21
Xenopus Egg Extracts
microfuidic systems, new adaptations to the biochemically
tractable cell-free egg extract system are being developed to
study other vital cellular processes. Innovations like lightinducible systems for initiating cell cycle progression are
being developed and hold great promise for even more f nely
tuned control of extract dynamics (Bischt et al., 2019).
ACKNOWLEDGMENTS
We are grateful to all investigators who have, over the last
40 years, helped reveal the awesome power of Xenopus egg
extracts for the study of complex vertebrate mechanisms.
We apologize in advance to the investigators whose work
in other systems informed the work done in Xenopus models; many citations were left out for brevity, as the Xenopus
model was the focus of this review. We are grateful to animal care staff, whose fne work ensures the continued viability of Xenopus as a model system, to our colleagues from
around the world who are generous with their expertise and
reagents, and of course to the frogs themselves. This work
was supported by NIH grant R01GM101250 to SR.
REFERENCES
Afanzar, O., Buss, G.K., Stearns, T., Ferrell, J.E., 2020. The nucleus
serves as the pacemaker for the cell cycle. Elife 9, e59989.
https://doi.org/10.7554/elife.59989
Amunugama, R., Willcox, S., Wu, R.A., Abdullah, U.B., El-Sagheer,
A.H., Brown, T., McHugh, P.J., Griff th, J.D., Walter, J.C.,
2018. Replication fork reversal during DNA interstrand
crosslink repair requires CMG unloading. Cell Reports 23,
3419–3428. https://doi.org/10.1016/j.celrep.2018.05.061
Arias, E.E., Walter, J.C., 2005. Replication-dependent destruction
of Cdt1 limits DNA replication to a single round per cell
cycle in Xenopus egg extracts. Genes & Development 19,
114–126. https://doi.org/10.1101/gad.1255805
Arias, E.E., Walter, J.C., 2006. PCNA functions as a molecular
platform to trigger Cdt1 destruction and prevent re-replication.
Nature Cell Biology 8, 84–90. https://doi.org/10.1038/ncb1346
Ayad, N.G., Rankin, S., Murakami, M., Jebanathirajah, J., Gygi, S.,
Kirschner, M.W., 2003. Tome-1, a trigger of mitotic entry, is
degraded during G1 via the APC. Cell 113, 101–113.
Ayad, N.G., Rankin, S., Ooi, D., Rape, M., Kirschner, M.W., 2005.
Identifcation of ubiquitin ligase substrates by in vitro expression cloning. Methods in Enzymology 399, 404–414. https://
doi.org/10.1016/s0076-6879(05)99028-9
Benbow, R.M., Ford, C.C., 1975. Cytoplasmic control of nuclear
DNA synthesis during early development of Xenopus laevis:
A cell-free assay. Proc National Acad Sci 72, 2437–2441.
https://doi.org/10.1073/pnas.72.6.2437
Bischt, J., LeValley, P., Noren, B., McBride, R., Kharkar, P., Kloxin,
A., Gatlin, J., Oakey, J., 2019. Light-inducible activation of
cell cycle progression in Xenopus egg extracts under microf uidic conf nement. Lab Chip 19, 3499–3511. http://doi.
org/10/1039/C9LC00569B
Blow, J., 1993. Preventing re-replication of DNA in a single cell
cycle: Evidence for a replication licensing factor. J Cell
Biology 122, 993–1002. https://doi.org/10.1083/jcb.122.5.993
Blow, J.J., Gillespie, P.J., Francis, D., Jackson, D.A., 2001. Replication origins in Xenopus egg extract are 5–15 kilobases apart
and are activated in clusters that fre at different times. J Cell
Biology 152, 15–26. https://doi.org/10.1083/jcb.152.1.15
Blow, J.J., Laskey, R.A., 1988. A role for the nuclear envelope in
controlling DNA replication within the cell cycle. Nature
332, 546–548. https://doi.org/10.1038/332546a0
Blow, J.J., Nurse, P., 1990. A cdc2-like protein is involved in the
initiation of DNA replication in Xenopus egg extracts. Cell
62, 855–862. https://doi.org/10.1016/0092-8674(90)90261-c
Cameron, G., Yardimci, H., 2021. Studying chromosome biology
with single-molecule resolution in Xenopus laevis egg extracts.
