DNA synthesis in a multi-enzyme system from Xenopus laevis eggs

Cytoplasm from unfertilized eggs of the frog Xenopus laevis was separated by DEAE-cellulose column chromatography into nine fractions. Supercoiled pXir 11 DNA molecules (pXir 11 is a Col El-based recombinant plasmid containing part of the Xenopus laevis 18S and 28S ribosomal genes and transcribed spacer region) were incubated with each fraction singly and in various combinations. After incubation for 4 hr at 26 degrees C, the pXir 11 DNA was reisolated and examined by electron microscopy. Using appropriate reaction conditions (pH 7.2, 10 mM Mg2+, 250 micron NTP, 50 50 micron dNTP, 50 MM KCl, fractions III and IV or VI), at least 5-10% of the input DNA was converted to theta structures (presumed intermediates in DNA replication).     

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.     

Mitochondria DNA synthesis in oocytes of Xenopus laevis

The process of attachment was studied in primary mouse kidney epithelial cell cultures by means of reflexion contrast microscopy, a method developed for studying the cell membrane-substrate relationship. The first in a series of events is simple adherence to the substrate, called close contact. This phenomenon is associated with the greatest extension of lamellar cytoplasm and the fewest number of cell nuclei/unit area. The nuclei of such cells are in close contact with the bottom portion of the cell membrane. Approx. 24 h after planting, as the cultures become more crowded, cells develop a different kind of attachment to the substrate—focal contacts—that are correlated with a decrease in lamellar cytoplasm. Cells detached from the substrate after close contact formation readily reattach, while cells detached after formation of focal contacts do not reattach. After incubation for periods greater than 5 days, the dense cultures degenerate and cells lose their attachment to the glass surface.     

Regulated replication of DNA microinjected into eggs of Xenopus laevis

Purified circular DNA of SV40 or polyoma virus has been injected into unfertilized eggs of Xenopus laevis. Injected DNA initiates and completes multiple rounds of semiconservative replication while observing cellular regulatory signals. Thus replication initiation of double-stranded templates is induced after the oocyte is matured in vitro by progesterone. Only one round of replication of injected DNA is observed in a single cell cycle. When protein synthesis is inhibited unreplicated molecules continue to initiate replication at an undiminished rate, but reinitiation on previously replicated molecules is completely and selectively abolished. The DNA sequence requirements for the replication of injected DNA have been investigated. A variety of procaryotic DNA molecules and circularized fragments of SV40 or polyoma DNA replicate, regardless of whether they contain the viral origin of DNA replication. These results suggest that a specialized DNA sequence is not essential for the initiation of semiconservative DNA replication in the Xenopus embryo, nor is a specialized sequence essential for the mechanism which prevents reinitiation on a molecule which has already replicated within a cell cycle. The possibility is discussed that viral origins of replication are not valid models for the eucaryotic chromosome but are adaptations for uncoupling viral replication from the mechanism which prevents reinitiation within a cell cycle.     

Metaphase protein phosphorylation in Xenopus laevis eggs.

Cytoplasmic extracts of metaphase (M-phase)-arrested Xenopus laevis eggs support nuclear envelope breakdown and chromosome condensation in vitro. Induction of nuclear breakdown is inhibited by AMPP(NH)P, a nonhydrolyzable ATP analog, but not by ATP or gamma-S-ATP, a hydrolyzable ATP analog, suggesting that protein phosphorylation may be required for M-phase nuclear events in vitro. By addition of [gamma-32P]ATP, we have identified in cytoplasmic extracts and in intact eggs at least six phosphoproteins that are present during M-phase but absent in G1/S-phase. These phosphoproteins also appear in response to partially purified preparations of maturation-promoting factor. A subset of these proteins are thiophosphorylated by gamma-S-ATP under conditions that promote nuclear envelope breakdown and chromosome condensation. Each of these proteins is phosphorylated on serine and threonine, and one, a 42-kilodalton protein, is also phosphorylated on tyrosine both in extracts and in intact eggs. These results indicate that activation of protein kinases accounts for at least part of the increased phosphorylation in M-phase and that both protein-serine-threonine kinases and protein-tyrosine kinases may play a role in controlling M-phase nuclear behavior.     

Periodic DNA synthesis in cell-free extracts of Xenopus eggs.

Cell-free extracts prepared from unfertilized eggs of Xenopus laevis support DNA synthesis on sperm pronuclei. Continuous labelling studies using [3H]dCTP and pulse labelling studies using [32P]dCTP demonstrate that synthesis occurs in short bursts of 40 min, which are punctuated by periods of 20-40 min during which no synthesis occurs. Density substitution experiments using bromodeoxyuridine demonstrate that this synthesis involves the initiation of replication and reveals that re-initiation events can occur following multiple bursts of replication. The periodic properties of these extracts are sensitive to protein synthesis inhibitors.     

Recombinant DNA formation in a cell-free system from Xenopus laevis eggs

A cell-free system is described which formed very high levels of recombinant DNA structures in 4 hr at 26°C. It consisted of a single fraction of a high speed supernatant prepared from an extract of unfertilized eggs of the frog Xenopus laevis. This fraction eluted at 0.16?0.18 M Tris homogenization buffer from a DEAE-cellulose column. When two partially homologous supercoiled DNA molecules of different contour lengths were incubated simultaneously in this system, high levels of heterologous figure eight DNA structures were formed and observed by electron microscopy. Subsequent cleavage of the newly formed figure eight structures with Bam HI and Eco RI restriction endonucleases gave rise to “α structures” and “χ structures.” The observed figure eight structures presumably represent the recombination intermediate predicted by the Holliday model for genetic recombination.     

