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| Extended Lecture Outline |
Chapter 13: DNA Replication And The Cell Cycle |
13.1 Genes are located on chromosomes.
a. After Mendel's work was brought to light in 1900, the physical structure and location of genes and the importance of chromosomes (Figure 13.1) became central objects of research in biology.
b. In 1902, Boveri and Sutton independently inferred that genes are located on chromosomes, after observing that chromosomes behave as the hereditary units postulated by Mendel.
c. The hereditary role of the nucleus, which contains the chromosomes, was demonstrated by Hammerling, with his experiments on Acetabularia algae cells (Figure 13.2).
d. Procaryotic cells do not have a well-defined nucleus (Figure 13.3).
13.2 Chromosomes are DNA—protein complexes.
a. All organisms' genomes are made of DNA.
b. Each unit of a double-stranded DNA molecule is a nucleotide pair, called a base pair (bp).
c. Genomes are commonly measured in kilobase pairs (kbp), or 1000 bp.
d. One turn of a DNA helix consists of 10 bps and is 3.4 nm long; one bp weighs 614 daltons.
e. Table 13.1 shows the lengths, in micrometers, of some representative genomes.
f. A eucaryotic genome is divided into several chromosomes, each of which is a long DNA molecule with attached proteins. Procaryotes have only one chromosome each, generally a circular DNA molecule with associated proteins.
g. DNA is therefore immensely compressed in a cell nucleus. In a human cell containing 46 chromosomes, the total length of DNA is about 1.8 meters, and is packed into a nucleus about 10 micrometers in diameter.
h. Staining a eucaryotic cell nucleus reveals chromatin, the substance which constitutes chromosomes, and which consists of DNA and structural proteins called histones.
i. Micrographs of chromatin fibers show uniformly spaced beads, called nucleosomes, which are DNA wound around histone complexes (Figure 13.4). The term "beads on a string" has been used to describe this nucleosome level of DNA packing.
j. Bacterial DNA is also combined with histones, but does not apparently form nucleosomes.
k. Chromosomes cannot be seen with a light microscope until they are condensed just prior to cell division.
l. A simplex chromosome becomes a duplex chromosome after replication during the S stage of interphase. Each duplex is made of two chromatids attached to each other at the centromere (Figure 13.5).
m. After the two chromatids of a duplex chromosome separate during cell division, each is again called a simplex chromosome.
13.3 DNA replicates semiconservatively.
a. The Watson—Crick DNA model suggested that DNA would replicate in a semiconservative fashion (Figure 13.6), rather than in a conservative fashion (Figure 13.6).
b. Other modes of replication (e.g. dispersive, Figure 13.6) were also considered possibilities.
c. About 30 years ago, Matthew Meselson and Franklin Stahl tested the semiconservative prediction and provided evidence to support it.
1. They produced a density gradient by centrifuging a cesium chloride solution for several days.
2. In such a gradient, molecules move to a point of equilibrium according to their density.
3. A dense DNA bacterial culture was grown with the heavy isotope 15N as a nitrogen source.
4. The dense DNA culture was transferred to an ordinary medium containing 14N.
5. It was predicted the bacteria would begin producing "hybrid" DNA, according to the semiconservative method, composed of double helixes with strands of different densities.
6. These hybrid molecules of intermediate density should centrifuge to an intermediate density level in a gradient, which they did.
7. A second round of replication by these bacterial DNA should still result in each duplex being composed of one heavy and one light strand, which should still centrifuge to an intermediate level in a gradient.
8. The results (Figure 13.7) were as predicted, and supported semiconservative replication.
d. Meselson and Stahl also showed that semiconservative replication involved entire strands (rather than partial strands) of new DNA being produced on each "old" strand template.
