DNA synthesis in a mitochondrial lysate of Xenopus laevis oocytes. H strand replication in vitro

Conditions for efficient replication in vitro of mitochondrial DNA L strand into H strand products have been established. Gel electrophoresis and hybridization analyses of the products show that neosynthesized H strands are progressively elongated from the D-loop region, and some of them are synthesized as full-length molecules. Evidence for initiation of these H strands de novo is presented. In contrast, there is no detectable L strand synthesis in vitro in this system. This may prove useful for analyzing the distinct molecular mechanisms operating at OH and OL. Use of specific inhibitors indicates that DNA synthesis in the mitochondrial lysate in vitro requires DNA polymerase gamma. These observations support the conclusion that replication in vitro in this system closely resembles the first steps of mitochondrial DNA replication in vivo.     

Adenosine 3': 5' Monophosphate Receptor Sites in Xenopus laevis Oocytes

Adenosine 3': 5' cyclic monophosphate (cyclic AMP) binds to proteins present in the 100,000 g supernatant fractions of full-grown oocytes of Xenopus laevis . Optimal pH for the binding is 4.0. Two receptor binding sites have been characterized by density gradient centrifugation as peaks with sedimentation constants of 4.6 S and 5.9 S. The apparent dissociation constants for the two cyclic AMP binding sites are 7 nM and 40 nM. The total cyclic AMP binding capacity of oocyte cytosol is 15.8 ± 2.2 fmoles/oocyte and remains constant during meiotic maturation induced by progesterone.     

Mitochondrial DNA-binding proteins that bind preferentially to supercoiled molecules containing the D-loop region of Xenopus laevis mtDNA

From the bulk of the Xenopus laevis mitochondrial proteins insoluble in 1% Triton X-100 + 1M NaCl, we have isolated, by DNA-cellulose chromatography, a protein fraction enriched in DNA-binding proteins. This fraction contains proteins showing a specific affinity for supercoiled DNA molecules containing the mitochondrial DNA displacement-loop region, as measured by filter binding and competition assays.     

Mapping of mitochondrial 4 S RNA genes in Xenopus laevis by electron microscopy

The distribution of sites hybridizing with mitochondrial 4 S RNA molecules on mitochondrial DNA of Xenopus laevis has been mapped in relation to the ribosomal RNA genes and EcoRI restriction endonuclease sites. RNA molecules linked to ferritin were employed for this purpose. We have obtained evidence for 15 4 S RNA sites on the H-strand and six sites on the L-strand of X. laevis mtDNA^∥. An indication of the possible existence of one additional site on the H-strand and four additional sites on the L-strand has been obtained. One 4 S RNA site is located in the gap between the two rRNA genes, and one site flanks each outside end of the rRNA genes. The other 4 S RNA sites are distributed almost evenly throughout both strands of the mtDNA. A comparison with the map of 4 S RNA sites on the mtDNA of HeLa cells (Angerer et al., 1976) suggests considerable evolutionary conservation of site organization.     

Unique features in the mitochondrial D-loop region of the European seabass Dicentrarchus labrax

We have cloned and sequenced the displacement-loop (D-loop) region of the mitochondrial DNA (mtDNA) from the European seabass Dicentrarchus labrax (Dl). This sequencing revealed the presence of four tandemly repeated elements (R1, R2, R3 and R4); the individual variation in mtDNA total length is entirely accounted for by their variable number. The individuals examined also possessed an imperfect copy of one of the tandem repeats (ΨR2). At least one termination-associated sequence (TAS) is present in each of the repeats and in two copies 5′ upstream from the tandem array as well. The alignment of the Dl D-loop region with D-loop sequences from four other Teleosts and one Chondrosteus showed the Dl sequence to be larger than that of other fish. The extraordinary length of the D1 D-loop sequence is also due to the 5′ and 3′ regions that are flanking the tandem array, the largest ones to date analyzed in fish. In this study, we also report the unique organization and localization of putative TAS and conserved-sequence block (CSB) elements, and the presence of a conserved 218-bp sequence in the D1 D-loop region.     

Evolution of the cetacean mitochondrial D-loop region.

We sequenced the mitochondrial DNA D-loop regions from two cetacean species and compared these with the published D-loop sequences of several other mammalian species, including one other cetacean. Nucleotide substitution rates, DNA sequence simplicity, possible open reading frames (ORFs), and potential RNA secondary structure were investigated. The substitution rate is an order of magnitude lower than would be expected on the basis of reports on human sequence variation in this region but are consistent with interspecific primate and rodent D-loop sequence variation and with estimates of substitution rates from whole mitochondrial genomes. Deletions/insertions are less common in the cetacean D-loop than in other vertebrate species. Areas of high sequence simplicity (clusters of short repetitive motifs) across the region correspond to areas of high sequence divergence. Three regions predicted to form secondary structures are homologous to such putative structures in other species; however, the presumptive structures most conserved in cetaceans are different from those reported for other taxa. While all three species have possible long ORFs, only a short sequence of seven amino acids is shared with other mammalian species, and those changes that had occurred within it are all nonsynonymous. We conclude that DNA slippage, in addition to point mutation, contributes to the evolution of the D-loop and that regions of conserved secondary structure in cetaceans and an ORF are unlikely to contribute significantly to the conservation of the central region.     