Essays Biochem. https://doi.org/10.1042/ebc20200026
Carpenter, P.B., Carpenter, P.B., Mueller, P.R., Mueller, P.R.,
Dunphy, W.G., Dunphy, W.G., 1996. Role for a Xenopus
Orc2-related protein in controlling DNA replication. Nature
379, 357–360. https://doi.org/10.1038/379357a0
Chang, J.B., Jr, J.E.F., 2013. Mitotic trigger waves and the spatial
coordination of the Xenopus cell cycle. Nature 500, 603–
607. https://doi.org/10.1038/nature12321
Chen, R.-H., Waters, J.C., Salmon, E.D., Murray, A.W., 1996.
Association of spindle assembly checkpoint component
XMAD2 with unattached kinetochores. Science 274, 242–
246. https://doi.org/10.1126/science.274.5285.242
Cheng, X., Ferrell, J.E., 2018. Apoptosis propagates through the
cytoplasm as trigger waves. Science 361, 607–612. https://
doi.org/10.1126/science.aah4065
Chevalier, S., Couturier, A., Chartrain, I., Guellec, R.L.,
Beckhelling, C., Guellec, K.L., Philippe, M., Ford, C.C.,
1996. Xenopus cyclin E, a nuclear phosphoprotein, accumulates when oocytes gain the ability to initiate DNA replication. J Cell Sci 109 (Pt 6), 1173–1184.
Coverley, D., Downes, C., Romanowski, P., Laskey, R., 1993.
Reversible effects of nuclear membrane permeabilization on
DNA replication: Evidence for a positive licensing factor. J Cell
Biology 122, 985–992. https://doi.org/10.1083/jcb.122.5.985
Cupello, S., Richardson, C., Yan, S., 2016. Cell-free Xenopus egg
extracts for studying DNA damage response pathways. Int J
Dev Biol 60, 229–236. https://doi.org/10.1387/ijdb.160113sy
Davey, N.E., Morgan, D.O., 2016. Building a regulatory network
with short linear sequence motifs: Lessons from the degrons
of the anaphase-promoting complex. Molecular Cell 64,
12–23. https://doi.org/10.1016/j.molcel.2016.09.006
Dimitrova, D.S., Gilbert, D.M., 1999. The spatial position and replication timing of chromosomal domains are both established
in early G1 phase. Molecular Cell 4, 983–993.
Douwel, D.K., Hoogenboom, W.S., Boonen, R.A., Knipscheer, P.,
2017. Recruitment and positioning determine the specif c
role of the XPF-ERCC1 endonuclease in interstrand crosslink repair. Embo J 36, 2034–2046. https://doi.org/10.15252/
embj.201695223
Elledge, S.J., Spottswood, M.R., 1991. A new human p34 protein kinase, CDK2, identifed by complementation of a
cdc28 mutation in Saccharomyces cerevisiae, is a homolog of Xenopus Eg1. Embo J 10, 2653–2659. https://doi.
org/10.1002/j.1460-2075.1991.tb07808.x
Evans, T., Rosenthal, E.T., Youngblom, J., Distel, D., Hunt, T.,
1983. Cyclin: A protein specifed by maternal mRNA in
sea urchin eggs that is destroyed at each cleavage division.
Cell 33, 389–396. https://doi.org/10.1016/0092-8674(83)
90420-8
Fang, G., Yu, H., Kirschner, M., 1998a. Direct binding of CDC20
protein family members activates the anaphase-promoting
complex in mitosis and G1. Molecular Cell 2, 163–171.
