DNA replication and cell cycle control in Xenopus egg extracts

In metazoans the endoplasmic reticulum (ER) changes during the cell cycle, with the nuclear envelope (NE) disassembling and reassembling during mitosis and the peripheral ER undergoing extensive remodeling. Here we address how ER morphology is generated during the cell cycle using crude and fractionated Xenopus laevis egg extracts. We show that in interphase the ER is concentrated at the microtubule (MT)-organizing center by dynein and is spread by outward extension of ER tubules through their association with plus ends of growing MTs. Fusion of membranes into an ER network is dependent on the guanosine triphosphatase atlastin (ATL). NE assembly requires fusion by both ATL and ER-soluble N-ethyl-maleimide–sensitive factor adaptor protein receptors. In mitotic extracts, the ER converts into a network of sheets connected by ER tubules and loses most of its interactions with MTs. Together, these results indicate that fusion of ER membranes by ATL and interaction of ER with growing MT ends and dynein cooperate to generate distinct ER morphologies during the cell cycle.

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DNA replication of mitotic chromatin in Xenopus egg extracts

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2336104100 ◽

2003 ◽

Vol 100 (23) ◽

pp. 13241-13246 ◽

Cited By ~ 18

Author(s):

T. A. Prokhorova ◽

K. Mowrer ◽

C. H. Gilbert ◽

J. C. Walter

Keyword(s):

Dna Replication ◽

Egg Extracts ◽

Xenopus Egg ◽

Xenopus Egg Extracts ◽

Mitotic Chromatin

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Efficient reactivation of Xenopus erythrocyte nuclei in Xenopus egg extracts

Journal of Cell Science ◽

10.1242/jcs.108.6.2187 ◽

1995 ◽

Vol 108 (6) ◽

pp. 2187-2196 ◽

Cited By ~ 3

Author(s):

L.J. Wangh ◽

D. DeGrace ◽

J.A. Sanchez ◽

A. Gold ◽

Y. Yeghiazarians ◽

...

Keyword(s):

Cell Cycle ◽

Somatic Cell ◽

Nuclear Transplantation ◽

Optimal Time ◽

Genome Replication ◽

Cell Nuclei ◽

Egg Extracts ◽

Xenopus Egg ◽

Xenopus Egg Extracts

Rapid genome replication is one of the hallmarks of the frog embryonic cell cycle. We report here that complete reactivation of quiescent somatic cell nuclei in Xenopus egg extracts depends on prior restructuring of the nuclear substrate and prior preparation of cytoplasmic extract with the highest capacity to initiate and sustain DNA synthesis. Nuclei from mature erythrocytes swell, replicate their DNA efficiently, and enter mitosis in frozen/thawed extracts prepared from activated Xenopus eggs, provided the nuclei are first treated with trypsin, heparin, and an extract prepared from unactivated, meiotically arrested, eggs. Optimal replicating extracts are prepared from large batches of unfertilized eggs that are synchronously activated into the cell cycle for 28 minutes (at 20 degrees C). Because the Xenopus cell cycle progresses so rapidly, extracts prepared just a few minutes before or after this time have substantially lower DNA synthetic capacities. At the optimal time and temperature, eggs have just reached the G1/S boundary of the first cell cycle. This fact was revealed by injecting and replicating an SV40 plasmid in intact unfertilized eggs as described previously. We estimate that under optimal conditions approximately 6.14 × 10(9) base pairs of DNA/per nucleus are synthesized in 30–40 minutes, a rate that rivals that observed in the zygotic nucleus. The findings reported here are one step in our long term effort to develop a new in vitro/in vivo approach to nuclear transplantation. Nuclear transplantation in amphibian embryos has been used to establish that the genomes of many types of differentiated somatic cells are pluripotent. But very few such nuclei have ever developed into advanced tadpoles or adult frogs, probably because somatic nuclei injected directly into activated eggs fail to reactivate quickly enough to avoid being damaged during first mitosis. We have already shown that unfertilized eggs can be injected prior to activation of the first cell cycle. Future experiments will reveal whether in vitro reactivated somatic cell nuclei transplanted into such eggs reliably reach advanced stages of development.

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The role of lamin LIII in nuclear assembly and DNA replication, in cell-free extracts of Xenopus eggs

Journal of Cell Science ◽

10.1242/jcs.98.3.271 ◽

1991 ◽

Vol 98 (3) ◽

pp. 271-279

Author(s):

J. Meier ◽

K.H. Campbell ◽

C.C. Ford ◽

R. Stick ◽

C.J. Hutchison

Keyword(s):

Dna Replication ◽

Membrane Structures ◽

Chromatin Decondensation ◽

Immunoblotting Analysis ◽

Nuclear Assembly ◽

Contrast Microscopy ◽

Egg Extracts ◽

Xenopus Egg ◽

Xenopus Egg Extracts

Xenopus egg extracts, which support nuclear assembly and DNA replication, were functionally depleted of lamin LIII by inoculating them with monoclonal anti-lamin antibodies. Phase-contrast microscopy and electron-microscopy studies indicated that lamin-depleted extracts supported efficient chromatin decondensation, and assembly of double membrane structures and nuclear pores on demembranated sperm heads. Immunofluorescence microscopy suggests that lamin-antibody complexes are transported across the nuclear membrane but do not assemble into a lamina. These findings were confirmed by immunoblotting analysis of isolated nuclei. Metabolic labelling studies with either biotin-11-dUTP or [32P]dCTP, revealed that nuclei lacking a lamina were unable to initiate DNA replication and that, although such nuclei could import proteins required for DNA replication (e.g. PCNA), these proteins were apparently not organized into replicon clusters.

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Absence of BLM leads to accumulation of chromosomal DNA breaks during both unperturbed and disrupted S phases

The Journal of Cell Biology ◽

10.1083/jcb.200402095 ◽

2004 ◽

Vol 165 (6) ◽

pp. 801-812 ◽

Cited By ~ 31

Author(s):

Wenhui Li ◽

Soo-Mi Kim ◽

Joon Lee ◽

William G. Dunphy

Keyword(s):

Dna Replication ◽

Chromosomal Abnormalities ◽

Sister Chromatid Exchanges ◽

Chromosomal Dna ◽

Dna Breaks ◽

Egg Extracts ◽

Xenopus Egg ◽

Xenopus Egg Extracts ◽

Chromatid Exchanges ◽

Chromosomal Defects

Bloom's syndrome (BS), a disorder associated with genomic instability and cancer predisposition, results from defects in the Bloom's helicase (BLM) protein. In BS cells, chromosomal abnormalities such as sister chromatid exchanges occur at highly elevated rates. Using Xenopus egg extracts, we have studied Xenopus BLM (Xblm) during both unperturbed and disrupted DNA replication cycles. Xblm binds to replicating chromatin and becomes highly phosphorylated in the presence of DNA replication blocks. This phosphorylation depends on Xenopus ATR (Xatr) and Xenopus Rad17 (Xrad17), but not Claspin. Xblm and Xenopus topoisomerase IIIα (Xtop3α) interact in a regulated manner and associate with replicating chromatin interdependently. Immunodepletion of Xblm from egg extracts results in accumulation of chromosomal DNA breaks during both normal and perturbed DNA replication cycles. Disruption of the interaction between Xblm and Xtop3α has similar effects. The occurrence of DNA damage in the absence of Xblm, even without any exogenous insult to the DNA, may help to explain the genesis of chromosomal defects in BS cells.

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