Histone-GFP fusion protein enables sensitive analysis of chromosome dynamics in living mammalian cells.
BACKGROUND
The amplification of oncogenes in cancer cells is often mediated by paired acentric chromatin bodies called double minute chromosomes (DMs), which can accumulate to a high copy number because of their autonomous replication during the DNA synthesis phase of the cell cycle and their subsequent uneven distribution to daughter cells during mitosis. The mechanisms that control DM segregation have been difficult to investigate, however, as the direct visualization of DMs in living cells has been precluded because they are far smaller than normal chromosomes. We have visualized DMs by developing a highly sensitive method for observing chromosome dynamics in living cells.


RESULTS
The human histone H2B gene was fused to the gene encoding the green fluorescent protein (GFP) of Aequorea victoria and transfected into human HeLa cells to generate a stable line constitutively expressing H2B-GFP. The H2B-GFP fusion protein was incorporated into nucleosomes without affecting cell cycle progression. Using confocal microscopy, H2B-GFP allowed high-resolution imaging of both mitotic chromosomes and interphase chromatin, and the latter revealed various chromatin condensation states in live cells. Using H2B-GFP, we could directly observe DMs in living cancer cells; DMs often clustered during anaphase, and could form chromosomal 'bridges' between segregating daughter chromosomes. Cytokinesis severed DM bridges, resulting in the uneven distribution of DMs to daughter cells.


CONCLUSIONS
The H2B-GFP system allows the high-resolution imaging of chromosomes, including DMs, without compromising nuclear and chromosomal structures and has revealed the distinctive clustering behavior of DMs in mitotic cells which contributes to their asymmetric distribution to daughter cells.
Kanda
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Gene Expression Laboratory, The Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, California 92037, USA.
Sullivan
K F
KF
Wahl
G M
GM
D000818:Animals; D001483:Base Sequence; D002453:Cell Cycle; D002877:Chromosomes, Human / Q000378:metabolism* / Q000648:ultrastructure*; D017931:DNA Primers / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006657:Histones / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D009707:Nucleosomes / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D017931:DNA Primers; D006657:Histones; D008164:Luminescent Proteins; D009707:Nucleosomes; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins