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

Abstract

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 α-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 α-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/.

We thank A. Belmont (University of Illinois) for giving us the multiple LacO/GFP-Lac repressor system, K. Hamazaki for GFP-HAC data analyses, K. Yoda for producing the anti-CENP-A antibody, N. Nozaki (Kanagawa Dental College) for producing anti-CENP-A and -B antibodies, and V. Larionov (NCI/NIH) for critical reading.

This work was supported by a grant-in-aid for Scientific Research on Priority Areas (B), Core Research for Evolutional Science and Technology (CREST), and Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Science, Sports, and Culture of Japan and by a grant-in-aid from the Cell Science Research Foundation. This research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.