Structure of the mammalian kinetochore |
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Authors: | Hans Ris Patricia L Witt |
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Institution: | (1) Department of Zoology, University of Wisconsin-Madison, 53706 Madison, Wisconsin, USA |
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Abstract: | The structure of the mammalian trilaminar kinetocnore was investigated using stereo electron microscopy of chromosomes in
hypotonie solutions which unraveled the chromosome but maintained microtubules. Mouse and Chinese hamster ovary cells were
arrested in Colcemid and allowed to reform microtubules after Colcemid was removed. Recovered cells were then swelled, lysed
or spread in hypotonic solutions which contained D2O to preserve microtubules. The chromosomes were observed in thin and thick sections and as whole mounts using high voltage
electron microscopy. Bundles of microtubules were seen directly attached to chromatin, indicating that the kinetochore outer
layer represents a differential arrangement of chromatin, continuous with the body of the chromosome. In cells fixed without
pretreatment, the outer layer could be seen to be composed of hairpin loops of chromatin stacked together to form a solid
layer. The hypotonically-induced unraveling of the outer layer was found to be reversible, and the typical 300 nm thick disk
reformed when cells were returned to isotonic solutions. Short microtubules, newly nucleated after Colcemid removal, were
found not to be attached to the kinetochore outer layer, but were situated in the fibrous corona on the external surface of
the outer layer. This was verified by observations of thick sections in stereo which made it possible to identify microtubule
ends within the section. Thus, kinetochore microtubules are nucleated within the fibrous corona, and subsequently become attached
to the outer layer.
We dedicate this paper to Wolfgang Beermann on the occasion of his 60th birthday in appreciation of many years of friendship
and his pioneering contributions in the field of chromosome biology |
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