Circular mitochondrial DNA from Xenopus laevis and Rana pipiens

Mitochondrial DNA from oocytes of Xenopus laevis and Rana pipiens and from the liver of Gallus domesticus was studied by electron microscopy using the Kleinschmidt technique. A high percentage of circular molecules, either highly twisted or open, was observed in all preparations. The mean contour length of circles from X. laevis was 5.40 , from R. pipiens 5.56 and from G. domesticus 5.26 . Highly twisted circles were found in greater abundance in a fresh preparation than in preparations left standing for 3 months. These molecules are considered to be the native form of mitochondrial DNA.     

A restriction map of Xenopus laevis mitochondrial DNA

The mitochondrial DNA from Xenopus laevis is a 17.4 x 10(3)-base-pair circular DNA molecule. The mapping of this DNA, using 19 different restriction endonucleases is reported here. The sites are as follows: 1 for BamHI, PstI, SacI, SalI, BalI; 2 for BglII, SacII, EcoRI, ClaI, 3 for XhoI, 4 for AvaI, XbaI, PvuII, 5 for HindIII, 6 for HhaI, BclI, HpaI, 10 for AvaII and 11 for HincII. The same sites (except for one of the two ClaI sites) are observed in the molecule cloned in pBR322 DNA. The fragments corresponding to 62 cleavage sites have all been ordered and precisely located. They provide suitable conditions for further investigations connected with the study of replication and nucleotide sequence determination of this molecule.     

Accumulation of mitochondrial DNA during oogenesis in Xenopus laevis

The mitochondrial DNA (mtDNA) content of Xenopus laevis oocytes at various stages of oogenesis has been determined by molecular hybridization with ³H-labeled complementary RNA (cRNA). The previtellogenic oocyte less than 250 μm in diameter (stage 1) contains 0.95 ± 0.47 ng of mtDNA. Accumulation of mtDNA proceeds until stage 4 (500–750 μm diameter oocyte), by which time a steady-state level of 4.28 ± 0.40 ng/oocyte is attained. Using the hybridization assay, the stage 6 (full-grown) Xenopus oocyte contains 4.51 ± 0.69 ng of mtDNA, compared to the previously reported value of 3.8 ng determined by direct measurement on the unfertilized egg. There appears to be a reasonable correlation, therefore, between the termination of mtDNA accumulation and the dispersal of the juxtanuclear, mitochondrial aggregate (Balbiani body) at the onset of vitellogenesis in Xenopus. It is concluded that the enormous complement of oocyte mitochondria is accumulated well before the end of oocyte growth and is maintained at a constant level during the remainder of oogenesis, through maturation, fertilization, and on into early development.     

Testicular and androgen dependence of skin gland morphology in the anurans,Xenopus laevis and Rana pipiens

In anuran amphibians, there is increasing evidence that exocrine glands dispersed throughout the general integument are secondary sex characters (SSC). Following the recent discovery of sexually dimorphic “breeding glands” in the dorsum of male Rana pipiens, we studied the effects of castration and testosterone treatment on the dorsal skin glands of male Xenopus laevis and R. pipiens to determine whether the dorsal breeding glands, or any other dorsal skin glands, are androgen dependent. The dorsal skin glands of X. laevis were unaffected by androgen status. By contrast, in R. pipiens, breeding, mucous, and seromucous glands responded to testosterone stimulation. Mucous glands were significantly (P < 0.05) larger in testosterone-treated frogs than in castrates. There was a large, but statistically insignificant, increase in the size of the dorsal breeding glands. Testosterone treatment also increased the epithelial cell height of breeding and seromucous glands (P < 0.05). In the skins of castrated and testosterone-treated frogs, there was a reciprocal relationship between the abundance of seromucous and breeding glands: in castrates, seromucous glands were abundant and breeding glands virtually absent, whereas in testosterone-treated frogs, breeding glands were abundant and seromucous glands less common. The total number of the two gland types was similar in both treatment groups. Glands that appeared to be intermediate in form between seromucous and breeding glands were observed in some frogs. These data suggest that seromucous glands may be the regressed form of breeding glands in the dorsal skin of R. pipiens and that the dorsal skin of R. pipiens is a SSC. © 1993 Wiley-Liss, Inc.     

A DNA helicase from Xenopus laevis ovaries

A DNA helicase was extensively purified from Xenopus laevis ovaries. The most purified fraction was free of DNA topoisomerase, DNA polymerase, and nuclease activities. The enzyme had a Stokes radius of 54 A and a sedimentation coefficient of 6-7.3 S, from which a native molecular weight of 140,000-170,000 was calculated. DNA helicase activity required Mg2+ or Mn2+ and was dependent on hydrolysis of ATP or dATP. Monovalent cations, K+ and Na+, stimulated DNA unwinding with an optimum at 130 mM. DNA-dependent ATPase activity copurified with the X. laevis DNA helicase. Double-stranded and single-stranded DNA were both cofactors for the ATPase activity, but single-stranded DNA was more efficient. The molecular weight, monovalent cation dependence, cofactor requirements, and elution from single-stranded DNA-cellulose suggest that the X. laevis DNA helicase is different from previously described eukaryotic DNA helicases.     

