LLOYD MATSUMOTO , HARUMI KASAMATSU , LAJOS PIKO , and JEROME

Mitochondrial DNA (mtDNA) replicative intermediates from S t r o n g y l o c e n t r o t u s p u r p u r a t u s oocytes were isolated by ethidium bromide-CsC1 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-CsC1 gradient, supporting the notion that displacement replication procedds 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. Mitochondria are among the cellular constituents which accumulate during oogenesis and are stored in the egg. As a result, animal eggs are greatly enriched in mitochondrial DNA (mtDNA). ~ Oo1 Abbreviations used in this paper: Cairns' form, replicative intermediate in which both daughter segments are double stranded; D-loop DNA, mitochondrial DNA with a single-stranded displacement loop of approximately 3% genome size; Exp-D DNA, replicative intermediate with an expanded D loop which may be totally or partially single stranded; KTE, 0.55 M KC1, 0.01 M cyte and egg mtDNA has been well characterized as to amount, location, circularity, and physicalchemical properties in several animal groups: amphibians (2, 3, 5), echinoderms (13, 14), and echiurids (4). However, there is relatively little information available on mitochondrial biogenesis and mtDNA replication in growing oocytes. In Tris-HCl, 0.001 M EDTA, pH 7.6; mtDNA, mitochondrial DNA; TE, 0.01 M Tris-HCl, 0.001 M EDTA, pH 7.6. The terminology adopted in this paper follows that of Robberson et al. (16). 146 THE JOURNAL OF CELL BIOLOGY . VOLUME 63, 1974 . pages 146-159 on A uust 2, 2006 w w w .jc.org D ow nladed fom vivo long-term labeling exper iments in the sea u rch in have d e m o n s t r a t e d i n c o r p o r a t i o n of [3H]thymidine into oocyte m t D N A (11, 14). Fine s t ructural studies of oogenesis in sea urchins (20) as well as de te rmina t ions of cytochrome c oxidase activity in developing oocytes of Urechis caupo (12) indicate tha t mi tochondr ia are synthesized cont inuously during oogenesis. The format ion of new mi tochondr ia in growing sea urchin oocytes occurs in the absence of nuclear D N A replication