p42MAPK Rsk



Myt1 V

Cdc2/cyclin B

Fig. 1. Simplified diagram of signaling during oocyte maturation. Through a receptor yet-to-be-unambiguously identified (2), progesterone leads to a transient decrease in cAMP levels and protein kinase A (PKA) activity. PKA directly inhibits the phosphatase Cdc25 and thus helps suppress Cdc2 activation (20). PKA also inhibits several other signaling events through unknown mechanisms (shown as dotted lines) The kinase Aurora-A phosphorylates CPEB (cytoplasmic polyadenylation element-binding protein) and thus promotes the polyadenylation and translational activation of maternal mRNA encoding the kinase Mos (21,22). This in turn triggers a kinase cascade through MAPK, Rsk, and ultimately Cdc2 (23). For more details, see ref. 1. PKI, the heat-stable inhibitor subunit of PKA, can be used to stimulate signaling in oocyte extracts (10).

associated receptor, mPR, has been identified in fish and frog oocytes (6). Once activated, Cdc2/cyclin B triggers cell cycle reentry, meiotic progression, and differentiation into a "mature" fertilizable egg. Thus, Xenopus oocyte maturation has become an important paradigm for the G2/meiosis I cell cycle transition, and it is also an important system to study nongenomic mechanisms of steroid hormone effects.

1.2. Advantages of Studying Signaling in Oocyte Extracts

Historically, oocyte maturation has been studied using intact oocytes; ovary fragments are surgically isolated, and individual oocytes are released after defolliculation by collagenase. Signaling events can be followed by biochemical analysis of oocytes taken at different times following progesterone stimulation; entry to meiosis is conveniently marked by a white spot that appears at the animal pole following breakdown of the nuclear (germinal vesicle) envelope. Intact oocytes can be manipulated by microinjection of messenger ribonucleic acid (mRNA), protein, or chemical inhibitors.

However, synchrony among different oocytes is often poor, even within a batch from the same frog. It can be difficult to achieve sufficient loss of proteins through antisense or RNAi approaches because oocytes contain large stockpiles of maternally supplied proteins. It is also difficult to gauge the cytosolic concentration of injected molecules because of variable leakage during injection and absorption by the yolk. Oocyte extracts are therefore attractive because they potentially offer synchronous signaling, immunodepletion of specific maternal proteins, and addition of precisely known concentrations of proteins or chemical inhibitors.

1.3. Difficulties Encountered With Oocyte Extracts

Extracts of Xenopus eggs can reliably complete one or more full cell division cycle in the test tube (7-9). For this reason, egg extracts have been developed to investigate many aspects of cell cycle regulation, including deoxyribonucleic acid (DNA) replication, replication licensing, spindle formation, mitotic entry, DNA damage and replication checkpoints, chromosome segregation, and proteolysis during mitotic exit.

By contrast, oocyte extracts have yet to achieve such widespread utility. Efforts to develop oocyte extracts that reproduce all events of the G2/meiosis I transition in vitro have met with two major obstacles. First, oocyte extracts tend to activate signaling spontaneously, probably because of transient decreases in cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) activity (see Fig. 1) or removal of inhibitors during extract preparation. Spontaneous activation can be suppressed by addition of the cAMP analog 8-[4-chlorophenylthio]-cAMP (10).

Second, it has not been possible so far to reproduce the complete signaling pathway from progesterone to Cdc2 activation. We have found that oocyte extracts can occasionally induce p42MAPK (p42 mitogen-activated protein kinase) activation in response to progesterone or testosterone, but only if the dose of 8-[4-chloro-phenylthio]-cAMP is reduced to a level that risks spontaneous activation (R. C. and J. V. R., unpublished data, 2003). The separation of cytosolic and lipid fractions during extract preparation may also tend to disrupt membrane-associated signaling components. Consistent with this notion, mPR and a small but significant fraction of the other presumed Xenopus progesterone receptor (XPR), are membrane associated (5,6).

