JCS02702 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Minichromosome was generated from input DNA, and did not incorporate detectable host chromosomal sequences. (A) FISH analysis of S026 cell line was performed using BAC probe (red) and mixture probe (green) of intra-Alu (278A/278B) and inter-Alu (517/TC65) PCR products amplified from HT1080 genome. Chromosomes were counterstained with DAPI (gray). Minichromosome overlapped with BAC signal was not detected with human genomic Alu sequence-specific probes. Scale bars are 2 mm. Arrow indicates minichromosome. (B) We investigated whether minichromosome in S026 cells incorporates any host chromosomal alphoid DNA other than a21-I. Labeled alphoid DNA amplified from HT1080 genome using a(1)18a/b, a(Y)a/b and CENP-B box primers (CB15a/b) were used as an pan-alphoid probe and a 25-fold excess amount of unlabeled p11-4 (a21-I) DNA was added to the mixture as a competitor (Masumoto et al., 1998). Pan-alphoid probe signal overlapping with the minichromosome detected with BAC probe (a) disappeared when unlabeled a21-I competitor DNA was added (b).

• Supplemental Figure 2 -

  Fig. S2. HAC formation including 7c5-SV/CMV BAC DNA. 7C5-SV/CMV BAC DNA and 7C5-basic BAC DNA in a ratio of 0.1:1 were mixed, introduced into HT1080 cells and selected with both BS and G418. Overall transformation efficiency was reduced simply correlating with the reduction of the DNA amount (ratio) of CMV-bgeo marker gene. However, de novo HAC forming activity was recovered up to 16% (3 of 19 analyzed cell lines, 7C5 mix in Table 1) among the transformants. The signals for 7C5-SV/CMV BAC DNA were only detected on the HAC overlapped with extrachromosomal signals detected by a BAC probe or alphoid probe (A). In addition, CENP-A, -B and -C signals were also detected on a HAC (B). These results indicate that the existence of 7c5-SV/CMV BAC molecules itself and the transcriptional activities from the both vector arms into the insert alphoid DNA themselves are not inhibitory for the de novo HAC formation activity if 7C5-basic BAC DNA can supply something missing from the left arm of 7c5-SV/CMV BAC by the transcriptional activation. (A) Metaphase chromosomes from the HAC cell lines generated from the co-transfection, were analyzed by FISH with CMV-bgeo probe (green) and BAC probe or a21-I probe (red). Arrows indicate the HAC. CMV-bgeo signals were not detected in any host chromosomal loci except on extra-minichromosome. (B) Metaphase chromosomes from the same HAC cell line, were analyzed by FISH with BAC probe (red) in combination with indirect immunofluorescence using antibodies against CENPs (green). Chromosomes were counterstained with DAPI (blue or gray).

• Supplemental Figure 3 -

  Fig. S3. Localization patterns of CENP-A and YFP-HP1a on HAC. Metaphase chromosomes were stained by immunofluorescence (A) with CENP-B (green) and anti-GFP antibodies (red) or (B) with CENP-A (red) and anti-GFP antibodies (green) in combination with FISH using BAC probe (red). Chromosomes were counterstained with DAPI (gray). Arrows indicate the HAC. (c) The merged image of BAC (red), YFP-HP1a (green) and DAPI (blue) signals. (d) The merged image of CENP-A (red), YFP-HP1a (green) and DAPI (blue) signals. YFP-HP1a signals were detected at the inner region of CENP-A signals on the HAC and at pericentromeric heterochromatin regions on host chromosomes. Scale bars show 10 mm.