Fang, G., Yu, H., Kirschner, M., 1998b. The checkpoint protein
MAD2 and the mitotic regulator CDC20 form a ternary
complex with the anaphase-promoting complex to control
anaphase initiation. Gene Dev 12, 1871–1883. https://doi.
org/10.1101/gad.12.12.1871
Xenopus Egg Extracts
microfuidic systems, new adaptations to the biochemically
tractable cell-free egg extract system are being developed to
study other vital cellular processes. Innovations like lightinducible systems for initiating cell cycle progression are
being developed and hold great promise for even more f nely
tuned control of extract dynamics (Bischt et al., 2019).
ACKNOWLEDGMENTS
We are grateful to all investigators who have, over the last
40 years, helped reveal the awesome power of Xenopus egg
extracts for the study of complex vertebrate mechanisms.
We apologize in advance to the investigators whose work
in other systems informed the work done in Xenopus models; many citations were left out for brevity, as the Xenopus
model was the focus of this review. We are grateful to animal care staff, whose fne work ensures the continued viability of Xenopus as a model system, to our colleagues from
around the world who are generous with their expertise and
reagents, and of course to the frogs themselves. This work
was supported by NIH grant R01GM101250 to SR.
REFERENCES
Afanzar, O., Buss, G.K., Stearns, T., Ferrell, J.E., 2020. The nucleus
serves as the pacemaker for the cell cycle. Elife 9, e59989.
https://doi.org/10.7554/elife.59989
Amunugama, R., Willcox, S., Wu, R.A., Abdullah, U.B., El-Sagheer,
A.H., Brown, T., McHugh, P.J., Griff th, J.D., Walter, J.C.,
2018. Replication fork reversal during DNA interstrand
crosslink repair requires CMG unloading. Cell Reports 23,
3419–3428. https://doi.org/10.1016/j.celrep.2018.05.061
Arias, E.E., Walter, J.C., 2005. Replication-dependent destruction
of Cdt1 limits DNA replication to a single round per cell
cycle in Xenopus egg extracts. Genes & Development 19,
114–126. https://doi.org/10.1101/gad.1255805
Arias, E.E., Walter, J.C., 2006. PCNA functions as a molecular
platform to trigger Cdt1 destruction and prevent re-replication.
Nature Cell Biology 8, 84–90. https://doi.org/10.1038/ncb1346
Ayad, N.G., Rankin, S., Murakami, M., Jebanathirajah, J., Gygi, S.,
Kirschner, M.W., 2003. Tome-1, a trigger of mitotic entry, is
degraded during G1 via the APC. Cell 113, 101–113.
Ayad, N.G., Rankin, S., Ooi, D., Rape, M., Kirschner, M.W., 2005.
Identifcation of ubiquitin ligase substrates by in vitro expression cloning. Methods in Enzymology 399, 404–414. https://
doi.org/10.1016/s0076-6879(05)99028-9
Benbow, R.M., Ford, C.C., 1975. Cytoplasmic control of nuclear
DNA synthesis during early development of Xenopus laevis:
A cell-free assay. Proc National Acad Sci 72, 2437–2441.
https://doi.org/10.1073/pnas.72.6.2437
Bischt, J., LeValley, P., Noren, B., McBride, R., Kharkar, P., Kloxin,
A., Gatlin, J., Oakey, J., 2019. Light-inducible activation of
cell cycle progression in Xenopus egg extracts under microf uidic conf nement. Lab Chip 19, 3499–3511. http://doi.
org/10/1039/C9LC00569B
Blow, J., 1993. Preventing re-replication of DNA in a single cell
cycle: Evidence for a replication licensing factor. J Cell
Biology 122, 993–1002. https://doi.org/10.1083/jcb.122.5.993
Blow, J.J., Gillespie, P.J., Francis, D., Jackson, D.A., 2001. Replication origins in Xenopus egg extract are 5–15 kilobases apart
and are activated in clusters that fre at different times. J Cell
Biology 152, 15–26. https://doi.org/10.1083/jcb.152.1.15
Blow, J.J., Laskey, R.A., 1988. A role for the nuclear envelope in
controlling DNA replication within the cell cycle. Nature
332, 546–548. https://doi.org/10.1038/332546a0
Blow, J.J., Nurse, P., 1990. A cdc2-like protein is involved in the
initiation of DNA replication in Xenopus egg extracts. Cell
62, 855–862. https://doi.org/10.1016/0092-8674(90)90261-c
Cameron, G., Yardimci, H., 2021. Studying chromosome biology
with single-molecule resolution in Xenopus laevis egg extracts.