Status of RNAs, localized in Xenopus laevis oocytes, in the frogs Rana pipiens and Eleutherodactylus coqui

Early development in the frog model, Xenopus laevis, is governed by RNAs, localized to the vegetal cortex of the oocyte. These RNAs include Xdazl RNA, which is involved in primordial germ cell formation, and VegT RNA, which specifies the mesoderm and endoderm. In order to determine whether orthologues of these RNAs are localized and have similar functions in other frogs, we cloned RpDazl and RpVegT from Rana pipiens, a frog that is phylogenetically distant from X. laevis. RNAs from both genes are localized to the vegetal cortex of the R. pipiens oocyte, indicating that the vegetal localization is likely the basal state. The animal location of EcVegT RNA in Eleutherodactylus coqui that we found previously (Beckham et al., 2003) is then a derived state, probably due to the great increase in egg size required for direct development of this species. To answer the question of function, we injected RpVegT or EcVegT RNAs into X. laevis embryos, and assayed animal caps for gene expression. Both of these RNAs induced the expression of endodermal, mesodermal, and organizer genes, showing that the function of RpVegT and EcVegT as meso-endodermal determinants is conserved in frogs. The RNA localizations and the function of VegT orthologues in germ layer specification may be synapomorphies for anuran amphibians.     

Regulated Formation of Extrachromosomal Circular DNA Molecules during Development in Xenopus laevis

Extrachromosomal circular DNA molecules of chromosomal origin have been detected in many organisms and are thought to reflect genomic plasticity in eukaryotic cells. Here we report a developmentally regulated formation of extrachromosomal circular DNA that occurs de novo in preblastula Xenopus embryos. This specific DNA population is not detected in the male or female germ cells and is dramatically reduced in later developmental stages and in adult tissues. The activity responsible for the de novo production of extrachromosomal circles is maternally inherited, is stored in the unfertilized egg, and requires genomic DNA as a template. The formation of circular molecules does not require genomic DNA replication but both processes can occur simultaneously in the early development. The production of extrachromosomal circular DNA does not proceed at random since multimers of the tandemly repeated sequence satellite 1 were over-represented in the circle population, while other sequences (such as ribosomal DNA and JCC31 repeated sequence) were not detected. This phenomenon reveals an unexpected plasticity of the embryonic genome which is restricted to the early developmental stage.     

A comparative study of the membrane potential from before fertilization through early cleavage in two frogs, Rana pipiens and Xenopus laevis

The membrane potential of Rana pipiens eggs (-55.0 mV +/- 11.2(16)) was more likely to recover from impalement and was always more negative than that of eggs of Xenopus laevis (-19.3 mV +/- 4.2(68)). It was also much more negative than previously reported. Essentially similar membrane resistance changes were measured in the two frog species through fertilization and cleavage. Small transient depolarizations only associated with the onset of the fertilization potential in Xenopus could be prevented by hyperpolarizing the egg membrane prior to fertilization. Repolarization was variable and longer in Rana and often accompanied by large transient spontaneous depolarizations. Insemination time, the time between fertilization and cleavage and the first cleavage division cycle, were all about twice as long in Rana. Xenopus egg cleavage was invariably accompanied by pronounced transient hyperpolarizations that were essentially absent in Rana.     

DNA ligase I from Xenopus laevis eggs.

We have purified the major DNA ligase from Xenopus laevis eggs and raised antibodies against it. Estimates from SDS PAGE indicate that this DNA ligase is a 180 kDa protein. This enzyme is similar to the mammalian type I DNA ligase which is presumed to be involved in DNA replication. We have also analysed DNA ligase activity during X. laevis early development. Unfertilized eggs contain the highest level of activity reflecting the requirement for a large amount of DNA replicative enzymes for the period of intense replication following fertilization. In contrast with previous studies on the amphibians axolotl and Pleurodeles, the major DNA ligase activity detected during X. laevis early development is catalysed by a single enzyme: DNA ligase I. And the presence of this DNA ligase I in Xenopus egg before fertilization clearly demonstrates that the exclusion process of two forms of DNA ligase does not occur during X. laevis early development.     

A DNA binding protein from Xenopus laevis oocyte mitochondria

A DNA binding protein has been isolated, by affinity chromatography on DNA cellulose, from mitochondria and from purified mitDNA-protein complexes from oocytes of Xenopus laevis. This 12,500 daltons protein is polymeric in its native form and binds to DNA with a high efficiency. It exhibits an apparently preferential binding to the single-stranded fiber of the D loop structures.