Two-dimensional gel analysis of the fate of oocyte nuclear proteins in the development of Xenopus laevis

Homologous recombination of linear DNA molecules in Xenopus laevis oocytes is very efficient. The predictions of molecular models for this recombination process were tested with substrates with terminal nonhomologies (nonhomologous sequences). It was found that nonhomologies on one or both ends of an otherwise efficient substrate substantially reduced the yield of recombination products. In the case of a single nonhomology, inhibition was observed for all lengths of nonhomology, from 60 to 1,690 bp, being most dramatic for the longer blocks. Examination of time courses of recombination showed that the blocks were largely kinetic; that is, substrates with short nonhomologies eventually yielded substantial levels of completed products. Intermediates that accumulated after the injection of end-blocked substrates were characterized by two-dimensional gel electrophoresis and hybridization with strand-specific oligonucleotide probes. These blocked intermediates were shown to have base-paired junctions, but resolution was prevented by the failure to remove the 3'-ending strand of the original nonhomology. Continuing exonuclease action created a single-strand gap adjacent to the position of the persistent nonhomology. In contrast, the strand that included the unblocked side of the junction could be sealed. These results are consistent with a nonconservative, resection-annealing mechanism of homologous recombination in the oocytes and suggest the absence of any activity that can efficiently remove 3' tails.

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Differential accumulation of oocyte nuclear proteins by embryonic nuclei of Xenopus

Development ◽

10.1242/dev.101.4.829 ◽

1987 ◽

Vol 101 (4) ◽

pp. 829-846 ◽

Cited By ~ 1

Author(s):

C. Dreyer

Keyword(s):

De Novo ◽

Germinal Vesicle ◽

Intrinsic Property ◽

Nuclear Proteins ◽

Cell Nuclei ◽

Germinal Vesicle Breakdown ◽

Early Stages ◽

Nuclear Antigens ◽

Gradual Process ◽

Oocyte Nuclear Proteins

Oocyte nuclear proteins of Xenopus are distributed into the cytoplasm of the maturing egg after germinal vesicle breakdown. Later they are found in all cell nuclei of the embryo. At early stages of development, different nuclear proteins behave differently. A class of ‘early shifting’ antigens is accumulated by pronuclei and cleavage nuclei, whereas others appear to be excluded from the nuclei at early stages but are shifted into the nuclei at blastula or during and after gastrulation. Accumulation of ‘late-shifting’ nuclear antigens is a gradual process and occurs during a period characteristic of each protein. Multiple artificial pronuclei can be formed after injection of sperm nuclei, erythrocyte nuclei or pure lambda-DNA into unfertilized eggs. The artificial pronuclei accumulate early- but not late-shifting proteins. Early-migrating proteins rapidly accumulate into the germinal vesicle after de novo synthesis in the oocyte, indicating that the efficiency of translocation into nuclei is an intrinsic property of each protein. Artificial extension of the length of the cell cycle before midblastula transition does not lead to accumulation of the late-shifting nuclear antigens investigated.

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