1.4. Examples of the Use of Oocyte Extracts

Despite the difficulties in reproducing progesterone-stimulated signaling, oocyte extracts have nevertheless proven to be a powerful system to investigate signaling events further downstream. Shibuya and colleagues developed oocyte extracts that can activate both p42MAPK and Cdc2 in response to specific stimuli (11). These stimuli include okadaic acid (a phosphatase inhibitor) and indestructible cyclin mutants used to demonstrate the existence of a positive-feedback loop from Cdc2 to p42MAPK. It was subsequently found that addition of purified Mos kinase to oocyte extracts can activate p42MAPK and Rsk (12,13) and even Cdc2 if the extracts are supplied with additional p42MAPK (14). Addition of the heat-stable inhibitor subunit of protein kinase A, PKI (protein kinase A inhibitor), was found to activate numerous signaling events known to occur in progesterone-stimulated intact oocytes, including the synthesis of Mos kinase and activation of p42MAPK, Plx1, and Cdc25 (15).

Signaling in oocyte extracts can be easily manipulated by the addition of purified proteins that antagonize the function of particular signaling molecules. For example, the addition of purified MAPK phosphatase inhibits p42MAPK in extracts (14), and puri fied p21(Cip1) inhibits Cdc2 (10). The suspected involvement of the kinase Raf in signaling from Mos to p42MAPK was ruled out using a dominant negative Raf protein (16).

Given appropriate antibodies, endogenous signaling proteins can be quantitatively removed from extracts by immunodepletion. For example, removal of Plx1 blocks PKI-stimulated activation of Cdc2 (10). Other reagents that fail to cross cell membranes can be added directly to extracts, thus widening the range of experimental opportunities. Finally, oocyte extracts have afforded an opportunity to study signaling proteins not normally involved in oocyte maturation. Examples include the oncogenic mammalian proteins H-Ras (V12) (12,17), and v-Src (18).

2. Materials

1. Materials for surgical removal of Xenopus oocytes (as described in Chapter 3): forceps, scissors, absorbent pads, anesthetic (MS-222, Sigma A5040, St. Louis, MO), stitches.

2. Dissecting microscope.

3. OR2 buffer: 5 mM HEPES/NaOH at pH 7.6 and 18°C, 82.5 mM NaCl, 2.5 mM KCl, 1 mM MgCl2. A 10X stock can be prepared in advance and stored at 4°C for several weeks. Filter sterilize the 1X buffer before use and warm to 18°C.

4. Materials to defolliculate oocytes: 10-cm glass Petri dishes extensively washed to remove soap residue, 50-mL polypropylene tubes (e.g., Falcon), collagenase type 1A (Sigma C9891), trypsin inhibitors (type 1-S, Sigma T9003), bovine serum albumin (Sigma A4503).

5. ND96 buffer: 5 mM HEPES/NaOH at pH 7.6 and 18°C, 96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2. A 10X stock can be prepared in advance and stored at 4°C for several weeks. Before use, filter sterilize the 1X buffer, add penicillin/streptomycin (Invitrogen/Gibco 15140-148, Carlsbad, CA) and warm to 18°C.

6. Refrigerate or keep at 18°C.

7. Extract buffer: 250 mM sucrose, 100 mM NaCl, 2.5 mM MgCl2, 20 mM HEPES/NaOH at pH 7.2.

8. Centrifugation equipment: tabletop clinical centrifuge, refrigerated ultracentrifuge with a swinging bucket rotor capable of 15,000g (e.g., Sorvall HB6 rotor, Asheville, NC), 13 x 51 mm ultracentrifuge tubes (Nalgene 3410-1351, Rochester, NY), and Versilube F-50 (a silicone oil; General Electric Corp., Westview, NY).

9. A mix of 10 mg/mL each of leupeptin (Sigma L2023), chymostatin (Sigma C7268), and pepstatin A (Sigma P4265) in dimethyl sulfoxide (DMSO); store at -20°C.

10. 20 mg/mL Stock of cytochalasin B (Sigma 6762) in DMSO; store at -20°C.

11. 2 mg/mL Stock of creatinine phosphate (Sigma C6507) in water; store at -70°C.

12. 100 mM EGTA [ethylene glycol bi's(b-aminoethyl ether)-N,N,W,W-tetraacetic acid; Sigma E4378], pH 5.2.

13. 1 mg/mL Ac-DEVD-CHO (caspase inhibitor; Biomol, Plymouth Meeting, PA) in DMSO; store at -20°C.

