Isolation of Large Quantities of Functional, Cytoplasm-Free Xenopus laevis Oocyte Nuclei

Lehman, Chris W.; Carroll, Dana

doi:10.1006/abio.1993.1275

Cited by 15 publications

(14 citation statements)

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“…First, the overall recombination activity was considerably lower in the extracts; processing was incomplete in the stage V and VI extracts, despite the high ratio of extract to substrate. This has been observed consistently (27,28) end-joined species, regardless of stage, were products of simple ligation, because they were all sensitive to XhoI digestion (Fig. 4A, PvuII + XhoI).…”

Section: Resultsmentioning

confidence: 96%

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Homologous and illegitimate recombination in developing Xenopus oocytes and eggs.

Lehman¹,

Clemens²,

Worthylake³

et al. 1993

Mol. Cell. Biol.

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Exogenous DNA is efficiently recombined when injected into the nuclei of Xenopus laevis oocytes. This reaction proceeds by a homologous resection-annealing mechanism which depends on the activity of a 5'-3' exonuclease. Two possible functions for this recombination activity have been proposed: it may be a remnant of an early process in oogenesis, such as meiotic recombination or amplification of genes coding for rRNA, or it may reflect materials stored for embryogenesis. To test these hypotheses, recombination capabilities were examined with oocytes at various developmental stages. Late-stage oocytes performed only homologous recombination, whereas the smallest oocytes ligated the restriction ends of the injected DNA but supported no homologous recombination. This transition from ligation to recombination activity was also seen in nuclear extracts from these same stages. Exonuclease activity was measured in the nuclear extracts and found to be low in early stages and then to increase in parallel with recombination capacity in later stages. The accumulation of exonuclease and recombination activities during oogenesis suggests that they are stored for embryogenesis and are not present for oocyte-specific functions. Eggs were also tested and found to catalyze homologous recombination, ligation, and illegitimate recombination. Retention of homologous recombination in eggs is consistent with an embryonic function for the resection-annealing mechanism. The observation of all three reactions in eggs suggests that multiple pathways are available for the repair of double-strand breaks during the extremely rapid cleavage stages after fertilization.In a number of earlier studies, we have shown that appropriately designed DNA substrates are recombined very efficiently after injection into the nuclei of large oocytes from Xenopus laevis (6,23,(33)(34)(35)39). Each oocyte has a large capacity for recombination, processing 109 substrate molecules to completed products in several hours (6). Recombination requires homology and DNA ends (6), and it appears to proceed exclusively by a nonconservative resectionannealing mechanism (also called single-strand annealing [22,35,39]). Unlike the situation in Xenopus eggs (37) and mammalian cells (42), no illegitimate end joining is observed in oocytes (6). Even products formed at low efficiency in the absence of substantial homologies depend on short sequence matches and result in small deletions (17).The resection-annealing process (Fig. 1)

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Section: Resultsmentioning

confidence: 96%

“…The extracts had been centrifuged, so only soluble GV proteins were measured. We have previously demonstrated that, for stage VI oocytes, the soluble protein represents a little more than 50% of the total nuclear protein (28). The mean protein concentrations for GVs from each oocyte stage are shown in Table 1.…”

Section: Methodsmentioning

confidence: 99%

Homologous and illegitimate recombination in developing Xenopus oocytes and eggs.

Lehman¹,

Clemens²,

Worthylake³

et al. 1993

Mol. Cell. Biol.

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“…Oocyte Extracts and Assays-Nuclear (germinal vesicle (GV)) extracts of Xenopus oocytes were prepared in I buffer (20 mM Tris (pH 7.5), 75 mM KCl, 7 mM MgCl2, 0.1 mM EDTA, and 2 mM dithiothreitol) as described previously (39,40). Both manual and bulk extracts have been used with comparable results.…”

Section: Methodsmentioning

confidence: 99%

“…32 P]dCTP (40). After incubation for 3 h at room temperature, the DNA was extracted and fractionated by electrophoresis on a 1% agarose gel and visualized by autoradiography of the dried gel.…”

Section: Methodsmentioning

confidence: 99%

“…The mammalian homologues of the enzyme have been identified repeatedly as components of the DNA replication apparatus. Although Xenopus oocytes and oocyte extracts are not capable of replication, they will synthesize a second strand on a single-stranded circular DNA template (40,47,48). The process is presumably analogous to lagging strand synthesis during chromosomal replication and/or to mechanisms of DNA repair.…”

Section: Fig 4 Yeast Complementation By Xfen-1mentioning

confidence: 99%

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Characterization of FEN-1 from Xenopus laevis

Bibikova

Chi

et al. 1998

Journal of Biological Chemistry

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cDNAs for the Xenopus laevis homologue of the endo/ exonuclease FEN-1 (DNase IV) have been cloned using a polymerase chain reaction strategy. Products were obtained from two nonallelic Xenopus genes (xFEN-1a and xFEN-1b) that differ from each other by 4.5% in amino acid sequence. Both are 80% identical to mammalian FEN-1 proteins and 55% identical to the yeast homologues. When expressed in Escherichia coli, the Xenopus enzymes showed flap endonuclease activity, a unique feature of this class of nucleases. In addition, expression from the Xenopus cDNAs complemented the temperature and methyl methanesulfonate sensitivity of a yeast rad27 deletion, which eliminates the endogenous FEN-1 gene product. Antiserum raised against xFEN-1 was used to show that the protein accumulates during the middle and late stages of oogenesis, in parallel with other DNA metabolic activities, and that it is localized to the oocyte nucleus. Flap endonuclease activity was demonstrated in oocyte nuclear extracts, and this was inhibited by the anti-xFEN-1 antiserum. The antiserum did not inhibit the major oocyte 5 3 3 exonuclease activity. DNA synthesis in oocyte extracts was blocked by the antiserum, and the nature of this inhibition suggests that xFEN-1 may be part of a large complex of replication factors. Chromatographic evidence was obtained for the existence of a complex that forms during DNA synthesis and includes proliferating cell nuclear antigen in addition to xFEN-1. These observations support a critical role for xFEN-1 in DNA replication, but indicate that another enzyme must be responsible for the exonuclease function required for homologous recombination in Xenopus oocytes.

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