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 distingu ish between micronuclei (MN) containing lagging chromosomes or acentri c fragments. Different frequencies of signal-positive MN induced by mi tomycin C (MMC) were obtained by Miller et al. (Mutagenesis, 6, 297-30 2, 1991) between CREST labelling and FISH with the mouse major-gamma-s atellite DNA probe (major probe). Both modes of identifying the presen ce of an entire chromosome in a MN are theoretically prone to misclass ification. Breaks induced in pericentric heterochromatin can produce f ragment-containing MN with a major signal. Alternatively, alterations of kinetochore proteins can produce CREST-negative MN containing laggi ng chromosomes. To improve the reliability of MN differentiation two a dditional 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 probe s. At 1 mg/kg of MMC the labelling frequencies of MN with CREST and th e minor probe were identical (18.5 and 19%, respectively) and the majo r 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 w ere not significantly different at either dose of MMC whereas the mino r and the major signal frequencies were significantly different. The C REST 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 ob tained in the present experiments the minor probe is recommended for F ISH detection of MN containing lagging chromosomes.