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An Artificially Constructed De Novo Human Chromosome Behaves Almost Identically to Its Natural Counterpart during Metaphase and Anaphase in Living Cells

Tomohiro Tsuduki ,^1,2,, Megumi Nakano,^1,3, Nao Yasuoka,¹ Saeko Yamazaki,¹ Teruaki Okada,¹ Yasuhide Okamoto,¹ and Hiroshi Masumoto^1,3*

Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan,¹ Graduate School of Agriculture, Shinshu University, 8304, Minamiminowa, Kamiina, Nagano 399-4598, Japan,² Laboratory of Biosystems and Cancer, National Cancer Institute, National Institutes of Health, Bldg. 37, Room 5040, 9000 Rockville Pike, Bethesda, Maryland 20892³

Received 27 February 2006/ Returned for modification 17 April 2006/ Accepted 25 July 2006

Human artificial chromosomes (HACs) are promising reagents for the analysis of chromosome function. While HACs are maintained stably, the segregation mechanisms of HACs have not been investigated in detail. To analyze HACs in living cells, we integrated 256 copies of the Lac operator into a precursor yeast artificial chromosome (YAC) containing -satellite DNA and generated green fluorescent protein (GFP)-tagged HACs in HT1080 cells expressing a GFP-Lac repressor fusion protein. Time-lapse analyses of GFP-HACs and host centromeres in living mitotic cells indicated that the HAC was properly aligned at the spindle midzone and that sister chromatids of the HAC separated with the same timing as host chromosomes and moved to the spindle poles with mobility similar to that of the host centromeres. These results indicate that a HAC composed of a multimer of input -satellite YACs retains most of the functions of the centromeres on natural chromosomes. The only difference between the HAC and the host chromosome was that the HAC oscillated more frequently, at higher velocity, across the spindle midzone during metaphase. However, this provides important evidence that an individual HAC has the capacity to maintain tensional balance in the pole-to-pole direction, thereby stabilizing its position around the spindle midzone.

Supplemental material for this article may be found at http://mcb.asm.org/.

T.T. and M.N. contributed equally to this work.

Present address: Kitayama Labes Co., Ltd. 8046-1, Nishiminowa, Ina 399-4501, Japan.