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Dev Cell
2005 Mar 01;83:435-42. doi: 10.1016/j.devcel.2004.12.008.
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Drosophila genome-scale screen for PAN GU kinase substrates identifies Mat89Bb as a cell cycle regulator.
Lee LA
,
Lee E
,
Anderson MA
,
Vardy L
,
Tahinci E
,
Ali SM
,
Kashevsky H
,
Benasutti M
,
Kirschner MW
,
Orr-Weaver TL
.
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Although traditional organism-based mutational analysis is powerful in identifying genes involved in specific biological processes, limitations include incomplete coverage and time required for gene identification. Biochemical screens using cell transfection or yeast two-hybrid methods are rapid, but they are limited by cDNA library quality. The recent establishment of "uni-gene sets" has made it feasible to biochemically screen an organism's entire genome. Radiolabeled protein pools prepared from the Drosophila Gene Collection were used in a Drosophila in vitro expression cloning ("DIVEC") screen for substrates of PAN GU kinase, which is crucial for S-M embryonic cell cycles. Ablation of one identified substrate, Mat89Bb, by RNAi produces a polyploid phenotype similar to that of pan gu mutants. Xenopus embryos injected with Mat89Bb morpholinos arrest with polyploid nuclei, and Mat89Bb RNAi in HeLa cells gives rise to multinucleated cells. Thus, Mat89Bb plays an evolutionarily conserved role as a crucial regulator of both cell cycle and development.
Figure 3. Downregulation of Mat89Bb Disrupts Development and Cell Cycles of Xenopus Embryos and Mitotic Divisions of Cultured Mammalian Cells(A) Injection of Mat89Bb morpholinos in Xenopus embryos leads to developmental delay, with gastrulation defects and polyploidization. (A) Embryos injected with Mat89Bb morpholinos at the one-cell stage appear morphologically to be at (right panel) stage 10.5, whereas age-matched siblings similarly injected with control morpholinos are at (left panel) stage 26. Embryos injected with Mat89Bb morpholinos ([B], bottom) show defects consistent with disruption of gastrulation (e.g., shortened anterior-posterior axis) that are rescued ([B], middle) by coinjection of human Mat89Bb-GFP RNA. ([B], top) An uninjected embryo is shown for comparison. (C) Embryos injected with (right panel) Mat89Bb morpholinos have a greater intensity of DNA staining than (left panel) control morpholino-injected embryos at a similar stage of development. (D and E) Animal caps dissected from (E) Mat89Bb morpholino-injected embryos have larger nuclei than caps from (D) control morpholino-injected embryos; a corresponding increase in cell size is apparent in caps kept flat under a coverslip bridge and viewed at higher magnification (compare insets). Each bar represents 50 μm.(F) RNA interference of human Mat89Bb in HeLa cells results in a multinucleated phenotype. (F and G) Low and (H and I) high magnification of stained HeLa cells fixed at day 6 postinfection. (G and I) Infection with lentivirus encoding Mat89Bb shRNA results in cells with multiple nuclei. (F and H) Cells infected with control virus encoding luciferase shRNA appear normal. DNA is blue, and actin is red. Each bar represents 10 μm.
