Table II.

Centromere protein assembly on introduced DNA

Input DNA
 (Chromosomal event)Cell line
 (MAC loss ratea)SpreadCentromere protein assemblyb (%)Multiplicity of BAC input DNA
CENP-ACENP-BCENP-CCENP-ESynthetic alphoidNeoPI intensity (DNA amount)c
pWTR11.32
(artificial chromosome)W0203 (0.00175)d40100100100100141618 (1.2 Mb)
W0206 (ND)d50100100++261030 (2.0 Mb)
W0210R-8 (0.00105)d50100100++231624 (1.6 Mb)
W0212 (<0.001)d50100100++362939 (2.6 Mb)
(ectopic integration)W0210R-150243812221714
Colocalization with CENP-Be50100100100
W1203R-8501022248207
Colocalization with CENP-Be25100100100
pMTR11.32f
(ectopic integration)M13195000001912
M13185000004329
pRF322B.192g
(ectopic integration)B010450012002016
B01063008004836
pRF322L.192
(ectopic integration)L01025000004238
L02035000003826
  • a MAC loss rate (R) was calculated using the following formula: N60 = N0 × (1 − R)60. N0 and N60 are the rates for MAC-containing cells in these cell lines at the time points of 0 and 60 d, respectively. During the 60 d, cells were cultured in the absence of G418, a selective drug.

  • b Percentage of centromere protein assembly was assessed based on colocalized signals from indirect immunofluorescence of centromere proteins and FISH analysis of BAC DNA probe.

  • c See Materials and methods.

  • d FISH analysis using inter-/intra-Alu, other alphoid, and Sat III probes indicated that all MACs from the four cell lines in this table were negative.

  • e Colocalization with CENP-B was determined from indirect immunofluorescence of CENP-B and other centromere proteins.

  • f In addition to the results for these two cell lines, results for another four cell lines obtained by transformation with pMTR11.32 also showed no centromere protein assembly on introduced DNA sites.

  • g In another four cell lines obtained by transformation with pRF322B, only CENP-B assembly was observed.