1. They heated the intermediate density DNA to separate the strands by breaking the hydrogen bonds.
2. The results were two density fractions, one heavy and one light (Figure 13.8)
3. They concluded that each new strand is either all heavy or all light, not a mixture.
e. Sidebar 13.1 emphasizes that creative reasoning, inference, and alternative hypotheses are all part of the logic of scientific experiments, as illustrated by the Meselson and Stahl experiments.
13.4 DNA is replicated by a protein complex that operates in different ways on the two strands of a double helix.
a. An existing DNA strand is a template for a new strand, as complementary nucleotides line up along the template strand, and are eventually linked into a new strand (Figure 13.9).
b. DNA replication is controlled by several enzymes.
1. A replisome is a protein complex that moves along a DNA template strand and carries out several replication activities (Figure 13.10).
2. Helicase, one enzyme in the replisome, unwinds the old double helix, and makes a replication point available on a template strand.
3. The point where two old strands begin to separate, and where the replisome acts, is called a replication fork.
4. DNA polymerase, another replisome enzyme, links new nucleotides into a new strand as the replisome moves along the template.
5. Single-strand binding proteins stabilize the single-stranded molecules and prevent them from reforming a double-stranded helix during replication.
c. DNA polymerases are only known to synthesize (or, add nucleotides) in the 5' -> 3' direction.
d. This implies only one strand of a double-helix can be replicated at one time, in one direction, since the template strands have opposite polarities.
e. However, the replisome does synthesize two strands, as it contains different DNA polymerases oriented in opposite directions and acting differently on each template strand.
1. The replisome moves along one template in the 3' -> 5' direction.
2. The 3' -> 5' template is replicated by one DNA polymerase and a continuous, leading strand, is formed.
3. At the same time, another DNA polymerase makes a second new strand, the lagging strand, in short, discontinuous pieces called Okazaki fragments (after R. Okazaki, who first observed them).
f. DNA polymerase cannot begin synthesis on a bare strand, but must work from an RNA primer, a strand that has already been started off the template by a primase enzyme, contained in the replisome.
g. Okazaki fragments each contain RNA primers at first; these are replaced with DNA by a DNA polymerase, and the DNA fragments are linked together by DNA ligase.
13.5 DNA synthesis requires energy furnished by nucleoside triphosphates.
a. The four nucleoside triphosphates (dATP, dCTP, dGTP, and dTTP) provide the energy needed to form bonds between new deoxyriboses.
b. Two terminal phosphates are split off each new nucleoside in the form of inorganic pyrophosphate; the breaking of these bonds provides the energy for new bonds between new deoxyriboses.
c. Figure 13.9 illustrates how each new nucleoside fits onto the template strand; hydrogen bonds hold these to the base of the template and thus ensure precise replication.
d. The inorganic pyrophosphate is hydrolized, preventing the disassembly of the genome by reversal of the replication process.
13.6 All growing cells proceed through a regular cycle.
a. The text uses the more complicated eucaryotic cell (as opposed to the procaryotic cell) to inclusively address cell cycle events (Figure 13.11).
b. Growing cells periodically undergo a nuclear division called mitosis, where the cell's chromosomes are first replicated, then partitioned equally to two new daughter nuclei.
c. Cytokinesis, division of the cell's cytoplasm, immediately follows mitosis in most cells.
d. Coenocytes are cells whose cytoplasm divides irregularly or only occasionally while mitosis proceeds regularly; these cells have multiple nuclei and are commonly found in fungi and algae.
e. Human body muscle cells, which do not undergo mitosis, form syncytia, which are cells with multiple nuclei formed by the fusion of cells.
f. Interphase is the part of the cell cycle that does not include mitosis or cytokinesis.
1. G1 is a growth phase, which greatly varies in length among various types of cells and among different organisms; the cell has very active growth and other functions during G1.
