[Differentiation of interphase nucleolus organizers in embryonic pig kidney cells (PK cell line)]

The prometaphase karyotype of cell line PK contains two heteromorphous pairs of nucleolus organizers that belong to chromosomes 8a and 8, and to 10L and 10s. It was proposed that such heteromorphism may promote chromosome differentiating of interphase nucleolus organizers (INOs) with linear configuration. To test this assumption, we used two-dimensional (2D) preparations of methanol fixed PK cells surface stretched without hypotonic treatment. It was shown that in these preparations the large bulk of interphase PK cells contained 3-4 necklace-like linear structures arranged in nucleolar domains. The observed structures were positive in phase contrast and after DAPI-staining. Complimentary rDNA-FISH revealed that these structures were INOs, the largest iNO in individual cells containing prominent terminal rDNA FISH/DAPI signal. In accordance with the data on prometaphase analysis, the latter INOs belong to chromosomes 8a. As reported by Smetana and coworkers (1999), proteins of the nucleolar fibrillar center reacted preferentially with silver in methanol fixed unwashed smears of human peripheral lymphocytes. It was established that the same specific silver reaction is characteristic most probably of 2D preparations of methanol fixed PK cells. Both silver stained and rDNA-FISH linearized INOs had necklace-like or banded structure with different degrees of resolution. Banded INOs consisted of transverse argyrophilic structures: dense bands and loose interbands. High resolved banded INOs revealed a longitudinal splitting (binemic structure) of interband zone. Necklace-like INOs consisted of argyrophilic beads nearly two-fold more narrow than argyrophilic bands, and uninemic or silver-negative interbead zones. Our findings evidence that necklace-like INOs are typical for G1 and S phase cells, whereas banded INOs are characteristic of G2 cells. Among high resolved linear INOs, we found four reproducible patterns of silver staining, which could be combined it two homologous groups. Because each given pattern is unique for individual PK cells, we concluded that the patterns under study were chromosome specific. Using prometaphase analysis data, we determined chromosome affiliation for each of the four tested patterns of INO silver staining. High resolved INOs, belonging to different chromosomes, were further compared with regard to their average length and the mean of argyrophilic bead number per individual INO, in addition to the length and argyrophilic bead number ratios calculated for different INO pairs of individual cells. Surprisingly, we found that both the ratios, detected for most heteromorphous pair of homologous chromosomes 8a and 8, made only 1.26 +/- 0.02. In comparison, the similar length ratio for nucleolus organizers in chromosomes 8a and 8, calculated for individual prometaphase cells, reached 2.92 +/- 0.30.