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Published October 1974 | Published
Journal Article Open

Mitochondrial DNA replication in sea urchin oocytes


Mitochondrial DNA (mtDNA) replicative intermediates from Strongylocentrotus purpuratus oocytes were isolated by ethidium bromide-CsCl density gradient centrifugation and examined by electron microscopy after formamide spreading. In some experiments, the mtDNA was radioactively labeled by exposing isolated oocytes to [3H]thymidine. Oocyte mtDNA replication appears to follow the displacement loop model outlined in mouse L cells. There are differences in detail. The frequency of D-loop DNA is much lower in oocytes, suggesting that the relative holding time at the D-loop stage is shorter. Duplex synthesis on the displaced strand occurs early and with multiple initiations. The frequency of totally duplex replicative forms, or Cairns' forms, is the highest reported for mtDNA. The differences may be related to the fact that oocyte mtDNA replication occurs in the absence of cell division and need not be coordinated with a cell cycle. Molecules with expanded D loops banded in the intermediate region between the lower and upper bands in an ethidium bromide-CsCl gradient, supporting the notion that displacement replication proceeds on a closed circular template which is subject to nicking-closing cycles. In mature sea urchin eggs, replicative forms are absent and virtually all the mtDNA is stored as clean circular duplexes. Some novel structural variants of superhelical circular DNA (molecules with denaturation loops and double branch-migrated replicative forms) are reported.

Additional Information

© 1974 by Rockefeller University Press. RUP grants the public the non-exclusive right to copy, distribute, or display the Work under a Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/ and http://creativecommons.org/licenses/by-nc-sa/3.0/legalcode. Received for publication 1 March 1974, and in revised form 3 May 1974. We thank Ms. Phyllis Hotchkin and Ms. Roxanne Meyer for technical assistance. This work was supported in part by United States Public Health Service, National Institutes of Health grants CA08014 from the National Cancer institute and GM15327 from the National Institute of General Medical Sciences. L. Matsumoto was supported by National Institutes of Health postdoctoral fellowship 1 FOCA53287-01 from the National Cancer Institute and H. Kasamatsu was the recipient of a postdoctoral fellowship from the Helen Hay Whitney Foundation. This is contribution no. 4845 from the Division of Chemistry and Chemical Engineering, California Institute of Technology.

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