Parthenogenesis in Xenopus eggs injected with centrosomes from synchronized human lymphoid cells

In Xenopus eggs, normal development requires the participation of the centrosome provided by the sperm. Injection of foreign centrosomes purified from exponentially growing mammalian cells enables the eggs to undertake parthenogenesis. In order to know whether such a complementation required centrosomes already committed to duplication, we have prepared centrosomes from human cells synchronized at different stages of the cell cycle (G0, G1, G2). We show that the three types of centrosome possess a similar parthenogenetic activity and conclude that duplication of heterologous centrosome can be triggered in Xenopus eggs.     

Tissue-specific expression of actin genes injected into Xenopus embryos

We have isolated a complete Xenopus borealis cardiac actin gene, which is normally expressed in the myotomes and heart of the embryo and tadpole. After injection into the zygote, this cloned gene becomes distributed throughout the embryo, but it is expressed almost wholly in the myotomes. The same wide distribution of injected DNA but spatially restricted pattern of expression is found with a fusion between the first two actin gene exons and the last exon of a mouse beta-globin gene. By contrast, a histone-globin fusion gene is expressed fairly uniformly in all regions. We discuss the special advantages of using Xenopus in studies of tissue-specific gene expression from injected, cloned genes in early development.     

Glutathione transferase isoenzymes from frog (Xenopus laevis) liver and embryo

The expression of glutathione transferase isoenzymes has been investigated in embryo and adult liver of the frog Xenopus laevis. By analysing the GST isoenzymes recovered from GSH-affinity chromatography in terms of electrophoretic mobility, HPLC elution profile, immunological reactivity, N-terminal amino acid sequence and mass spectrometry molecular mass no significant difference in the GST subunit composition between embryos and liver was found. In both tissues the same three subunits, showing similarity to mu, alpha and sigma class GSTs, are present. These results, together with those previously reported for toad (Bufo bufo), strongly support the notion that the transition from an aquatic environment to a terrestrial atmosphere containing high oxygen concentration has accompanied specific GST gene expression.     

Initiation of replication at specific origins in DNA molecules microinjected into unfertilized eggs of the frog Xenopus laevis

Initiation of DNA replication at specific origins was observed by electron microscopy after microinjection of pXlr11, pXlr14 or Col E1 plasmid DNA molecules into unfertilized eggs of the frog, Xenopus laevis. These results are in apparent contradiction with published reports (Harland and Laskey, Cell 21, 761-771, 1980; Laskey and Harland, Cell 24, 283-284, 1981) that specific origin sites were not used in Xenopus laevis eggs. We suggest that eucaryotic origins exist that both increase the probability of replication of contiguous sequences and determine the site at which replication is most likely to begin.     

Activation of frog (Xenopus laevis) eggs by inositol trisphosphate. I. Characterization of Ca2+ release from intracellular stores

Iontophoresis of inositol 1, 4, 5-triphosphate into frog (Xenopus laevis) eggs activated early developmental events such as membrane depolarization, cortical contraction, cortical granule exocytosis, and abortive cleavage furrow formation (pseudocleavage). Inositol 1, 4-bisphosphate also triggered these events, but only at doses approximately 100-fold higher, whereas no level of fructose-1, 6-bisphosphate tested activated eggs. Using Ca2+-selective microelectrodes, we observed that activating doses of inositol 1, 4, 5-trisphosphate triggered a Ca2+ release from intracellular stores that was indistinguishable from that previously observed at fertilization (Busa, W. B., and R. Nuccitelli, 1985, J. Cell Biol., 100:1325-1329), whereas subthreshold doses triggered only a localized Ca2+ release at the site of injection. The subthreshold IP3 response could be distinguished from the major Ca2+ release at activation with respect to their dose-response characteristics, relative timing, sensitivity to external Ca2+ levels, additivity, and behavior in the activated egg, suggesting that the Xenopus egg may possess two functionally distinct Ca2+ pools mobilized by different effectors. In light of these differences, we suggest a model for intracellular Ca2+ mobilization by sperm-egg interaction.     

The stability and translation of sea urchin histone messenger RNA molecules injected into Xenopus laevis eggs and developing embryos

Embryonic sea urchin histone mRNA was injected into eggs and developing zygotes of Xenopus. The functional stability of the mRNA was monitored by separating newly synthesized sea urchin histones from those of Xenopus. Just as when injected into Xenopus oocytes, sea urchin H1, H2A, and H2B mRNA molecules have a functional half-life of about 3 hr in the developing embryo. This suggests that the endogenous Xenopus histone mRNA is also unstable and has a number of implications for the amount of histone mRNA that is stored in the oocyte and the time at which histone genes should become active in development. The injected mRNA is translated with little, if any, greater efficiency in the egg than in the oocyte. However, Xenopus histone synthesis increases about 20- to 50-fold during the transition from oocyte to egg. The injection experiments therefore suggest that this increase is brought about primarily by the mobilization of stored mRNA, rather than an increase in the efficiency of histone synthesis.     

DNA synthesis in ocytes and eggs of Xenopus laevis injected with DNA.

Single-stranded calf thymus DNA injected into preovulation oocytes, postovulation oocytes or eggs of Xenopus laevis induces synthesis of double-stranded DNA of similar base composition. In contrast, native (double-stranded) calf thymus DNA injected into oocytes does not stimulate DNA synthesis, though it does do so in eggs. The buoyant density of normal or IUdR-substituted newly-synthesized DNA on neutral or alkaline CsCl gradients suggests that the injected DNA is replicated.The amount of synthesis induced by injecting single-stranded DNA is five times greater in eggs than in oocytes. The maximum synthesis observed in eggs injected with native DNA is 50 pg/hr; this is sufficient for nuclear DNA replication in uninjected fertilised eggs, but not in midcleavage. However in vitro studies (reported elsewhere) indicate the presence of a large store of DNA polymerase activity in eggs. We conclude that only a small proportion of the total DNA polymerase activity in an egg is available for DNA synthesis during the first 2 hr of development.     

Cytostatic factor (CSF) activity in cytosols extracted from Xenopus laevis eggs

Cytostatic factor (CSF), found in the cytoplasm of unfertilized eggs of amphibians, causes metaphase arrest when microinjected into cleaving blastomeres. Although CSF from Rana pipiens eggs has been extracted and characterized, little is known about CSF extracted from eggs of other species. We investigated the conditions required to preserve CSF activity in cytosols extracted from Xenopus laevis eggs and found that it was necessary to expose the eggs to CO2 prior to extraction and that the extraction buffer must contain sodium beta-glycerophosphate. CSF activity disappeared after 24 h of storage at 2 degrees C. Cytological examination showed that the arrested blastomeres injected with cytosols had been arrested at metaphase and contained a spindle lacking polar asters, in which highly condensed chromosomes were embedded.