Key Concepts Of Section 211

Overview of the Cell Cycle and Its Control

■ The eukaryotic cell cycle is divided into four phases: M (mitosis), G1 (the period between mitosis and the initiation of nuclear DNA replication), S (the period of nuclear DNA replication), and G2 (the period between the completion of nuclear DNA replication and mitosis) (see Figure 21-1).

■ Cyclin-CDK complexes, composed of a regulatory cy-clin subunit and a catalytic cyclin-dependent kinase subunit, regulate progress of a cell through the cell cycle (see Figure 21-2). Large multisubunit ubiquitin ligases also polyubiquitinate key cell-cycle regulators, marking them for degradation by proteasomes.

■ Diffusible mitotic cyclin-CDK complexes cause chromosome condensation and disassembly of the nuclear envelope in G1 and G2 cells when they are fused to mitotic cells. Similarly, S-phase cyclin-CDK complexes stimulate DNA replication in the nuclei of G1 cells when they are fused to S-phase cells.

■ The isolation of yeast cell-division cycle (cdc) mutants led to the identification of genes that regulate the cell cycle (see Figure 21-4).

■ Amphibian and invertebrate eggs and early embryos from synchronously fertilized eggs provide sources of extracts for biochemical studies of cell-cycle events.

fertilizing them simultaneously by addition of sperm (or treating them in ways that mimic fertilization), researchers can obtain extracts from cells at specific points in the cell cycle for analysis of proteins and enzymatic activities.

In the following sections we describe critical experiments that led to the current model of eukaryotic cell-cycle regulation summarized in Figure 21-2 and present further details of the various regulatory events. As we will see, results obtained with different experimental systems and approaches have provided insights about each of the key transition points in the cell cycle. For historical reasons, the names of various cy-clins and cyclin-dependent kinases from yeasts and vertebrates differ. Table 21-1 lists the names of those that we discuss in this chapter and indicates when in the cell cycle they are active.

21.2 Biochemical Studies with Oocytes, Eggs, and Early Embryos

A breakthrough in identification of the factor that induces mitosis came from studies of oocyte maturation in the frog Xenopus laevis. To understand these experiments, we must first lay out the events of oocyte maturation, which can be duplicated in vitro. As oocytes develop in the frog ovary, they replicate their DNA and become arrested in G2 for 8 months during which time they grow in size to a diameter of 1 mm, stockpiling all the materials needed for the multiple cell divisions required to generate a swimming, feeding tadpole. When stimulated by a male, an adult female's ovarian cells secrete the steroid hormone progesterone, which induces the G2-arrested oocytes to enter meiosis I and progress through meiosis to the second meiotic metaphase (Figure 21-5). At this stage the cells are called eggs. When fertilized by sperm, the egg nucleus is released from its metaphase II arrest and completes meiosis. The resulting haploid egg pronucleus then fuses with the haploid sperm pronucleus, producing a diploid zygote nucleus. DNA replication follows and the first mitotic division of early embryogenesis begins. The resulting embryonic cells then proceed through 11 more rapid, synchronous cell cycles generating a hollow sphere, the blastula. Cell division then

Oocyte Meiosis I Egg arrested Male Female First arrested in G2 in meiosis II pronucleus pronucleus cleavage

Oocyte Meiosis I Egg arrested Male Female First arrested in G2 in meiosis II pronucleus pronucleus cleavage

▲ EXPERIMENTAL FIGURE 21-5 Progesterone stimulates meiotic maturation of Xenopus oocytes in vitro. Step 1 : Treatment of G2-arrested Xenopus oocytes surgically removed from the ovary of an adult female with progesterone causes the oocytes to enter meiosis I. Two pairs of synapsed homologous chromosomes (blue) connected to mitotic spindle microtubules (red) are shown schematically to represent cells in metaphase of meiosis I. Step 2| : Segregation of homologous chromosomes and a highly asymmetrical cell division expels half the chromosomes into a small cell called the first polar body. The oocyte immediately commences meiosis II and arrests slows, and subsequent divisions are nonsynchronous with cells at different positions in the blastula dividing at different times.

Maturation-Promoting Factor (MPF) Stimulates Meiotic Maturation of Oocytes and Mitosis in Somatic Cells

When G2-arrested Xenopus oocytes are removed from the ovary of an adult female frog and treated with progesterone, they undergo meiotic maturation, the process of oocyte maturation from a G2-arrested oocyte to the egg arrested in metaphase of meiosis II (see Figure 21-5). Microinjection of cytoplasm from eggs arrested in metaphase of meiosis II into G2-arrested oocytes stimulates the oocytes to mature into

▲ EXPERIMENTAL FIGURE 21-6 A diffusible factor in arrested Xenopus eggs promotes meiotic maturation. When

~5 percent of the cytoplasm from an unfertilized Xenopus egg arrested in metaphase of meiosis II is microinjected into a G2-arrested oocyte (step 1a| ), the oocyte enters meiosis I (step 2 ) and proceeds to metaphase of meiosis II (step 3 ), generating a mature egg in the absence of progesterone. This process can be in metaphase to yield an egg. Two chromosomes connected to spindle microtubules are shown schematically to represent egg cells arrested in metaphase of meiosis II. Step 3 : Fertilization by sperm releases eggs from their metaphase arrest, allowing them to proceed through anaphase of meiosis II and undergo a second highly asymmetrical cell division that eliminates one chromatid of each chromosome in a second polar body. Step 4| : The resulting haploid female pronucleus fuses with the haploid sperm pronucleus to produce a diploid zygote, which undergoes DNA replication and the first mitosis of 12 synchronous early embryonic cleavages.

eggs in the absence of progesterone (Figure 21-6). This system not only led to the initial identification of a factor in egg cytoplasm that stimulates maturation of oocytes in vitro but also provided an assay for this factor, called maturation-promoting factor (MPF). As we will see shortly, MPF turned out to be the key factor that regulates the initiation of mitosis in all eukaryotic cells.

Using the microinjection system to assay MPF activity at different times during oocyte maturation in vitro, researchers found that untreated G2-arrested oocytes have low levels of MPF activity; treatment with progesterone induces MPF activity as the cells enter meiosis I (Figure 21-7). MPF activity falls as the cells enter the interphase between meiosis I and II, but then rises again as the cells enter meiosis II and remains high in the egg cells arrested in metaphase II. Following repeated multiple times without further addition of progesterone, showing that egg cytoplasm contains an oocyte maturation-promoting factor (MPF). Microinjection of G2-arrested oocytes provided the first assay for MPF activity (step 1b) at different stages of the cell cycle and in different organisms. [See Y. Masui and C. L. Markert, 1971, J. Exp. Zool. 177:129.]



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