Essays Biochem. https://doi.org/10.1042/ebc20200026
Carpenter, P.B., Carpenter, P.B., Mueller, P.R., Mueller, P.R.,
Dunphy, W.G., Dunphy, W.G., 1996. Role for a Xenopus
Orc2-related protein in controlling DNA replication. Nature
379, 357–360. https://doi.org/10.1038/379357a0
Chang, J.B., Jr, J.E.F., 2013. Mitotic trigger waves and the spatial
coordination of the Xenopus cell cycle. Nature 500, 603–
607. https://doi.org/10.1038/nature12321
Chen, R.-H., Waters, J.C., Salmon, E.D., Murray, A.W., 1996.
Association of spindle assembly checkpoint component
XMAD2 with unattached kinetochores. Science 274, 242–
246. https://doi.org/10.1126/science.274.5285.242
Cheng, X., Ferrell, J.E., 2018. Apoptosis propagates through the
cytoplasm as trigger waves. Science 361, 607–612. https://
doi.org/10.1126/science.aah4065
Chevalier, S., Couturier, A., Chartrain, I., Guellec, R.L.,
Beckhelling, C., Guellec, K.L., Philippe, M., Ford, C.C.,
1996. Xenopus cyclin E, a nuclear phosphoprotein, accumulates when oocytes gain the ability to initiate DNA replication. J Cell Sci 109 (Pt 6), 1173–1184.
Coverley, D., Downes, C., Romanowski, P., Laskey, R., 1993.
Reversible effects of nuclear membrane permeabilization on
DNA replication: Evidence for a positive licensing factor. J Cell
Biology 122, 985–992. https://doi.org/10.1083/jcb.122.5.985
Cupello, S., Richardson, C., Yan, S., 2016. Cell-free Xenopus egg
extracts for studying DNA damage response pathways. Int J
Dev Biol 60, 229–236. https://doi.org/10.1387/ijdb.160113sy
Davey, N.E., Morgan, D.O., 2016. Building a regulatory network
with short linear sequence motifs: Lessons from the degrons
of the anaphase-promoting complex. Molecular Cell 64,
12–23. https://doi.org/10.1016/j.molcel.2016.09.006
Dimitrova, D.S., Gilbert, D.M., 1999. The spatial position and replication timing of chromosomal domains are both established
in early G1 phase. Molecular Cell 4, 983–993.
Douwel, D.K., Hoogenboom, W.S., Boonen, R.A., Knipscheer, P.,
2017. Recruitment and positioning determine the specif c
role of the XPF-ERCC1 endonuclease in interstrand crosslink repair. Embo J 36, 2034–2046. https://doi.org/10.15252/
embj.201695223
Elledge, S.J., Spottswood, M.R., 1991. A new human p34 protein kinase, CDK2, identifed by complementation of a
cdc28 mutation in Saccharomyces cerevisiae, is a homolog of Xenopus Eg1. Embo J 10, 2653–2659. https://doi.
org/10.1002/j.1460-2075.1991.tb07808.x
Evans, T., Rosenthal, E.T., Youngblom, J., Distel, D., Hunt, T.,
1983. Cyclin: A protein specifed by maternal mRNA in
sea urchin eggs that is destroyed at each cleavage division.
Cell 33, 389–396. https://doi.org/10.1016/0092-8674(83)
90420-8
Fang, G., Yu, H., Kirschner, M., 1998a. Direct binding of CDC20
protein family members activates the anaphase-promoting
complex in mitosis and G1. Molecular Cell 2, 163–171.
Fang, G., Yu, H., Kirschner, M., 1998b. The checkpoint protein
MAD2 and the mitotic regulator CDC20 form a ternary
complex with the anaphase-promoting complex to control
anaphase initiation. Gene Dev 12, 1871–1883. https://doi.
org/10.1101/gad.12.12.1871