It was once believed that microbes generated spontaneously. For example, meat was believed to rot because microbes spontaneously formed from the blood. As spontaneous generation became increasingly controversial, the following experiments were performed:

These results support that airborne microbes were settling into the broth. However, some people argued that growth did not occur in sealed flasks because there was not enough circulation. Pasteur proposed the U-Tube experiment, using a specialized flask he developed. Shown belows is a diagram of Pasteur’s U-Tube flask:

The flask is partially filled with nutrient broth. The broth is sterilized via boiling. The flask is incubated for 72 hrs. Result: no growth. The flask is tipped so that the broth spills into the dip in the neck. The flask is incubated for 72 hrs. Result: growth observed. This experiment works because no microbes can fall into the broth. The microbes are captured in the dip in the neck. When the flask is tipped, though, and the neck is washed with nutrient broth, the broth gets polluted. That is why growth is observed in the second part of the experiment.

Endospores are produced by sporulation. Endospores are differentiated cells highly ressistant to heat, chemicals & radiation. They are strongly refractile, impermeable to most dyes. Outermost layer is the exosporium, a thin protein covering. Within this are spore coats composed of spore-specific porteis. Then the cortex, composed of loosly corss-linked peptidoglycan. Then the core or spore protplast. Core wall, cm, cm, nucleoid, ribosomes & other cellular essentials. Differs from vegetative cell based on structure found outside core wall. Characteristic of endospores is dipicolinic acid. Located in the core. Endospores enriched in calclium ions, combined with dipicolinic acid. Repressents 10% of dry wight. Intercalates in DNA & stabliizes it to heat denaturation. Calcium dipicolinate reduces H₂O content of core. Dehydrations confers resistance to chemicals like H₂O₂ (hydrogen peroxide) & causes enzymes remaining in core to become inactive. pH of core one unit lower than vegetative cell cytoplasm. Small acid-soluble proteins in high concentration (SASPs). Made during sporulation & have 2 functions: bind tightly to DNA & protet it from UV, dessication & dry heat. SASPs make DNA the A form. They also function as a carbon & NRG source for the outgrowth of a new vegetative cell from endospore, process caled germination.

Microtubule organizing centers (aka centrosomes) are composed of asters at each end, with centrosomes spanning between them. The tubules that connect to the chromosome kinetochore are called kinetochore microtubules, while the tubules which interact with each other are polar microtubules (aka non-kinetochore microtubules). Microtubules are composed of α- and β-tubulin monomers polymerized to form hollow tubes. Depolymerization shortens the microtubules, while the chromosomes simultaneously migrate toward the asters, driving apart the spindle poles.

Cycles in M-Phase Promoting Factor (MPF) activity control mitosis. As a protein kinase, MPF likely acts via phosphorylation of the major histone protein H1 and the major nuclear envelope protein lamin. This leads to the degradation of the nuclear envelope and condensation of chromatin into chromosomes in anticipation of mitosis. Found in all organisms, MPF is a heterodimer composed of cyclin-dependent kinase (cdk1) and cyclin B.

Cycles of MPF activity are based on synthesis and degradation of cyclin B. During the M phase (mitosis) the heterodimer is functional and drives cells into metaphase. Cyclin B is degraded, inactivating MPF once cells go into mitosis. During the S phase, newly synthesized Cyclin B from maternal mRNA leads to formation of a new functional MPF heterodimer.

Cytoplasm from mammalian cells arrested in metaphase of mitosis by treatment with drugs that inhibit the polymerization of microtubules (e.g. colchicine or nocodazole) had high oocyte maturation promoting factor (MPF).

When the ctoplasm from Xenopus eggs was injected into the cytoplasm of mammalian cells in G1, it caused the mammalian cells to undergo the events of early mitosis: chomosome condensation and nuclear envelope break down.

Also, when the cytoplasm from mammalian cells arrested in mitotic metaphase by treatment with micrtotubule inhibitors was injected into the cytoplasm of mammalian cells in G1, it caused the mammalian cells to undergo the events of early mitosis: chomosome condensation and nuclear envelope break down.

This is like the mitosis promoting activity first observed in the cell fusion experiments.

So, Xenopus ooctye MATURATION PROMOTING FACTOR = mammalian MITOSIS PROMOTING FACTOR.

(Fortunatley, “MPF” is the abbreviation for both Maturation Promoting Factor (revealed by injection into Xenopus oocytes) and Mitosis Promoting Factor (revealed by fusing an M-phase cell to a cell in G1).

