Table II. Centromere protein assembly on introduced DNA.

Input DNA (Chromosomal event)	Cell line (MAC loss ratea)	Spread	Centromere protein assemblyb (%)				Multiplicity of BAC input DNA
			CENP-A	CENP-B	CENP-C	CENP-E	Synthetic alphoid	Neo	PI intensity (DNA amount)c
pWTR11.32
(artificial chromosome)	W0203 (0.00175)d	40	100	100	100	100	14	16	18 (1.2 Mb)
	W0206 (ND)d	50	100	100	+	+	26	10	30 (2.0 Mb)
	W0210R-8 (0.00105)d	50	100	100	+	+	23	16	24 (1.6 Mb)
	W0212 (<0.001)d	50	100	100	+	+	36	29	39 (2.6 Mb)
(ectopic integration)	W0210R-1	50	24	38	12	22	17	14
	Colocalization with CENP-Be	50	100	−	100	100
	W1203R-8	50	10	22	24	8	20	7
	Colocalization with CENP-Be	25	100	−	100	100
pMTR11.32f
(ectopic integration)	M1319	50	0	0	0	0	19	12
	M1318	50	0	0	0	0	43	29
pRF322B.192g
(ectopic integration)	B0104	50	0	12	0	0	20	16
	B0106	30	0	8	0	0	48	36
pRF322L.192
(ectopic integration)	L0102	50	0	0	0	0	42	38
	L0203	50	0	0	0	0	38	26

^aMAC 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.

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

^cSee Materials and methods.

^dFISH 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.

^eColocalization with CENP-B was determined from indirect immunofluorescence of CENP-B and other centromere proteins.

^fIn 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.

^gIn another four cell lines obtained by transformation with pRF322B, only CENP-B assembly was observed.