More Than Numbers David Yonggi Cho
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The number of foci per nucleus. We counted the number of foci per nucleus by using a fluorescence microscope. The results are shown in Fig. 5. The number of foci per nucleus was significantly increased by Pvu II, Rsa I and Bam HI. We also observed the foci with no colocalized focal proteins by using the double labeling with 53 BP1. The results are shown in Fig. 6. The number of the foci with no colocalized foci was significantly increased by Pvu II. The foci-positive nuclei increased more than the foci-negative nuclei.
The main purpose for this study was to observe the effect of restriction endonuclease on the distribution of focal proteins. Some of the foci colocalized with focal proteins and the others were not colocalized. If the foci formation were dependent on the presence of the focal proteins, the number of foci positive cells should increase in proportion to the number of colocalized foci positive cells. But, on the contrary, we observed that the number of foci positive cells was independent of the number of colocalized focal proteins. These results suggest that the nuclei with foci may be derived from other cells in addition to the cells with localized proteins. So we also carried out a double labeling experiment with 53BP1 to identify the nuclei with foci and the colocalization of the foci with the focal proteins. The results indicated that two of the foci (Figs. 3A, B, J, K and N) colocalized with the focal proteins. However, the number of nuclei with foci as well as the number of foci positive nuclei increased remarkably. These results suggest that the foci positive nuclei are not derived from the cells with localized proteins. On the other hand, we did not observe any colocalized foci in the nuclei with no foci. These results indicate that when a nucleus containing a number of localized proteins is subjected to a restriction endonuclease, the nuclei with the localized proteins show foci of damage induced by the restriction endonuclease. Moreover, we observed that the damage foci induced by the restriction endonuclease were heterogeneous in the form of numerous fine granules (Fig. 4D).
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