Oocytes remain arrested in G2 until they mature and addition of a sperm nucleus (via fertilization when in vivo). This is a great model for figuring out what the egg must have synthesized beforehand for mitosis to occur.

Polyubiquitination marks eukaryotic proteins for degradation by proteasomes. Three enzymes are required for the Ubiquitin to work: E1, the Ubiquitin-activating enzyme; E2, the Ubiquitin-conjugating enzyme; and E3, the Ubiquitin ligase. SCF and APC/C are ubiquitin protein ligase complexes that that control three major transitions in the cell cycle: onset of S-phase through degradation of Sic1 by SCF; initiation of anaphase via degradation of securin by APC-Cdc20; exit from mitosis via degradation of cyclin B’s by APC-Cdh2. APC has several substrates that must be degraded at different times in the cycle; thus, its activity is directed by specificity factors that bind it. SCF only degrades Sic1 and thus its activity is regulated only by phosphorylation of its substrate.

	Mitotic Events
Process	Genomic		Cellular
Prophase	The genome condenses. Visible chromosomes form. Each chromosome has 2 sister chromatids bound at the centromere by cohesin.		Spindle fibers (aka spindle poles) emanate from the centromere. The two centrosomes form G2 sprout microtubules by polymerizing free-floating tubulin. The microtubules repel each other, pushing the centrosomes to opposite ends of the cell. This microtubular network is the start of the mitotic spindle. The nuclear double-membrane begins to reabsorb into the endoplasmic reticulum; when this finishes in prometaphase, the cell no longer has a nucleus.
Prometaphase	Each sister chromatid forms a kinetochore at its centromere as a place for microtubules to latch onto the chromosome (thus, there are two kinetochores per centromere). With the nuclear envelope gone, microtubules invade the nuclear space and (once the centromere’s spindles are sufficiently long) clasp onto the kinetochore. The kinetochore has an ATP-dependent motor that is activated by the microtubules. Kinetochore motors push each chromosome along the microtubules toward the metaphase plate. The metaphase plate is an imaginary plane that is perpendicular to the plane between the two centrosomes. Microtubules not bound to a kinetochore find and bind other “free” microtubules from the opposite centrosome; this forms the mitotic spindle.
Metaphase	The chromosomes have aligned such that their centromeres convene on metaphase plate. This precise alignment is due to opposing kinetochores functioning like equally strong people in a tug of war. Some species’ chromosomes do not align, but instead move randomly back and forth between the poles before roughly lining up at the metaphase plate. The mitotic spindle checkpoint refers to an arresting signal emitted by any kinetchores still unattached to microtubules. This is important because proper chromosomes separation in anaphase requires attachment of every kinetochore to many microtubules. Anaphase commences only once every kinetochore has attached to a cluster of microtubules, with every chromosome thus lined up along the metaphase plate.
Anaphase	Sister chromatids separate, resulting in two distinct populations of genetic material (one at each centrosome) that are identical. This occurs in two steps sometimes labeled early anaphase and late anaphase. Also, cytokinesis begins in anaphase and ends in telophase. Cohesin is cleaved, and sister chromatids thus become sister chromosomes. Sister chromosomes are pulled apart toward their attached centrosome via shortening of kinetochore microtubules. The non-kinetochore microtubules elongate, pushing the centrosomes (and the set of chromosomes to which they are attached) apart to opposite ends of the cell.
Telophase	The nonkinetochore microtubules continue to lengthen, elongating the cell even more. Sister chromosomes attach to opposite ends of the cell. Fragments of the parental nuclear envelope attach around each set of separated sister chromatids. Each set of nuclei, now surrounded by a new nuclear envelope, unfold back into chromatin. The cleavage furrow forms.
Cytokinesis	Often, (mistakenly) thought to be the same process as telophase cytokinesis, if slated to occur, is usually well under way by this time. In animal cells, a cleavage furrow develops where the metaphase plate used to be, pinching off the separated nuclei. In plant cells, vesicles derived from the Golgi apparatus move along microtubules to the middle of the cell, coalescing into a cell plate that develops into a cell wall, separating the two nuclei. Each daughter cell has a complete copy of the genome of its parent cell. Mitosis is complete.
Growth 1	Each chromosome is copied during S phase, forming two identical sister chromatids that then separate into the future daughter cells during anaphase. Thus, each generation has exactly as much DNA as its predecessor.		Upon completion of cytokinesis, the two daughter cells are identical to their predecessor and enter G1, an interphase. Most cells spend the majority of the cell cycle in G1; some animal cells never undergo mitosis.