2. S is the phase following G1, during which the cell replicates its DNA.
3. G2 follows S, and includes more normal metabolic activities and preparation for division.
4. Some mature, differentiated cells pause during G1 and enter a G0 resting phase, after which most do not divide.
13.7 The cell cycle is driven by the synthesis and degradation of special proteins.
a. Genetic analysis and some of the organisms and terms used for it, are discussed in the first paragraph of this section.
b. The cell cycle is controlled by certain protein levels known as checkpoints.
c. The checkpoint system has apparently evolved to ensure that key events of cell growth and division happen at appropriate times in the life of the cell.
d. There are at least two checkpoints in the cycle; each is passed when the appropriate proteins have been made (Figure 13.12).
1. SPF, or S-phase promoting factor, allows the cell to pass the S phase.
2. MPF, or mitosis-promoting factor, allows the cell to enter mitosis.
e. Each checkpoint protein is made of a cyclin and a protein kinase, as examples of the many specialized protein kinases addressed in Chapter 11.
f. To enter the S phase, the cell must pass the Start point, at which time it has a sufficient level of SPF, as indicated by several proteins that effectively measure cell size.
g. After S, the cell enters G2, during which mitotic proteins are made and MPF is measured before mitosis can begin.
h. Inhibitors can prevent DNA synthesis and still allow the cell cycle proteins to be made, activated, and deactivated.
13.8 Mitosis is a mechanism for dividing chromosomes into two identical sets.
a. Mitosis results in each daughter nucleus receiving one complete copy of the genome from the parent cell.
b. Mitosis is a continuous process, but is divided for illustrative purposes, into four or five phases.
c. Interphase is the non-mitotic phase where nuclear chromatin is uncondensed.
d. Prophase is marked as:
1. the nuclear material begins to condense and first becomes visible as chromosomes,
2. the nucleus enlarges,
3. the centrosome (Section 11.11) divides into two centers that begin migrating to opposite poles of the cell,
4. the centrosomes begin forming the spindle between them.
e. Prometaphase (not always included as a separate phase) begins as the nuclear envelope begins to disintegrate and the mitotic spindle begins attaching to the kinetochore in the centromere region of each chromosome.
f. Metaphase is marked by the arrival of the chromosomes at the central metaphase plate.
g. Anaphase begins as the centromeres break apart and chromatids of formerly duplex chromosomes separate and move to opposite poles of the cell as simplex chromosomes.
h. Telophase begins when the simplex chromosomes arrive at opposite poles and begin to uncoil; new nuclear envelopes are also starting to form, and the spindle disintegrates.
i. Nuclear division is followed by cytokinesis, preceding the next interphase for each new cell.
j. The mitotic spindle that moves chromosomes contains three types of microtubules (Figure 13.13).
1. Astral microtubules lead to the cell membrane.
2. Kinetochore microtubules attach to the chromosomes.
3. Polar microtubules extend from each centrosome and overlap in the middle of the cell.
k. Spindle microtubules are much more unstable than interphase microtubules, as they continuously add and remove tubulin subunits at opposite ends; they become stable once they are attached to a kinetochore (Figure 13.14).
l. Movements during anaphase are due to:
1. the cell's elongation,
2. polar microtubules pushing against one another,
3. shortening of the kinetochore microtubules (Figure 13.14).
m. Section 11.12 covers microtubule motor proteins.
n. Each daughter nucleus receives one simplex chromosome from each original duplex chromosome that divides; the chromosomes divide and move to opposite poles independently of one another.
o. The probably haphazard divisions of primitive cells must have resulted in the evolutionary preservation of mechanisms which regulate the cell cycle and ensure relatively error-free cell division.
p. Chapter 26 addresses mitosis in primitive eucaryotes.
13.9 Procaryotic and eucaryotic chromosomes are replicated in different ways.
a. Procaryotic cells have no mitotic apparatus, and do not engage in mitosis.
b. The circular procaryotic DNA molecule, attached inside the plasma membrane (Figure 6.31), replicates during much of the cell cycle by means of two replication forks.
c. Figure 13.15 shows a generalized bacterial cell cycle.
d. Figure 13.16 illustrates replication in a bacterial cell.
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