14. 25 mg/mL stock of 8-[4-chlorophenythio]-cAMP (a cAMP analog; Sigma C3912) in DMSO; store at -20°C.

15. Dilution buffer: extract buffer (see above) with 1 mM sodium orthovanadate, 10 mM sodium fluoride, 5 mM EGTA at pH 5.2, 40 mM P-glycerophosphate. This solution should be filter sterilized and stored at 4°C. Immediately before use, add a protease inhibitor cocktail (e.g., Complete EDTA-free, Roche, Indianapolis, IN) and 10 |lg/mL phenylmethylsulfonyl fluoride (Sigma P7626).

16. Signaling stimulus as necessary, for example: heat-stable inhibitor of protein kinase A (PKI; Sigma P0300), progesterone (Sigma P0130), or recombinant Mos protein (15).

3. Methods

The methods outline (1) harvest of oocytes, (2) the preparation of cytosolic extracts, and (3) use of the extracts to study signaling.

3.1. Harvesting Xenopus Oocytes

To obtain a final extract volume of 500 |L, between 3000 and 5000 oocytes should be harvested. It is inadvisable to combine oocytes from more than one frog because poor-quality oocytes from one frog are prone to lyse prematurely and may trigger apoptosis in the entire extract. The procedure for harvesting oocytes is described in detail in Chapter 3, but for completeness is briefly summarized here.

1. Anesthetize a female frog, make a lateral incision, and observe a small ovary fragment under the dissecting microscope. Oocytes with a pale or speckled animal pole generally give poor-quality extract, so the incision should be repaired and the frog allowed to recover. If the oocytes are good quality, surgically remove as much of the ovaries as possible into OR2 buffer.

2. Cut apart the ovary lobes and wash four or five times with 50 mL OR2 buffer to remove as much blood and tissue debris as possible.

3. Defolliculate the oocytes by gentle swirling in two or three 10-cm glass Petri dishes (rinsed free of soap residue), each containing 10 mL OR2 buffer with 5 mg/mL collagenase, 1 mg/mL trypsin inhibitors, and 1 mg/mL bovine serum albumin. Incubate for 90 min or until about half the oocytes are detached from the ovary (do not exceed 2 h). Using a 50-mL polypropylene tube, wash the oocytes several times in 50 mL OR2 buffer and then several times in 50 mL ND96 buffer. Be careful not to agitate the oocytes. See Note 1 for alternative methods.

4. Select stage VI oocytes (the largest) using a sizing mesh or by manual transfer to a fresh 10-cm glass dish containing 10 mL ND96 buffer. For transferring oocytes, use a sawed glass pipet that has been blunted in a flame to give a smooth aperture larger than an oocyte. Remove any lysed or apoptotic oocytes (which have a marbled appearance) and wash further in ND96 buffer.

5. Allow the oocytes to recover for at least 8 h (or overnight) at 18° C.

3.2. Preparation of Cytosolic Oocyte Extracts

It is important to keep the oocytes at 18°C and the extract at 4°C from the time of centrifugation, so make sure all the buffers, tubes, and centrifuges are appropriately cooled in advance.

1. Using a sawed, blunted glass pipet, remove any lysed or apoptotic oocytes and wash twice in ND96 buffer (18°C) by gentle inversions in a 50-mL polypropylene tube.

2. Wash oocytes twice in extract buffer (18°C) and then twice in extract buffer containing 5 |g/mL each of leupeptin, pepstatin, and chymostatin. Continue to remove lysed oocytes and take care not to agitate the tube. See Note 2 for an alternative buffer.

3. Fill as many ultracentrifuge tubes (13 x 51 mm) as necessary with oocytes, remove excess extract buffer, and add 200 |L Versilube F-50. Place the tubes inside 15-mL glass Corex tubes (to act as adaptors) and spin in a tabletop clinical centrifuge at 150g for 1 min, then 600g for 30 s. Versilube F-50 is a silicone oil that displaces the buffer surrounding the oocytes, resulting in tighter packing. See Note 2 for an alternative centrifu-gation procedure.

