The Study of Cell Division Control and
2 DNA Replication in Xenopus Egg Extracts
Allison M. Jevitt and Susannah Rankin
CONTENTS
2.1. Introduction .................................................................................................................................................................. 13
2.2. In Vitro Recapitulation of Cell Cycle Events ............................................................................................................... 13
2.3. Protein Degradation Drives the Cell Cycle .................................................................................................................. 15
2.3.1. Pausing the Cell Cycle .................................................................................................................................... 16
2.4. Systems Biology of the Cell Cycle .............................................................................................................................. 16
2.5. DNA Replication Control ............................................................................................................................................ 17
2.6. Ordering Events in DNA Replication .......................................................................................................................... 18
2.7. Origins and Timing ...................................................................................................................................................... 20
2.8. Summary and Future Directions .................................................................................................................................. 20
Acknowledgments .................................................................................................................................................................. 21
References .............................................................................................................................................................................. 21
2.1. INTRODUCTION
The eggs of the African clawed frog, Xenopus laevis, have
played critical roles in our understanding of the regulation
of nuclear events and cell cycle control. Some of the earliest
studies involved the use of Xenopus eggs to reprogram nuclei
from an adult frog. Following injection of somatic nuclei
into the enucleated egg, a considerable portion of the eggs
would go on to cleave and some even go on to form adult
frogs (Gurdon, 1962). These striking results challenged the
prevailing notion that cells could not “dedifferentiate,” that
is, go from a differentiated cell type to a thoroughly pluripotent embryonic state. The second conclusion that could be
drawn from this experiment was that the nuclear changes that
occurred upon differentiation were largely epigenetic; factors
were present in the egg that could reprogram the nuclei, leading to changes in gene expression and thus cell fate. In this
extreme case, nuclei from somatic epithelial cells were reprogrammed simply through incubation in egg cytoplasm. This
notion had fundamental implications for several f elds of
study and led to signifcant efforts to optimize the preparation
of cytoplasmic extracts for further in vitro experimentation.
By the mid 1970s, it was becoming clear that the individual steps involved in cell division, including chromosome
condensation, spindle assembly, nuclear envelope dynamics, and DNA replication, were somehow interdependent.
Genetic experiments from Leland Hartwell and colleagues
demonstrated the existence of the “cell division cycle,” a
regulatory system that controls the events of cell division
(Hartwell et al., 1974). They identifed a set of temperaturesensitive yeast mutants that arrested with the characteristic morphology of each step of cell division. This implied
that an intrinsic regulatory system ensured timely, ordered
completion of all the steps of cell division. The genetics
were clear, but the mechanisms remained to be elucidated.
2.2. IN VITRO RECAPITULATION OF CELL
CYCLE EVENTS
In parallel with genetic analyses in fungal models, investigators worked to develop biochemically tractable methods to
characterize the stages of cell division. In groundbreaking
experiments, Rao and Johnson showed that the cytoplasm
from one cell could impose cell cycle control on nuclei from
a cell fusion partner ( Rao and Johnson, 1970 ). This observation inspired efforts to develop new experimental systems
to monitor and manipulate the steps of cell division in vitro.
Previous experiments suggested that amphibian eggs might
provide an excellent source of material from which to build
such a system: frog eggs are relatively large; laid in abundance; moderately soft and easy to lyse; and stockpiled with
material required for multiple, rapid rounds of cell division
in the absence of new transcription.
