DIFFERENTIATION OF MICRONUCLEI IN MOUSE BONE-MARROW CELLS - A COMPARISON BETWEEN CREST STAINING AND FLUORESCENT IN-SITU HYBRIDIZATION WITH CENTROMERIC AND TELOMERIC DNA PROBES

Immunofluorescent staining (CREST) of kinetochore proteins and in situ hybridization (FISH) with centromeric DNA probes are able to distinguish between micronuclei (MN) containing lagging chromosomes or acentric fragments. Different frequencies of signal-positive MN induced by mitomycin C (MMC) were obtained by Miller et al. (Mutagenesis, 6, 297-302, 1991) between CREST labelling and FISH with the mouse major-gamma-satellite DNA probe (major probe). Both modes of identifying the presence of an entire chromosome in a MN are theoretically prone to misclassification. Breaks induced in pericentric heterochromatin can produce fragment-containing MN with a major signal. Alternatively, alterations of kinetochore proteins can produce CREST-negative MN containing lagging chromosomes. To improve the reliability of MN differentiation two additional DNA probes, the mouse minor satellite DNA probe (minor probe) and the telomere repeat (5'-TTAGGG-3')(7), have been used and double labelling has been employed with minor/major and minor/telomere probes. At 1 mg/kg of MMC the labelling frequencies of MN with CREST and the minor probe were identical (18.5 and 19%, respectively) and the major probe showed a higher labelling rate (30.5%). Using double-labelling the difference between minor and major probe responses was confirmed (17 and 31.5%, respectively). At 5 mg/kg of MMC, CREST labelling gave the lowest (6%), the minor probe gave intermediate (10 and 11.5% after single- and double-labelling, respectively) and the major probe gave the highest signal frequencies (16.5 and 15% after single- and double-labelling, respectively). The CREST and the minor signal frequencies were not significantly different at either dose of MMC whereas the minor and the major signal frequencies were significantly different. The CREST and the major signal frequencies differed significantly as in the original observation by Miller et al. (1991). The telomere probe did not add to the accuracy of MN differentiation. Based on the results obtained in the present experiments the minor probe is recommended for FISH detection of MN containing lagging chromosomes.