Fig. 3. Minichromosome re-formation in the cell line 7C5HT1-19. (A) The proportion of cells containing minichromosomes (solid line) or the integrated alphoid YAC site (dashed line). 7C5HT1-19 cells were cultured in selective (square) or nonselective (circle) medium for the number of days indicated. More than 50 metaphase cells were analyzed for each observation. (B) 7C5HT1-19 metaphase chromosomes after 80 days in culture in either nonselective (left) or selective (middle and right) medium. FISH probes specific to the whole of human chromosome 16 (green) and to YAC arm sequences (red) were used. Arrowheads indicate alphoid YAC DNA. Scale bar: 2 µm. (C) FISH signal intensities with the chromosome 16 probe on re-formed minichromosomes relative to that of the host chromosome 16. The diamond indicates the average intensity for the short arm of chromosome 16. Bars indicate maximum and minimum values. (D) Assembly of CENPs on re-formed minichromosomes. Indirect immunofluorescence and simultaneous staining by FISH was performed to detect CENPs (green) and the YAC arm (red). Arrowheads indicate minichromosomes. Scale bar: 2 µm. (E) Stability of re-formed minichromosomes in 7C5HT1-19 cells after 60 days in nonselective medium. (F) RT-PCR analysis of bsr transcripts. (Upper) Portions of RT-PCR products amplified with primers specific to the bsr gene and the human ß-actin gene (1, 1/2, 1/4 and 1/10, 1/20, 1/40, respectively) were electrophoresed in agarose gel. (lower) Ratios of transcriptional products of bsr to those of the 7C5HT1-19 cell line at day 0 quantitated by real-time RT-PCR. Both analyses showed the same transcription levels of bsr. The relative copy number of bsr genes was quantitated by real-time PCR.