Foundational work by Gurdon and colleagues showed
that Xenopus egg cytoplasm had the capacity to induce
DNA replication in somatic nuclei that were micro-injected
into Xenopus eggs (Graham et al., 1966). The induction
of DNA replication in nuclei that had been isolated from
post-mitotic tissues suggested that egg cytoplasm contains
critical regulators of DNA replication and, interestingly,
that the species from which the nuclei were isolated was
relatively unimportant: it worked as well with mouse nuclei
as frog nuclei. The Gurdon micro-injection experiment was
essentially moved into the test tube by Benbow and Ford in
1975. They successfully prepared a concentrated cytosolic
DOI: 10.1201/9781003050230-3
13
2 DNA Replication in Xenopus Egg Extracts
Allison M. Jevitt and Susannah Rankin
CONTENTS
2.1. Introduction .................................................................................................................................................................. 13
2.2. In Vitro Recapitulation of Cell Cycle Events ............................................................................................................... 13
2.3. Protein Degradation Drives the Cell Cycle .................................................................................................................. 15
2.3.1. Pausing the Cell Cycle .................................................................................................................................... 16
2.4. Systems Biology of the Cell Cycle .............................................................................................................................. 16
2.5. DNA Replication Control ............................................................................................................................................ 17
2.6. Ordering Events in DNA Replication .......................................................................................................................... 18
2.7. Origins and Timing ...................................................................................................................................................... 20
2.8. Summary and Future Directions .................................................................................................................................. 20
Acknowledgments .................................................................................................................................................................. 21
References .............................................................................................................................................................................. 21
2.1. INTRODUCTION
The eggs of the African clawed frog, Xenopus laevis, have
played critical roles in our understanding of the regulation
of nuclear events and cell cycle control. Some of the earliest
studies involved the use of Xenopus eggs to reprogram nuclei
from an adult frog. Following injection of somatic nuclei
into the enucleated egg, a considerable portion of the eggs
would go on to cleave and some even go on to form adult
frogs (Gurdon, 1962). These striking results challenged the
prevailing notion that cells could not “dedifferentiate,” that
is, go from a differentiated cell type to a thoroughly pluripotent embryonic state. The second conclusion that could be
drawn from this experiment was that the nuclear changes that
occurred upon differentiation were largely epigenetic; factors
were present in the egg that could reprogram the nuclei, leading to changes in gene expression and thus cell fate. In this
extreme case, nuclei from somatic epithelial cells were reprogrammed simply through incubation in egg cytoplasm. This
notion had fundamental implications for several f elds of
study and led to signifcant efforts to optimize the preparation
of cytoplasmic extracts for further in vitro experimentation.
By the mid 1970s, it was becoming clear that the individual steps involved in cell division, including chromosome
condensation, spindle assembly, nuclear envelope dynamics, and DNA replication, were somehow interdependent.
Genetic experiments from Leland Hartwell and colleagues
demonstrated the existence of the “cell division cycle,” a
regulatory system that controls the events of cell division
(Hartwell et al., 1974). They identifed a set of temperaturesensitive yeast mutants that arrested with the characteristic morphology of each step of cell division. This implied
that an intrinsic regulatory system ensured timely, ordered
completion of all the steps of cell division. The genetics
were clear, but the mechanisms remained to be elucidated.
2.2. IN VITRO RECAPITULATION OF CELL
CYCLE EVENTS
In parallel with genetic analyses in fungal models, investigators worked to develop biochemically tractable methods to
characterize the stages of cell division. In groundbreaking
experiments, Rao and Johnson showed that the cytoplasm
from one cell could impose cell cycle control on nuclei from
a cell fusion partner ( Rao and Johnson, 1970 ). This observation inspired efforts to develop new experimental systems
to monitor and manipulate the steps of cell division in vitro.
Previous experiments suggested that amphibian eggs might
provide an excellent source of material from which to build
such a system: frog eggs are relatively large; laid in abundance; moderately soft and easy to lyse; and stockpiled with
material required for multiple, rapid rounds of cell division
in the absence of new transcription.
Foundational work by Gurdon and colleagues showed
that Xenopus egg cytoplasm had the capacity to induce
DNA replication in somatic nuclei that were micro-injected
into Xenopus eggs (Graham et al., 1966). The induction
of DNA replication in nuclei that had been isolated from
post-mitotic tissues suggested that egg cytoplasm contains
critical regulators of DNA replication and, interestingly,
that the species from which the nuclei were isolated was
relatively unimportant: it worked as well with mouse nuclei
as frog nuclei. The Gurdon micro-injection experiment was
essentially moved into the test tube by Benbow and Ford in
1975. They successfully prepared a concentrated cytosolic
DOI: 10.1201/9781003050230-3
13