This is called open mitosis, and it occurs in most multicellular organisms. Some protists, such as algae, undergo a variation called closed mitosis where the microtubules are able to penetrate an intact nuclear envelope.

An obvious advantage of proteolysis for controlling passage through these critical points in the cell cycle is that protein degradation is an irreversible process, ensuring that cells proceed irreversibly in one direction through the cycle.

Mitosis is the division of a parent cell into two daughter cells that are identical to itself. A cell does this to increase population size and to increase genetic diversity. The parent cell duplicates each of its chromosomes before mitosis; these duplicate chromosomes are called sister chromatids, and are attached at their centromere. In animals and plants, the nuclear envelope degrades during mitosis. Microtubules emanate from opposite ends of the cell to latch onto the sister chromatids, then shorten and pull the sister chromatids apart into sister chromosomes. The cell elongates and the nuclear envelope reforms. By now, cytokinesis is already underway, and by the time it finishes the parent cell has been split in half into two daughter cells that are identical to it. This is similar to binary fission in prokaryotes, but is different in that prokaryotes lack a nucleus and have a single chromosome without a centromere.

In binary fission, a parent cell divides to form two daughter cells. Thus, bacterial population increase exponentially. Note the table below, demonstrating how quickly a population of bacteria can explode from just a single cell. The generation (first, second, third, etc) is denoted n and the number of cells at a given time is N (or N₀ at time zero).

The table below describes important formulas regarding population dynamics. They are valid for any value of x, as long as it is kept constant throughout the problem. Also, t is elapsed time and t_d is the time require for one complete cell division (for a cell to double).

Cyclin-CDK		Nick-Name	Overview
Mitotic-Cyclin+CDK		MPF	Triggers the formation of mitotic spindle. Promotes mitosis i.e. chromatin condensation. Causes nuclear envelope breakdown by phosphorylating the lamins that form an intermediate filament-type network (nuclear lamina) underlying the inner nuclear membrane. The three lamins present in the nuclear lamina, lamin A,B & C, are phosphorylated by MPF at serine amino residues. This leads to depolymerisation of nuclear lamina & breakdown of nuclear envelope into small vesicles.
Vertebrate	S. cerevisae	S. pombe	Role	Overview
Cyclin B	Cdc28	Cdc13	Mitotic Cyclin	Mitotic cyclins must be degraded by APC/C-Cdh2 for chromosomes to decondense; however, this degradation is not needed for sister chromatids to separate during anaphase. Mitotic cyclins all have a conserved sequence at their N-terminus (Arg-X-X-Leu-Gly-X-Ile-Gly-X) that is recognized by APC/C-Cdh2; without this sequence, mitotic cyclins are not degraded. Cyclin-B+CDK1 is MPF in Xenopus and S. pombe.
CDK1	Cdc28	Cdc2	Mitotic CDK	MPF is Cyclin-B+CDK1 in Xenopus and S. pombe.
Cyclin-CDK		Nick-Name	Overview
Mid-G1-cyclin+CDK		SPF	Known as the s-phase promoting factor, as it is required to initiate DNA synthesis. Activates transcription factors for genes encoding the S phase cyclins and Sic1, and for genes needed for dNTP and DNA synthesis. Also, the G1 cyclin-CDK inactivates Cdh2, which would have destructed the S phase and mitotis cyclins.
Vertebrate	S. cerevisae	S. pombe	Role	Overview
Cyclin D	Cln3	Cdc13	Mid G1 Cyclin	Transcription “growth” factors activating Cyclin D and CDK4/6 are encoded by delayed response genes.
CDK4 & CDK6			Mid G1 CDK	Transcription “growth” factors activating Cyclin D and CDK4/6 are encoded by delayed response genes.

A cyclin-CDK consists of a regulatory subunit (the cyclin) and a kinase subunit (the cyclin-dependent kinase, aka CDK). CDKs trigger cell cycle events and regulate transcription and mRNA processing (except CDK9, which has a totally unrelated function). CDKs phosphorylate Serine and Threonine residues, but have little or no activity unless bound by a cyclin (hence their name). Cdc28p (the predominant yeast cyclin-dependent kinase involved in cell cycle control) is highly homologous to Cdk2.

High Copy Suppressor Experiment

Cdc28 (found in budding yeast) is highly homologous to Cdc2, a CDK found in S. pombe (fission yeast). Thus, it was theorized that Cdc28 is a CDK itself; thus, it must have a corresponding cyclin. If Cdc28 is a CDK, then in wild-type cells there must be a G₁ cyclin that bound Cdc28 to form an S-phase promoting factor (aka SPF).