Figure 5

ASPM^{IVS25+1G > T} mutation produces a novel splice variant with decreased efficiency for spindle pole localisation. A. Schematic of the ASPM protein showing the location of the ASPM^{IVS25+1G > T} splice site mutation (filled arrows with an upper diamond). Domains of the protein are indicated as described in the legend to Figure 1. Location of epitopes of anti-ASPM antibodies are shown beneath the protein. B. Fibroblasts at metaphase immunostained using 216-1 anti-ASPM antibody (N-terminal) and the 279-3 anti-ASPM antibody, raised against a C-terminal peptide sequence. Note the reduced ASPM localization at the mitotic spindle poles of ASPM^{IVS25+1G > T} cells. Scale bar = 10 μm. C. Chart representing integral intensity of ASPM 216-1 antibody staining at the spindle poles of ASPM^wtand ASPM^{IVS25+1G > T}fibroblast cells. Horizontal lines indicate the average integral intensity of the immunostaining. Standard error bars are present. ASPM^{IVS25+1G > T} was at statistically significantly lower level (asterisks) at the spindle poles than ASPM^wt when compared using a paired two tailed t-test. P = < 0000.1. D. Immunoblotting of fibroblast control (WT) and ASPM^{IVS25+1G > T}(Patient) lysates with 217-2 anti-ASPM antibody and anti β-actin. Equivalent levels of stable ASPM protein are expressed by each cell culture, indicating the IVS25+1G > T mutation does not induce nonsense mediated decay. E. Sequence analysis of exon 25-exon 26 control ASPM^wtand Patient ASPM^{IVS25+1G > T}cDNA. The point of removal of the nine nucleotides due to the IVS25+1G > T mutation is indicated by an asterisk. F. IVS25+1G > T mutation removes the exon 25 splice donor site and instigates the utilisation of an in frame splice donor site nine nucleotides downstream. The resultant ASPM^{IVS25+1G > T} protein is lacking just three amino acids (3326-3328).