Table 2a.
CENPs assembly and centromere function on introduced alphoid DNAs: HAC formation cell lines
| Input DNA size (vector) | Cell lines | Multiplicity of input DNA | CENPs assembly (%)a | de novo HACb | Loss ratec | |||||
|---|---|---|---|---|---|---|---|---|---|---|
| Left arm (copies) | Right arm (copies) | Total size of alphoid (kb)d | A | B | C | E | ||||
| Linear constructs | ||||||||||
| 70 kb (α7C5htel) | HT1–2e | 34 | 35 | 2500 | 100 | 100 | 100 | 100 | Yes | 0.0086 |
| 50 kb (del.24) | del.24HT3–14 | 35 | 42 | 1900 | 100 | 100 | 100 | 100 | Yes | 0.0049 |
| 30 kb (del.20) | del.20HT1–5 | 74 | 68 | 2100 | 100 | 100 | 100 | 100 | Yes | 0.00010 |
| del.20HT5–2 | 160 | 160 | 4800 | 100 | 100 | nt | 100 | Yes | nt | |
| Circular constructs | ||||||||||
| 60 kb (pWTR11.32) | W0210R-8f | − | − | 1400 | 100 | 100 | 100 | 100 | Yes | 0.0011 |
| 240 kb (pWTR11.128) | W11.128HT1–14 | − | − | 1500 | + | + | nt | nt | Yes | nt |
| aPercentage of centromere protein assembly was assessed based on colocalized signals from indirect immunostaining of CENP and FISH analysis of vector probes (n>20). | ||||||||||
| bNon-acquisition of host chromosomal DNA fragments was examined by FISH analysis using inter-/intra-Alu, other alphoid probes. | ||||||||||
| cThe number of HACs in 30–50 metaphase cells was scored by FISH using YAC probes and 11-mer probes at day 0 (N0) or at day 60 (N60) without selection. Values were calculated using the following formula: N60=N0 × (1–R)60. | ||||||||||
| dTotal size of alphoid DNA in multimer of input alphoid YAC was calculated by multiplying alphoid DNA size of input alphoid YAC by copy number of average of left arm and right arm. | ||||||||||
| eThese cell lines were produced by Ikeno et al (1998). | ||||||||||
| fThese cell lines were produced by Ohzeki et al (2002) | ||||||||||
| nt, not tested; +, assembly detected with ChIP assay. | ||||||||||