4. Remove the Versilube F-50 and transfer tubes to an ultracentrifuge swinging bucket rotor chilled to 2°C. Spin at 15,000g for 15 min. The oocyte lysate will separate into an upper yellow lipid layer, a central straw-colored cytosolic layer, and a lower black yolk protein layer.

5. Place the tubes in an ice-cooled water bath. One by one, remove the cytosolic layer by piercing the side of tube with a 20-gage needle connected to a 1-mL syringe. Take care to aspirate slowly (it should take at least 20 s to remove the entire layer) but do not be concerned by minor contamination of the lipid or yolk layers. Before expelling the extract from the syringe, remove the needle to reduce shearing. Pool the cytosolic layers into a single ultracentrifuge tube and place in an ice-cooled bath.

6. Per 1 mL of the pooled cytosolic layer, quickly add the following: 1 ||L 1 M MgCl2, 2.5 |L 10 mg/mL leupeptin/chymostatin/pepstatin mix, 0.5 |L 20 mg/mL cytochalasin B, 1 |L 2 mg/mL creatinine phosphate, 1 |L 100 mM EGTA at pH 5.2, 0.6 |L 1 mg/mL Ac-DEVD-CHO, and 2 |L 25 mg/mL 8-[4-chlorophenythio]-cAMP. Mix gently using a sawed pipet tip. For a discussion of the necessity for each of these reagents, see Note 3.

7. Spin again at 15,000g for 15 min at 2°C. The extract will again separate into lipid, cyto-solic, and yolk layers.

8. Slowly remove the cytosolic layer with a 20-gage needle (see step 5), again removing the needle before expelling extract from the syringe. Aliquot the extract into 1.5-mL tubes, either for immediate use or for freezing on liquid nitrogen for storage at -70°C (see Note 4).

3.3. Using Oocyte Extracts to Study Signaling

The 50-|L extract aliquots can be stimulated and manipulated in a variety of ways, some of which are listed next. At time-points following stimulus, typically hourly from 0 to 6 h, take 5-|L aliquots of the extract into 45-|L dilution buffer. For analysis by polyacrylamide gel electrophoresis, these diluted samples can be immediately mixed with sodium dodecyl sulfate sample buffer and boiled. For immunoblotting, loading the equivalent of 0.5 |L undiluted oocyte extract per lane will be appropriate for most antibodies.

Diluted samples can also be used for enzyme activity assays, for example, histone H1 kinase assays to monitor Cdc2 activity. To maintain phosphorylation and activity of certain kinases in the diluted samples (including Aurora-A but not p42MAPK), it is necessary to include the phosphatase inhibitor okadaic acid in the dilution buffer (final concentration 0.5 |M).

In published experiments using PKI-stimulated oocyte extracts, synthesis of Mos protein and the activation of p42MAPK, Plx1, Cdc25, and Cdc2/cyclin B all occur around 3 to 4 h (10); actual timing may vary according to temperature and extract quality.

The following list includes the more common manipulations of oocyte extracts. When designing an experiment, it is extremely important to minimize the dilution of extract by making stock reagents as concentrated as possible. It is also important to include controls for the potential effect of adding solvents to the extracts.

3.3.1. Stimuli

Progesterone (Sigma P0130): we have initiated p42MAPK phosphorylation in oocyte extracts using a range of doses from 0.01 to 10 mg/mL (~0.03-30 mM) (R. C. and J. V. R., unpublished). A 10-mg/mL stock in anhydrous ethanol does not lose potency for at least 3 mo at -20°C. Progesterone is also available in a cyclodextrin-encapsulated water-soluble form (Sigma P7556), but we have not thoroughly investigated its ability to stimulate oocyte extracts. Take great care to avoid contaminating laboratory equipment with progesterone because it strongly adheres to plastics.

Mos protein: 100 mg/mL (15). Bacterially expressed Mos protein is inactive but is activated on incubation with oocyte extract.

Rat p42MAPK (ERK2) to establish more robust signaling between Mos and Cdc2 (14): 44 mg/mL. This compares to the endogenous Xenopus p42MAPK concentration of 25 mg/mL. Avoid any commercial ERK2 preparations that are coexpressed in bacteria with constitutively active mitogen-activating protein kinase kinase (MEK).

