w:Xenopus egg extract

The first frog egg extract was reported in 1983 by Lohka and Masui.{{cite journal | author = Lohka MJ, Masui Y| title = Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components| journal = Science| volume = 220 | pages = 719–721 | year = 1983 | issue = 4598| doi = 10.1126/science.6601299| pmid = 6601299| bibcode = 1983Sci...220..719L}} This pioneering work used eggs of the Northern leopard frog Rana pipiens to prepare an extract. Later, the same procedure was applied to eggs of Xenopus laevis, becoming popular for studying cell cycle progression and cell cycle-dependent cellular events.{{cite journal | author = Lohka MJ, Maller JL. | title = Induction of nuclear envelope breakdown, chromosome condensation, and spindle formation in cell-free extracts | journal = J. Cell Biol.| volume = 101 | pages = 518–523 | year = 1985 | issue = 2 | doi = 10.1083/jcb.101.2.518 | pmid =3926780| pmc = 2113692 }} Extracts derived from eggs of the Japanese common toad Bufo japonicus{{cite journal | author = Ohsumi K, Katagiri C | title = Characterization of the ooplasmic factor inducing decondensation of and protamine removal from toad sperm nuclei: involvement of nucleoplasmin | journal = Dev. Biol.| volume = 148 | pages = 295–305 | year = 1991| issue = 1 | doi = 10.1016/0012-1606(91)90338-4 | pmid =1936566}} or of the Western clawed frog Xenopus tropicalis{{cite journal | author = Brown KS, Blower MD, Maresca TJ, Grammer TC, Harland RM, Heald R | title = Xenopus tropicalis egg extracts provide insight into scaling of the mitotic spindle | journal = J. Cell Biol.| volume = 176 | pages = 765–770 | year = 2007 | issue = 6 | doi = 10.1083/jcb.200610043 | pmid =17339377| pmc = 2064050 }} have also been reported.

The cell cycle of unfertilized eggs of X. laevis is arrested highly synchronously at metaphase of meiosis II. Upon fertilization, the metaphase arrest is released by the action of Ca²⁺ ions released from the endoplasmic reticulum, thereby initiating early embryonic cell cycles that alternates S phase (DNA replication) and M phase (mitosis).{{cite journal | author = Masui Y| title = The elusive cytostatic factor in the animal egg| journal = Nat. Rev. Mol. Cell Biol.| volume = 1 | pages = 228–232 | year = 2000 | issue = 3| doi = 10.1038/35043096| pmid = 11252899| s2cid = 5303121}}

File:extracts2E.png

Unfertilized eggs in a buffer containing the Ca2+ chelator EGTA (ethylene glycol tetraacetic acid) are packed into a centrifuge tube. After removing excess buffer, the eggs are crushed by centrifugation (~10,000 g). A soluble fraction that appears between the lipid cap and the yolk is called an M phase extract. This extract contains a high level of cyclin B-Cdk1. When demembranated sperm nuclei are incubated with this extract, it undergoes a series of structural changes and is eventually converted into a set of M phase chromosomes with bipolar spindles.

When CaCl₂ is added to an M-phase Xenopus egg extract at a concentration sufficient to override residual EGTA (typically several hundred micromolar), it triggers rapid inactivation of maturation promoting factor (MPF). This occurs through cyclin B degradation by the proteasome and induces cell cycle progression from metaphase to anaphase, and eventually into S phase. The resulting extract is referred to as an S-phase extract or interphase extract. Upon the addition of sperm chromatin to an S-phase extract, nuclear assembly is initiated: membrane vesicles accumulate around decondensed chromatin, fuse to form a nuclear envelope, and produce fully functional nuclei. Active nuclear transport occurs across the nuclear envelope, and DNA replication is initiated within the reconstituted nuclei. Because these extracts contain abundant mRNA and ribosomes, protein translation also takes place. Thus, this cell-free system can faithfully recapitulate many cellular events characteristic of proliferating cells. A notable exception is transcription, which does not occur in this system. This reflects the natural state of the Xenopus egg and early embryo, where transcription is largely repressed from the meiotic stages through to the blastula stage after fertilization.{{cite journal | author = Newport J, Kirschner M | title = A major developmental transition in early Xenopus embryos: I. characterization and timing of cellular changes at the midblastula stage | journal = Cell| volume = 30 | pages = 675–686 | year = 1982 | issue = 3 | doi = 10.1016/0092-8674(82)90272-0 | pmid =6183003}}

File:Nucleus-chromosomes.png

Cycling extract is a cell-free system derived from Xenopus eggs that autonomously undergoes repeated S phase and M phase cycles. It is prepared by artificially inducing intracellular responses that mimic fertilization, typically through calcium ionophore treatment or electrical stimulation of unfertilized eggs, followed by mechanical crushing of the eggs.{{cite journal | author = Murray AW| title = Cell cycle extracts| journal = Methods Cell Biol.| volume = 36 | pages = 581–605 | year = 1991 | doi = 10.1016/S0091-679X(08)60298-8| pmid = 1839804}} This extract has been instrumental in elucidating fundamental mechanisms of cell cycle regulation. For example, it revealed that entry into M phase depends on the translation of cyclin B, which in turn activates maturation promoting factor (MPF).{{cite journal | author = Murray AW, Kirschner MW| title = Cyclin synthesis drives the early embryonic cell cycle| journal = Nature| volume = 339 | pages = 275–280 | year = 1989 | issue = 6222| doi = 10.1038/339275a0| pmid = 2566917 | bibcode = 1989Natur.339..275M}} Cycling extracts are primarily used to study the regulation of cell cycle progression.