Axis determination in polyspermic Xenopus laevis eggs

Polyspermic Xenopus laevis eggs can be identified easily because of regions of pigment accumulation and white stripes, which arise by a nocodazole-sensitive process. Eggs containing up to four sperm are capable of forming a single embryonic axis. Dispermic eggs display two regions of pigment accumulation, one around each sperm entry point (SEP), and one white stripe between the SEPs. Such eggs with a 180 degree separation between the SEPs were bisected before first cleavage along the white stripe, creating dorsal and ventral halves in many cases. Each half cleaved and formed a tadpole. When eggs were bisected early in the period of cytoplasmic reorganization (0.5-0.6 normalized time), each half could form a complete tadpole. When eggs were bisected after the period of reorganization (0.8-0.9), often one half formed a tadpole with a complete head but reduced or absent tail and the other half formed a tadpole with a complete tail but reduced or absent head. These results demonstrate that sperm cooperate to give a single embryonic axis in polyspermic eggs and the development of dorsal and ventral egg halves differs after egg reorganization before first cleavage.     

Soluble cytokeratins in Xenopus laevis oocytes and eggs

Xenopus oocytes contain a radial network of cytokeratins which seems to fragment during meiosis reinitiation (maturation). The mature egg contains only a cortical network of cytokeratins. We have looked for the presence of soluble cytokeratins in oocytes and unfertilized eggs and have found them in both cases. However, the proportion of soluble to insoluble cytokeratins is slightly higher in the egg than in the oocyte. Soluble cytokeratins incorporate 35S-methionine at a high rate in the oocyte but to a lesser extent in the egg. This suggests that they are biosynthetic intermediates in the oocyte. In the egg, at least a fraction of the soluble cytokeratins may arise from the fragmentation of the polymer which seems to occur during the maturation process. Insoluble cytokeratins are strongly labeled with 32P both in oocytes and eggs. On the other hand only the soluble keratins of the egg incorporate 32P. Since the isoelectric point of soluble and insoluble cytokeratins is the same in oocytes and eggs, their absolute level of phosphorylation probably remains relatively constant. This suggests that: i) phosphate turnover is very slow in oocyte soluble cytokeratins, ii) phosphorylation is not a major way of changing the structural state of cytokeratins in amphibian oocytes and eggs.     

Involvement of single-stranded tails in homologous recombination of DNA injected into Xenopus laevis oocyte nuclei.

Homologous recombination of DNA molecules injected into Xenopus laevis oocyte nuclei is extremely efficient when those molecules are linear and have overlapping homologous ends. It was previously shown that a 5'----3' exonuclease activity in oocytes attacks injected linear DNAs and leaves them with single-stranded 3' tails. We tested the hypothesis that such tailed molecules are early intermediates on the pathway to recombination products. Substrates with 3' tails were made in vitro and injected into oocytes, where they recombined rapidly and efficiently. In experiments with mixed substrates, molecules with 3' tails entered recombination intermediates and products more rapidly than did molecules with flush ends. Molecules endowed in vitro with 5' tails also recombined efficiently in oocytes, but their rate was not faster than for flush-ended substrates. In most cases, the 5' tails served as templates for resynthesis of the 3' strands, regenerating duplex ends which then entered the normal recombination pathway. In oocytes from one animal, some of the 5' tails were removed, and this was exacerbated when resynthesis was partially blocked. Analysis by two-dimensional gel electrophoresis of recombination intermediates from 5'-tailed substrates confirmed that they had acquired 3' tails as a result of the action of the 5'----3' exonuclease. These results demonstrate that homologous recombination in oocytes proceeds via a pathway that involves single-stranded 3' tails. Molecular models incorporating this feature are discussed.     

Mitochondrial DNA synthesis in large and mature oocytes of Xenopus laevis

Deoxynucleosides are incorporated into mitochondrial DNA (mtDNA) of large oocytes; the rate of incorporation is about 2% of the mtDNA amount per 24 hr. When oocytes have been induced to mature in vitro with human chorionic gonadotropin (HCG), uptake and actual incorporation of thymidine decrease, although phosphorylation is enhanced. An examination of mtDNA replication shows that HCG treatment induces an increase in the relative synthesis of E-strands and an accumulation of D-loops. A similar effect is obtained by ethidium bromide treatment. Thus, gonadotropin appears to delay E-strand elongation and to synchronize mtDNA molecules at the begining of their replication cycle.     

Biochemical characterization of lysosomes in unfertilized eggs of Xenopus laevis.

Relations between lysosomes and yolk platelets of amphibian eggs have been suggested. This work demonstrates the presence of acid hydrolases in oocytes induced to ovulate in vitro. About 40% of the acid hydrolases are found in a sedimentable fraction, and, in accordance with the lysosomal concept, they display structural latency. Biochemical data did not indicate any association between lysosomal enzymes and yolk platelets. The mechanism of yolk resorption is discussed and it is suggested that the fusion of lysosomes and yolk platelets might be one of the mechanisms involved in yolk digestion.