Mapping the nucleotide and isoform-dependent structural and dynamical features of Ras proteins

Ras GTPases are conformational switches controlling cell proliferation, differentiation, and development. Despite their prominent role in many forms of cancer, the mechanism of conformational transition between inactive GDP-bound and active GTP-bound states remains unclear. Here we describe a detailed analysis of available experimental structures and molecular dynamics simulations to quantitatively assess the structural and dynamical features of active and inactive states and their interconversion. We demonstrate that GTP-bound and nucleotide-free G12V H-ras sample a wide region of conformational space, and show that the inactive-to-active transition is a multiphase process defined by the relative rearrangement of the two switches and the orientation of Tyr32. We also modeled and simulated N- and K-ras proteins and found that K-ras is more flexible than N- and H-ras. We identified a number of isoform-specific, long-range side chain interactions that define unique pathways of communication between the nucleotide binding site and the C terminus.     

Nucleotide sequence of a Xenopus laevis mitochondrial DNA fragment containing the D-loop, flanking tRNA genes and the apocytochrome b gene

Extensive corrections of the nucleotide sequence of the Xenopus laevis mitochondrial (mt) displacement (D) loop and surrounding genes [Wong et al., Nucl. Acids Res. 11 (1983) 4977-4995] are reported, including addition of two stretches of nucleotides and 60 scattered modifications. The additional sequences presented here correspond to the apocytochrome b gene, the tRNAGlu gene and part of URF6. This allows us to propose a conformational model for the X. laevis apocytochrome b protein and also permits comparisons with mammalian mtDNA. The D-loop sequence is poorly conserved except for sequences involved in the regulation of the mt genome (conserved sequence blocks and the DNA polymerase stop sequences). On the other hand, all genes show marked conservation both of their nucleotide sequence and their respective location on the mt genome. Organization of the genetic information described for mammalian mtDNA also holds for the X. laevis mtDNA. This result strongly suggests that all animal vertebrate mtDNAs have followed the same evolutionary pathway.     

Changes in D-loop frequency and superhelicity among the mitochondrial DNA molecules in relation to organelle biogenesis in oocytes of Xenopus laevis

Changes in mitochondrial DNA (mtDNA) replication activity are known to occur during oogenesis in Xenopus laevis. Electron microscopic and electrophoretic analyses carried out on mtDNA molecules at different vitellogenic stages show that 1. The frequency of displacement loop (D-loop) forms is correlated with the intensity of mitochondrial biogenesis. 2. Most of the native molecules as well as the D-loop carrying ones are superhelical. 3. Four families of different superhelicity may be distinguished and D-loops are found only in the most superhelical ones. To account for the changes in the frequencies of the D-loop forms and of the different topological types during cell differentiation, it is suggested that the initiation of a new replication occurs only on the most superhelical molecules and that some control of superhelicity may exist in mitochondria.     

Mapping neurogenesis onset in the optic tectum of Xenopus laevis

Neural progenitor cells have a central role in the development and evolution of the vertebrate brain. During early brain development, neural progenitors first expand their numbers through repeated proliferative divisions and then begin to exhibit neurogenic divisions. The transparent and experimentally accessible optic tectum of Xenopus laevis is an excellent model system for the study of the cell biology of neurogenesis, but the precise spatial and temporal relationship between proliferative and neurogenic progenitors has not been explored in this system. Here we construct a spatial map of proliferative and neurogenic divisions through lineage tracing of individual progenitors and their progeny. We find a clear spatial separation of proliferative and neurogenic progenitors along the anterior‐posterior axis of the optic tectum, with proliferative progenitors located more posteriorly and neurogenic progenitors located more anteriorly. Since individual progenitors are repositioned toward more anterior locations as they mature, this spatial separation likely reflects an increasing restriction in the proliferative potential of individual progenitors. We then examined whether the transition from proliferative to neurogenic behavior correlates with cellular properties that have previously been implicated in regulating neurogenesis onset. Our data reveal that the transition from proliferation to neurogenesis is associated with a small change in cleavage plane orientation and a more pronounced change in cell cycle kinetics in a manner reminiscent of observations from mammalian systems. Our findings highlight the potential to use the optic tectum of Xenopus laevis as an accessible system for the study of the cell biology of neurogenesis. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1328–1341, 2016     