High-speed supernatant (HSS) is a fraction obtained by ultracentrifuging a conventional Xenopus egg extract at 100,000–200,000 × g, which removes membrane components and ribosomes, leaving a solution enriched in soluble proteins. Although HSS lacks the capacity to support nuclear assembly or protein translation, it can partially recapitulate chromatin structural changes in a cell cycle–dependent manner.{{cite journal | author = Lohka MJ, Maller JL. | title = Induction of nuclear envelope breakdown, chromosome condensation, and spindle formation in cell-free extracts | journal = J. Cell Biol.| volume = 101 | pages = 518–523 | year = 1985 | issue = 2 | doi = 10.1083/jcb.101.2.518 | pmid =3926780| pmc = 2113692 }} It is particularly suitable for protein purification.

Nucleoplasmic extract (NPE) is prepared from Xenopus egg extracts by first assembling nuclei in S-phase extract through the addition of a high concentration of sperm chromatin (~10,000 nuclei per μL). The reaction mixture is then centrifuged without dilution to separate the nuclei, which form a distinct layer at the top. This nuclear fraction is collected and further centrifuged at high speed, yielding a soluble supernatant (nucleoplasm) and a pellet containing nuclear membranes and chromatin. The supernatant is referred to as the nucleoplasmic extract (NPE). When DNA is pre-incubated in S-phase HSS and then NPE is added, DNA replication can be initiated without the need for nuclear envelope formation—a significant distinction from standard S-phase extract protocols, where replication initiation requires nuclear assembly.{{cite journal | author = Walter J, Sun L, Newport J| title = Regulated chromosomal DNA replication in the absence of a nucleus| journal = Mol. Cell| volume = 1 | pages = 519–529 | year = 1998 | issue = 4| doi = 10.1016/S1097-2765(00)80052-0| pmid = 9660936| doi-access = free}} This system has enabled high-resolution analysis of replication initiation mechanisms. Moreover, NPE supports efficient replication of plasmid DNA and other non-sperm-derived templates. Leveraging this property, researchers have also used NPE to investigate DNA repair pathways using exogenously damaged DNA substrates.{{cite journal | author = Räschle M, Knipscheer P, Enoiu M, Angelov T, Sun J, Griffith JD, Ellenberger TE, Schärer OD, Walter JC| title = Mechanism of replication-coupled DNA interstrand crosslink repair | journal = Cell| volume = 134| pages = 969–980| year = 2008 | issue = 6 | doi = 10.1016/j.cell.2008.08.030 | pmid = 18805090}}