3.3.2. Pharmacological Agents

Cycloheximide (protein synthesis inhibitor; Sigma C6255): 100 mg/mL (10). A 10-mg/mL stock solution in water can be stored indefinitely at -20°C.

Roscovitine (Cdc2 and Cdk2 inhibitor; Sigma R7772): 50 mM (19). Avoid using higher doses, which may cause nonspecific inhibition of a broader range of kinases. UO126 (MEK inhibitor; Promega V1121, Madison, WI): 50 mM (10). A 20-mM stock solution in dimethyl sulfoxide is stable at -20°C for approx 2 wk.

3.3.3. Immune Depletions

For immune depletions from oocyte extracts, incubate 50 |L extract with 5 ||L antibody-coupled beads for 1 h at 4°C (10).

4. Notes

1. Qian and coworkers (10) used calcium-free Barth's solution in place of OR2. They defolliculated oocytes by a 2-h treatment with 0.5 mg/mL dispase and then added collagenase for an additional hour. In place of ND96, they used 0.65X Dulbecco's modified Eagle's medium with 25 mM HEPES at pH 7.5 and penicillin/streptomycin. Previous studies from the same lab obtained oocytes by manual defolliculation (15,18). This method avoids the need for oocyte recovery after collagenase treatment but requires dexterity and yields fewer oocytes.

2. VanRenterghem and coworkers (15,18) were able to achieve p42MAPK activation in response to PKI, v-Src, or cyclin A using a cruder extract preparation. Oocytes were homogenized in 6 volumes of a buffer containing 20 mM Tris-HCl at pH 7.2, 15 mM MgCl2, 80 mM ¿-glycerophosphate, 20 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, and 3 |lg/mL leupeptin and then centrifuged at 12,000g for 5 min. The supernatant was diluted twofold in 20 mM Tris-HCl at pH 7.2, 1 mM dithiothreitol prior to stimulation.

3. When extracts are incubated in the absence of stimulus, signaling pathways will often be activated spontaneously. On such occasions, p42MAPK threonine phosphorylation is typically observed after 2 h (R. C. and J. V. R., unpublished observations, 2003). Several factors may influence such "spontaneous activation." It is certainly important to observe strictly the required temperatures during extract preparation and to perform the extract procedure as rapidly as possible. Any apoptotic oocytes still present at the time of cen-trifugation will lyse and trigger apoptotic signaling; it is therefore essential to include Ac-DEVD-CHO in the extract. Finally, any transient decrease in the activity of cAMP-dependent PKA may be sufficient to trigger the oocyte maturation signaling pathway. To avoid this, the cAMP analog 8-[4-chlorophenythio]-cAMP is included at 50 |g/mL

(100 ||M) in the extracts to maintain high PKA activity. While attempting to reproduce hormone-induced signaling, we have found it necessary to reduce the concentration of 8-[4-chlorophenythio]-cAMP to 0.5 |g/mL or lower (R. C. and J. V. R., unpublished observations, 2003).

4. Although frozen extract is capable of reproducing some aspects of signaling (11), it is advisable to use fresh extract whenever possible. Freezing destroys the extract's capacity to synthesize proteins, and de novo synthesis of Mos and perhaps other signaling proteins is necessary for oocyte maturation in intact oocytes.


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11 Shibuya, E. K., Polverino, A. J., Chang, E., Wigler, M., and Ruderman, J. V. (1992) Onco-genic ras triggers the activation of 42-kDa mitogen-activated protein kinase in extracts of quiescent Xenopus oocytes. Proc. Natl. Acad. Sci. USA 89, 9831-9835.

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21 Mendez, R., Hake, L. E., Andresson, T., Littlepage, L. E., Ruderman, J. V., and Richter, J. D. (2000) Phosphorylation of CPE binding factor by Eg2 regulates translation of c-mos mRNA. Nature 404, 302-307.

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23 Palmer, A., Gavin, A. C., and Nebreda, A. R. (1998) A link between MAP kinase and p34(cdc2)/cyclin B during oocyte maturation: p90(rsk) phosphorylates and inactivates the p34(cdc2) inhibitory kinase Myt1. EMBO J. 17, 5037-5047.

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