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Mitochondrial D N A (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 soocytes were isolated by ethidium bromide-CsC1 density gradient centrifugation and examined by electron microscopy after formamide spreading. In some experiments, the m t D N A was radioactively labeled by exposing isolated oocytes to [3H]thymidine. Oocyte m t D N A 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 m t D N A 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 m t D N A is stored as clean circular duplexes. Some novel structural variants of superhelical circular D N A (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). ~ Oo-1 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).

The occurrence and types of complex forms and replicative intermediates of mitochondrial DNA (mtDNA) were investigated in tissues from C57BL/6J mice aged 10-11 months or 29-30 months. Total mtDNA from brain, heart, kidney and liver was isolated in ethidium bromide-CsCl gradients and examined by electron microscopy after aqueous or formamide spreading. Contour length measurements indicated no difference in the monomer size of mtDNA according to either tissue or donor age. The frequencies of catenated mtDNA, ranging from 4 to 8%, varied significantly according to tissue but changed relatively little as a result of donor age. The main age-related effect observed in this study was a significant increase in the frequency of circular dimers, from about 0.05% in adult tissues to 0.3% in kidney, 0.5% in liver, 0.6% in heart and 1.9% in brain of senescent mice. The frequency of D-loop DNA varied from 30 to 60% and that of larger replicative intermediates from 1 to 10%, suggesting differences in the rate of mtDNA replication according to tissue. The frequencies and types of the various replicative intermediates were unaffected by donor age.

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We have prepared a partial gene library of sheared DNA from the fungus fly, Sciara coprophila, by dA-T tailing and insertion into pBR322. Two ribosomal DNA clones which differ from the usual ribosomal DNA organization in this organism were studied in detail. Clone pBc 1L-1 has an intervening sequence of 1.4 kb, and clone pBc 6D-6 has an intervening sequence of 0.9 kb. These intervening sequences occur in about the same position in 28S rDNA, but do not appear to share sequence homology with one another. Previously we found that 90% of Sciara ribosomal DNA is homogenous and lacks an intervening sequence, and our present data explains the size heterogeneity found in most of the remaining 10%. We have found no evidence of size heterogeneity in the nontranscribed spacer.

The majority of haploid frogs are non-viable and few survive past early larval stages. While animals vary with respect to specific abnormalities, they succumb to the variety of defects which collectively comprise the haploid syndrome. This poor viability has been ascribed to unmasked lethal and semi-lethal genes or to alterations in the nucleocytoplasmic ratio. This investigation was designed to test these hypotheses by determining the uniformity of anomalies in clones of isogenic haploid embryos.Animal hemisphere nuclei of stage 10 androgenetic haploids of Rana pipiens were transpIanted into activated and enucleated eggs of uniform size. Haploid embryos were selected from eggs whose cleavage times coincided with those of the fertilized controls. Genetically identical haploid embryos displayed differences i n the variety as well as the severity of abnormalities. Thus, specific lethal genes are not responsible for the variability in defects seen in the haploid syndrome. We ascribe haploid inviability and variations within isogenic clones to a lack of heterozygosity of the haploid nucleus with resultant inability of the haploid nucleus to function in a heterogeneous environment, i.e., the egg cytoplasm.During embryonic development of haploid frog embryos, the majority of the organisms are non-viable. Few haploids survive past early larval stages. Cleavage of haploid embryos occurs at a normal rate; thereafter, development is characterized by delayed differentiation and stunted growth. As development progresses a variety of defects arise in several organs. These defects have been described in detail (Porter, '39; Briggs, '49; Miyada, '60; Volpe and Dasgupta, '62) for androgenetic and gynogenetic haploids. Briefly, the abnormalities are in the form of shortened neural plates, changes in body proportions, microcephalic heads, edematous abdomens, and poorly developed circulatory systems. While abnormalities vary from embryo to embryo in a group of haploids, both in their nature and degree of severity, collectively these deficiencies are referred to as the haploid syndrome. The syndrome is essentially similar in androgenetic and gynogenetic haploids.Hertwig ('13) proposed altered nucleocytoplasmic ratio as being responsible for the poor viability of haploid embryos.Briggs ('49) the development of androgenetic haploids produced from large and small eggs of R a n a pipiens. Haploids which developed from smaller eggs lived longer and exhibited deficiencies to a less severe extent. Subtelny ('58) rigorously tested Hertwig's 19 13 proposal by transplanting haploid nuclei into activated and enucleated eggs of Rana pipiens. Some of these eggs cleaved later than fertilized controls. These embryos were demonstrated to be diploids. It was reasoned that the haploid nucleus divided once while the cytoplasm failed to do so. Thus, these embryos were considered to be homozygous diploids whose nucleo-cytoplasmic ratio was not altered. Subtelny's rationale was that if these homozygous diploids survived, then haploid deaths cou...