• Purification of M-phase promoting factor (MPF){{cite journal | author = Lohka MJ, Hayes MK, Maller JL| title = Purification of maturation-promoting factor, an intracellular regulator of early mitotic events| journal =Proc. Natl. Acad. Sci. USA | volume = 85| pages = 3009–3013| year = 1988 | issue = 9| doi = 10.1073/pnas.85.9.3009| pmid = 3283736| pmc = 280132| bibcode = 1988PNAS...85.3009L| doi-access = free}}
• Elucidation of the role of synthesis and degradation of cyclin B in cell cycle progression{{cite journal | author = Murray AW, Kirschner MW| title = Cyclin synthesis drives the early embryonic cell cycle| journal = Nature| volume = 339 | pages = 275–280 | year = 1989 | issue = 6222| doi = 10.1038/339275a0| pmid = 2566917 | bibcode = 1989Natur.339..275M}} {{cite journal | author = Murray AW, Solomon MJ, Kirschner MW| title = The role of cyclin synthesis and degradation in the control of maturation promoting factor activity| journal = Nature| volume = 339 | pages = 280–286 | year = 1989 | issue = 6222| doi = 10.1038/339280a0| pmid = 2566918| bibcode = 1989Natur.339..280M| s2cid = 4319201}}
• Discovery that degradation of a protein(s) other than cyclin B is necessary for initiating chromosome segregation{{cite journal | author = Holloway SL, Glotzer M, King RW, Murray AW| title = Anaphase is initiated by proteolysis rather than by the inactivation of maturation-promoting factor| journal = Cell| volume = 73 | pages = 1393–1402 | year = 1993 | issue = 7| doi = 10.1016/0092-8674(93)90364-v| pmid = 8391932| s2cid = 26338475| doi-access = free}}
• Discovery of a mechanism of spindle assembly that depends on chromatin, but not centrosomes{{cite journal | author = Heald R, Tournebize R, Blank T, Sandaltzopoulos R, Becker P, Hyman A, Karsenti E| title = Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts| journal = Nature| volume = 382 | pages = 420–425 | year = 1996 | issue = 6590| doi = 10.1038/382420a0| pmid = 8684481| bibcode = 1996Natur.382..420H| s2cid = 4238425}}
• Proposal of a DNA replication licensing system{{cite journal | author = Blow JJ, Laskey RA| title = A role for the nuclear envelope in controlling DNA replication within the cell cycle| journal = Nature| volume = 332 | pages = 546–548| year = 1988 | issue = 6164| doi = 10.1038/332546a0| pmid = 3357511| bibcode = 1988Natur.332..546B| s2cid = 4313693}} and identification of its responsible factor{{cite journal | author = Kubota Y, Mimura S, Nishimoto S, Takisawa H, Nojima H| title = Identification of the yeast MCM3-related protein as a component of Xenopus DNA replication licensing factor| journal = Cell| volume = 81 | pages = 601–609| year = 1995 | issue = 4| doi = 10.1016/0092-8674(95)90081-0| pmid = 7758114| s2cid = 18797719| doi-access = free}}
• Identification of importin α/β responsible for nuclear transport{{cite journal | author = Görlich D, Prehn S, Laskey RA, Hartmann E| title = Isolation of a protein that is essential for the first step of nuclear protein import | journal = Cell| volume = 79| pages = 767–778| year = 1994 | issue = 5 | doi = 10.1016/0092-8674(94)90067-1 | pmid = 8001116| s2cid = 7539929 }}
• Discovery of the condensin complex essential for mitotic chromosome assembly{{cite journal | author = Hirano T, Mitchison TJ| title = A heterodimeric coiled-coil protein required for mitotic chromosome condensation in vitro| journal = Cell| volume = 79 | pages = 449–458 | year = 1994 | issue = 3| doi = 10.1016/0092-8674(94)90254-2| pmid = 7954811| s2cid = 24140495}}{{cite journal | author = Hirano T, Kobayashi R, Hirano M| title = Condensins, chromosome condensation protein complexes containing XCAP-C, XCAP-E and a Xenopus homolog of the Drosophila Barren protein | journal = Cell| volume = 89 | pages = 511–521| year = 1997 | issue = 4 | doi = 10.1016/s0092-8674(00)80233-0 | pmid = 9160743| s2cid = 15061740 | doi-access = free }}
• Identification of the cohesin complex essential for sister chromatid cohesion{{cite journal | author = Losada A, Hirano M, Hirano T| title = Identification of Xenopus SMC protein complexes required for sister chromatid cohesion| journal = Genes Dev. | volume = 12 | issue = 13 | pages = 1986–1997| year = 1998 | doi = 10.1101/gad.12.13.1986| pmid = 9649503| pmc = 316973}}

More recently, the egg extracts have been used to study reprogramming of differentiated nuclei,{{cite journal | author = Ganier O, Bocquet S, Peiffer I, Brochard V, Arnaud P, Puy A, Jouneau A, Feil R, Renard JP, Méchali M| title = Synergic reprogramming of mammalian cells by combined exposure to mitotic Xenopus egg extracts and transcription factors | journal = Proc Natl Acad Sci USA| volume = 108| pages =17331–17336| year = 2011 | issue = 42 | doi = 10.1073/pnas.1100733108 | pmid = 21908712| pmc = 3198361 | doi-access = free }} physical properties of spindles{{cite journal | author = Shimamoto Y, Maeda YT, Ishiwata S, Libchaber AJ, Kapoor TM| title = Insights into the micromechanical properties of the metaphase spindle | journal = Cell| volume = 145| pages = 767–778| year = 2011 | issue = 7 | doi = 10.1016/j.cell.2011.05.038 | pmid = 21703450| pmc = 3124677 }} and nuclei,{{cite journal | author = Hara Y, Merten CA| title = Dynein-based accumulation of membranes regulates nuclear expansion in Xenopus laevis egg extracts | journal = Dev Cell| volume = 33| pages =562–575| year = 2015 | issue = 5 | doi = 10.1016/j.devcel.2015.04.016 | pmid = 26004509| doi-access = free}} and theoretical understanding of cell cycle control.{{cite journal | author = Pomerening JR, Kim SY, Ferrell JE Jr| title = Systems-level dissection of the cell-cycle oscillator: bypassing positive feedback produces damped oscillations | journal = Cell| volume = 122| pages =565–578| year = 2005 | issue = 4 | doi = 10.1016/j.cell.2005.06.016 | pmid =16122424| s2cid = 11835940 | doi-access = free}}

• Yoshio Masui
• cell cycle
• Cdk1
• cyclin
• DNA replication
• nuclear transport
• spindle apparatus
• condensin
• cohesin

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Category:Cell cycle

Category:Mitosis

Category:DNA replication