if overexpressed. Here we show that this is based on dominant-negative effects since RNA interference gives neurogenic phenotypes indistinguishable from E(spl)-C mutations. Furthermore, a third member of the m4/mα gene family, named bbu/tom, behaves differently with respect to RNA expression patterns, its regulation by Notch signaling and loss of function phenotypes.