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Table 1.

Percentage of mutant phenotypes in GFP-DdNek2 and GFP-K33Rmutants exhibiting high expression of the GFP-fusion protein

GFP-DdNek2 (n=316)GFP-K33R (n=283)GFP (control) (n=311)
1. One centrosome per nucleus 33.2% 33.4% 94.5% 
2. Centrosomal and/or nuclear defects and one centrosome per nucleus 15.8% 22.9% 4.2% 
3. Multiple GFP foci and one centrosome per nucleus 6.6% 9.7% n.d. 
4. Supernumerary MTOCs 27.8% 25.4% 1.3% 
5. Supernumerary MTOCs and multiple GFP foci 17.1% 8.5% n.d. 
Cells with a centrosomal GFP fluorescence above a gray level of level 110 at a 256 level 8-bit scale (Hamamatsu CCD camera C5985, 1 second exposure,100×NA 1.3 lens) were evaluated (∼25% of all cells in each clone). The five main categories of phenotype are listed in the left column. The first category represents normal cells. Among the cells of the second category,approximately two thirds exhibited deformed centrosomes and approximately one third exhibited signs, such as additional small DNA-masses (visible in DAPI stainings) or enlarged nuclei, of aneuploidy. The third category comprises cells with multiple GFP foci (characterized by the absence of γ-tubulin labeling). Many of these cells also possess deformed centrosomes (about two thirds) and signs of aneuploidy (about one fifth). Category four contains cells with supernumerary MTOCs (characterized by the presence ofγ-tubulin); some cells showed signs of aneuploidy (∼1/10). Cells in category five exhibit a combination of the phenotypes of categories three and four. Control cells transformed with the GFP-fusion vector without an insert showed that the mutant phenotypes were not caused by the transformation procedure or by expression of GFP alone.    
GFP-DdNek2 (n=316)GFP-K33R (n=283)GFP (control) (n=311)
1. One centrosome per nucleus 33.2% 33.4% 94.5% 
2. Centrosomal and/or nuclear defects and one centrosome per nucleus 15.8% 22.9% 4.2% 
3. Multiple GFP foci and one centrosome per nucleus 6.6% 9.7% n.d. 
4. Supernumerary MTOCs 27.8% 25.4% 1.3% 
5. Supernumerary MTOCs and multiple GFP foci 17.1% 8.5% n.d. 
Cells with a centrosomal GFP fluorescence above a gray level of level 110 at a 256 level 8-bit scale (Hamamatsu CCD camera C5985, 1 second exposure,100×NA 1.3 lens) were evaluated (∼25% of all cells in each clone). The five main categories of phenotype are listed in the left column. The first category represents normal cells. Among the cells of the second category,approximately two thirds exhibited deformed centrosomes and approximately one third exhibited signs, such as additional small DNA-masses (visible in DAPI stainings) or enlarged nuclei, of aneuploidy. The third category comprises cells with multiple GFP foci (characterized by the absence of γ-tubulin labeling). Many of these cells also possess deformed centrosomes (about two thirds) and signs of aneuploidy (about one fifth). Category four contains cells with supernumerary MTOCs (characterized by the presence ofγ-tubulin); some cells showed signs of aneuploidy (∼1/10). Cells in category five exhibit a combination of the phenotypes of categories three and four. Control cells transformed with the GFP-fusion vector without an insert showed that the mutant phenotypes were not caused by the transformation procedure or by expression of GFP alone.    
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