Ribosomal-protein synthesis is not autogenously regulated at the translational level in Xenopus laevis

Whether ribosomal-protein synthesis in Xenopus laevis is autogenously controlled at the translational level as is known to occur in prokaryotes has been studied. For this purpose ribosomal (r) proteins were prepared from X. laevis ribosomal subunits and group fractionated by ion-exchange chromatography. They were then added to an in vitro translation system directed by an oocyte mRNA fraction which contains template activity for r proteins. The synthesized radioactive products were analyzed by 2D gel electrophoresis and compared with controls. Similarly in vivo experiments were performed by microinjection of the fractionated proteins into the cytoplasm of Xenopus oocytes followed by incubation with [35S]methionine for different times. In all the experiments no evident effect of r proteins on the translation of their own mRNA was observed.     

Changes in mitochondrial respiration during the development of Xenopus laevis

Mitochondria isolated from Xenopus laevis embryos at various developmental stages show a good oxidative capacity and an acceptable respiratory control provided that certain requirements are fulfilled. The rates of respiration with pyruvate and Krebs' cycle intermediates, especially with citrate and isocitrate, are very low during cleavage stages and increase after gastrulation. Glutamate in the presence of malate is the only substrate to be readily oxidized during early development and its rate of oxidation decreases after gastrulation. These results, together with the altered sensitivity of embryonic mitochondria towards azide, support the view that the oxidative metabolism undergoes important changes around gastrulation and is associated with mitochondrial differentiation.     

Polymorphic sequence in the D-loop region of equine mitochondrial DNA

The D-loop regions in equine mitochondrial DNA were cloned from three thoroughbred horses by polymerase chain reaction (PCR). The total number of bases in the D-loop region were 1114bp, 1115bp and 1146bp. The equine D-loop region is A/T rich like many other mammalian D-loops. The large central conserved sequence block and small conserved sequence blocks 1, 2 and 3, that are common to other mammals, were observed. Between conserved sequence blocks 1 and 2 there were tandem repeats of an 8bp equine-specific sequence TGTGCACC, and the number of tandem repeats differed among individual horses. The base composition in the unit of these repeats is G/C rich as are the short repeats in the D-loops of rabbit and pig. Comparing DNA sequences between horse and other mammals, the difference in the D-loop region length is mostly due to the difference in the number of DNA sequences at both extremities. The similarities of the DNA sequences are in the middle part of the D-loop. In comparison of the sequences among three thoroughbred horses, it was determined that the region between tRNA^Pro and the large central conserved sequence block was the richest in variation. PCR primers in the D-loop region were designed and the expected maternal inheritance was confirmed by PCR-RFLP (restriction fragment length polymorphism).     

RNA 3' cleavage and polyadenylation in oocytes and unfertilized eggs of Xenopus laevis

Xenopus laevis histone H4 and H1 genes were transcribed in vitro to generate artificial precursor mRNAs (pre-mRNAs). These pre-mRNAs were microinjected into oocytes, matured oocytes, and unfertilized eggs of Xenopus laevis and their 3' cleavage and polyadenylation were investigated. In the oocyte nucleus both H4 and H1 pre-mRNAs were 3' cleaved but were not detectably polyadenylated. In the oocyte cytoplasm there was neither 3' cleavage nor polyadenylation of these histone pre-mRNAs. When injected into either matured oocytes or unfertilized eggs, the pre-mRNAs underwent 3' cleavage but this was inefficient when compared to the oocyte nucleus. In addition approximately 50% of the remaining uncleaved pre-mRNA was subject to a polyadenylation activity which added A tails of approximately 70 A residues. In contrast, artificial mouse beta-globin pre-mRNAs were not detectably 3' cleaved or polyadenylated in either microinjected oocytes or unfertilized eggs.     

The nucleotide sequence of oocyte 5S DNA in Xenopus laevis. II. The GC-rich region

The primary sequence of the GC-rich half of the repeating unit in X. laevis 5S DNA has been determined in both a single plasmid-cloned repeating unit and in the total population of repeatig units. The GC-rich half of the repeating unit contains a single long duplication of 174 nucleotides. The duplicated segment commences 73 nucleotides preceding the 5' end of the gene and terminates at nucleotide 101 of the gene. The duplicated portion of the gene, termed the pseudogene, differs by 10 nucleotides from the corresponding portion of the gene, and the remaining duplicated sequence of 73 nucleotides differs by 13 nucleotides. The plasmid-cloned repeating unit differs from the dominant sequence in the total population repeating units by 6 nucleotides in the GC-rich region. Evidence is provided that most of the CpG dinucleotides in 5S DNA are at least partially methylated.