Fig. 6

Functional identification of centromeric sequences in M. murinus. a, a’: Female mouse lemur metaphase chromosomes (blue) were hybridized with Mm53 (green), showing that the 53 bp sequence, Mm53, was present at every centromere except for the two metacentric X chromosomes (arrows). Gray-scale image shows the Mm53 fluorescent signal alone, illustrating the vast difference in abundance among the mouse lemur chromosomes. b–b”: Combined immunostaining for the essential centromere protein CENP-A and FISH with the Mm53 probe showed that CENP-A was present at every mouse lemur chromosome, including the two X chromosomes (insets in b). Gray scale images of fluorescent signals for Mm53 (b’) and CENP-A (b”) are separated out to emphasize relatively equal amounts of CENP-A at each chromosome, despite varying amounts of Mm53 centromeric sequence. The two X chromosomes have functional centromeres but lack Mm53, indicating that the X centromere is defined by a novel sequence that remains unidentified. Multiple colocalization analyses (k1k2 overlap coefficient and Manders’ colocalization coefficient (MCC), without and with thresholding) were performed on individual metaphases (n = 10 for each dot plot) to measure colocalization of red (CENP-A) and green (Mm53) signals. These analyses emphasized that a high proportion of CENP-A overlapped with Mm53