Embryonic Cell Nuclei Research Articles

115 kDa protein from Xenopus laevis embryos recognized by antibodies directed against the Xenopus homeoprotein XIHbox 1.

Using antibodies against homeoprotein XIHbox 1 from Xenopus laevis, we have detected a new embryonic protein with a much larger molecular weight, 115 kDa. Antibodies fractionated according to their affinity for 3 different domains of the XIHbox 1 protein were used to show that this new protein is related to the C-terminal region of XIHbox 1 protein, downstream from the homeodomain. By immunohistochemistry, the protein was shown to be localized in nuclei of embryonic cells. On SDS-polyacrylamide gels, the 115 kDa protein appears as a set of closely spaced bands whose pattern varies with the stage of development and with the parental origin of the embryos. The protein could be extracted from embryos in a multiprotein complex of approximately 600 kDa. In contrast, the 18 and 27 kDa proteins predicted from the sequence of cloned cDNA to be transcribed and translated from the XIHbox 1 gene could not be detected, suggesting that they are rare or unstable in embryos. These data suggest that the new protein is involved in the development of Xenopus embryos, with a function possibly related to that of the homeoprotein XIHbox 1.

Identification of an amphibian oocyte nuclear protein as a candidate for a role in embryonic DNA replication.

Monoclonal antibody B24/3 recognizes a nuclear protein of 104 kD in germinal vesicles of newt oocytes. Immunohistostaining of oocytes at different stages of growth shows an accumulation of B24 protein throughout oogenesis. During development B24 protein is located inside embryonic cell nuclei from the onset of cleavage onwards. It gradually decreases from gastrulation and disappears at the tailbud stage. The NvB24 17.1 clone was isolated from an ovary expression library of the newt Notophthalmus viridescens and then sequenced: the open reading frame is capable of encoding a polypeptide of 744 amino acids. Northern blot experiments have shown that the 17.1 clone recognizes a single transcript of about 3 Kb in the ovary. In situ hybridization experiments showed that B24 mRNA transcription starts from previtellogenic oocytes, and is followed by the appearance and gradual accumulation of B24 protein in germinal vesicles of medium and large size oocytes. Keeping in mind the sequence similarity shown by the B24 protein to the mouse P1 protein as well as to the budding yeast Mcm3 and fission yeast cdc21 proteins, B24 protein can be speculated to play a role in the events of DNA replication during early amphibian embryogenesis. As B24 antigen is located in the sphere organelles both inserted on the lampbrush chromosomes and free in the oocyte nucleoplasm, an additional possible role of B24 protein could be related to assembling and/or storing snRNPs during oogenesis.

Different conformations of ribosomal DNA in active and inactive chromatin in Xenopus laevis

The chromatin structure of the ribosomal DNA in Xenopus laevis was studied by micrococcal nuclease digestions of blood, liver and embryonic cell nuclei. We have found that BglI-restricted DNA from micrococcal nuclease-digested blood cell nuclei has an increased electrophoretic mobility compared to the undigested control. Micrococcal nuclease digestion of liver cell nuclei causes a very slight shift in mobility, only in the region of the spacer containing the “Bam Islands”. In contrast, the mobility of ribosomal DNA in chromatin of embryonic cells, under identical digestion conditions, remains unaffected by the nuclease activity. Denaturing gels or ligase action on the nuclease-treated DNA abolishes the differences in the electrophoretic mobility. Ionic strength and ethidium bromide influence the relative electrophoretic migration of the two DNA fragment populations, suggesting that secondary structure may play an important role in the observed phenomena. In addition, restriction analysis under native electrophoretic conditions of DNA prepared from blood, liver and embryonic cells shows that blood cell DNA restriction fragments always have a faster mobility than the corresponding fragments of liver and embryo cell DNA. We therefore propose that nicking activity by micrococcal nuclease modifies the electrophoretic mobility of an unusual DNA conformation, present in blood cell, and to a lesser extent, in liver cell ribosomal chromatin. A possible function for these structures is discussed. The differences of the ribosomal chromatin structures in adult and embryonic tissues may reflect the potential of the genes to be expressed.

Onset of paternal and maternal Gpi-1 expression in preimplantation mouse embryos

The initial activation of the glucose phosphate isomerase gene, Gpi-1, was studied in mouse embryos produced by transplanting pronuclei between two strains of mice differing in alleles for this enzyme. Protein isozymes encoded by the embryonic cell nuclei were first detected on Day 4 of embryogenesis, and the maternal and paternal genes are seen to be activated simultaneously. Comparison of isozymes produced by these nuclear-transfer embryos and by F 1 embryos from these two strains suggests the absence of oocyte mRNA for GPI-1 at the time when these genes are first activated. Thus, the GPI-1 present is derived from newly transcribed mRNA contributed by both maternal and paternal genes. The relative proportion of maternal cytoplasmic GPI-1 enzyme declines from Day 3 to Day 6, such that on Day 6, almost no oocyte GPI-1 is detected.

Cellular localization of DNA polymerase activities in full-grown oocytes and embryos of Xenopus laevis

In full-grown oocytes of Xenopus laevis more than 80 % of the total DNA polymerase activity is found in the germinal vesicle (nucleus) and only about 8% in the cytoplasm. The intracellular distribution of the multiple DNA polymerase forms has been studied in oocytes and in embryonic cells. The oocyte nucleus contains a major DNA polymerase species, sedimenting at about 7S, and a minor species sedimenting at about 5S. These enzymes are comparable, respectively, with the DNA polymerases α and β described in other biological systems. In the oocyte cytoplasm, besides a small amount of the 7S form, an 8–9S DNA polymerase activity is also detectable. In the nuclei of embryonic cells, in addition to the DNA polymerase forms present in the oocyte nucleus, a new major form which seems specific for the eggs and embryos is detectable by DEAE chromatography.

Cloning in Chickens

Briggs and King (1952) perfected a method for transferring nuclei of embryonic cells into enucleated eggs of the frog. Gurdon (1960) described a method by which he obtained many genetically identical frogs by the direct and serial transplantation of the nuclei of dissociated endoderm cells to the enucleated eggs of a single female. The cells from which the donor nuclei were obtained were taken from a blastula or gastrula embryo. Gurdon showed that all frogs so produced from the cells of a single blastula or gastrula were identical. However the most remarkable feature brought to light by these experiments was the total power of differentiation possessed by the single cell nucleus when it was separated from its companion cells and placed in an enucleated egg. It occurred to the authors that certain cells of the chicken embryo might possess this power of differentiation to some degree. Therefore the main concern…

The induction of DNA synthesis by frog egg cytoplasm

The early effects are described of injecting nuclei from embryonic, larval, and adult cells of Xenopus laevis into unfertilized eggs of the same species. 3H-thymidine was injected into eggs immediately, or at intervals, after the injection of nuclei. Autoradiography of the sectioned eggs showed whether the injected nuclei had synthesized DNA after exposure to egg cytoplasm. The great majority of nuclei from gastrula endoderm cells, as well as from adult liver, brain, and blood cells, synthesized DNA at some time during the 90 minutes following injection into eggs. In the case of the liver, brain, and blood cell nuclei this constitutes a very considerable change in function, since not more than 1% of these cells incorporate 3H-thymidine in a 90-minute period in vivo, and the procedure used to prepare nuclei from these cells did not itself induce DNA synthesis. This change in nuclear function is induced by exposing nuclei to normal egg cytoplasm. The cytoplasmic influence is not Class specific since over 90% of mouse liver nuclei are also induced to synthesize DNA after injection into frogs' eggs. Gastrula, liver, brain, and blood cell nuclei injected into eggs commence DNA synthesis later, and continue it for longer, than the egg nucleus of a fertilized egg. DNA synthesis was observed in transplanted embryonic cell nuclei which were destined to promote abnormal as well as normal development. Liver, brain, and blood cell nuclei swell up to 60 times after injection into eggs, and this increase in volume takes place at the same rate as that at which nuclei are induced to synthesize DNA. These nuclei were not observed to enter mitosis during the 90 minutes immediately following injection into unfertilized eggs.

The transplantation of nuclei between two species of Xenopus

The following combinations of nucleus and cytoplasm have been obtained by transplanting embryonic cell nuclei into enucleated and unfertilized eggs: Xenopus tropicalis nuclei in Xenopus laevis cytoplasm; X. laevis nuclei in X. tropicalis cytoplasm; and X. laevis nuclei transferred back to their own kind of cytoplasm after about 12 divisions in foreign X. tropicalis cytoplasm. In each case transplant embryos were arrested in development at a stage characteristic for the combination concerned. The serial transplantation of nuclei between these species showed that the change induced in nuclei by foreign cytoplasm has the following properties: it is heritable through mitosis; it is specific in that all nuclei of one species are always changed in the same way by cytoplasm of the other species; some nuclear functions are changed more than others; the change is complete after 8 nuclear divisions and is not enhanced by further exposure to the foreign cytoplasm; lastly it is not reversed after 70 divisions of the nucleus in its original species of cytoplasm. It was shown that egg cytoplasm contains substances which have a specific effect on the function and replication of foreign nuclei. The nature of these substances is not known. The possibility is discussed that the nucleus of irradiated recipient eggs may affect the development which these eggs promote after nuclear transplantation. No evidence was found that it has any effect, and some experiments indicate that it does not.