Lighten Up – Appendix
Appendix
“Lighten Up”
Gene Regulation
The control of gene expression in an individual cell is of profound importance to the
entire organism. On an organismal level, each cell has a specific and unique role, and only
certain portions of the entire content of DNA are needed to carry out these unique functions.
Loss of control on the cellular level can ultimately lead to a transformed cell, also known as a
cancer cell.
The control of gene expression in a eukaryotic cell can occur at many different levels.
The central dogma in biology states: DNA is transcribed into mRNA, which in turn is translated
into protein (polypeptide). This statement allows for control to occur at the level of transcription
and translation, and indeed the cell does exert controls at these points. However, it helps to
expand this picture to get a better idea of the control of eukaryotic gene expression. (See
Figure 1.)
First, the three-dimensional architecture of the DNA is important. The total length of
DNA in a single human cell is 105 times the diameter of the cell. The DNA is packaged using
specialized proteins into complex structures, which are easily visualized as chromosomes during
mitosis. One level of control of gene expression is determined by the accessibility of a given
stretch of DNA within this complex structure. Folding and unfolding, and other changes to the
structure of DNA (methylation) can affect gene expression. Once a stretch of DNA is accessible
to proteins, other mechanisms of control come into play.
At this point, it is important to define a gene. A gene can be defined as the entire
sequence of nucleic acid that is necessary for the synthesis of a functional polypeptide or RNA
molecule. This definition includes not only the coding region (amino acid sequence of the
polypeptide), but all of the DNA sequences needed to get a particular stretch of DNA
transcribed. Researchers have identified many sequences in both prokaryotic and eukaryotic
organisms that affect the transcription of a specific gene or genes. Specific, and often multiple,
regulatory proteins interact with these different types of regulatory elements. In eukaryotic cells
regulatory sequences include: transcription-control regions (promoters) which control the
initiation of transcription by RNA polymerase, promoter-proximal elements which lie within
100-200 base pairs upstream of the promoter and may be cell-type specific, sequences which
boost the activity of any nearby gene (enhancers), and GC-rich regions which are found
preceding genes which do not contain the TATA box promoter or initiator-element promoters.
The importance of specific nucleotide sequences upstream of the coding regions of genes
was identified by creating constructs consisting of varying amounts of the upstream nucleotide
sequence linked to a gene whose product could be easily assayed (reporter gene). Two common
reporter genes are the lacZ gene of E. coli , and the lucerifase gene from fireflies.
Once an RNA transcript is made, it must be processed into mature mRNA that can be
translated into a polypeptide. Additional controls are exerted at the level of RNA processing in
the eukaryotic cell. The nucleotide sequence of a gene in a eukaryotic cell contains intervening
sequences (introns) as well as the amino acid coding sequences (exons), and the introns must be
removed from the primary RNA transcript and the exons spliced together. In some instances,
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differential splicing patterns can produce different proteins from the same initial RNA transcript,
providing the cell with increased genetic flexibility. A mature mRNA species also contains a 5'
cap structure and a poly-A tail. The 3' poly-A tail increases the stability of the mRNA and
protects it from degradation. Finally, the mature mRNA must cross the nuclear membrane to be
translated in the cytoplasm.
Some mRNAs contain sequences in their 3' untranslated regions which direct them to
certain cytoplasmic locations. The cytoskeleton of the cell is involved in this localization.
Translation of the mRNA also involves many initiation factors in eukaryotic cells. Some mRNA
species are said to be masked, meaning they can not be translated, until a specific initiation factor
is available. mRNA species also have different half-lives of stability within the cytoplasm.
Most eukaryotic mRNAs have half-lives of several hours, but some are very unstable. This level
of regulation of mRNA stability has a dramatic effect on the amount of the encoded protein that
can be synthesized. Finally, many polypeptides must be modified, for example by cleavage or
acylation or glycosylation, to attain their fully functional state.
Cell Growth and the Cell Cycle
Different eukaryotic cells grow and divide at differing rates. Some cells, such as nerve
and striated muscle cells do not divide at all; others, such as fibroblasts, divide only when they
receive specialized chemical signals at a wound site. A system for growing cells outside of the
organism, tissue culture, has aided in our understanding of the mechanism used by cells to
regulate their growth. The cell cycle of growth and division has been thoroughly analyzed in
actively growing mammalian cells in tissue culture. A representative mammalian cell with a
generation time of 16 hr is shown in Figure 2. The cell cycle is divided into 4 phases. The phase
in which the DNA is copied is termed the S phase (DNA synthesis), and it is separated from the
M phase (mitosis) in which the DNA is allocated between the developing mother and daughter
cells, and cell division actually occurs. Two separation phases are termed G1 and G2,
respectively. "G" stands for gap, and indicates a measurable space of time between the S and M
phases of the cell cycle. A G0 phase is also indicated in Figure 2, and this refers to cells such as
the nerve cell which maintain themselves, but do not grow and divide. These cells remain
arrested in G0 phase for their lifetime under normal circumstances. Studies of such diverse
organisms as yeast and developing frog eggs (Xenopus) have shown that all eukaryotic cells
grow and divide by fundamentally similar molecular mechanisms. The elucidation of these
mechanisms has also been aided by an understanding of the mechanisms by which oncogenic
virus families transform normal cells into cancer cells.
Most of the molecular mechanisms that control a cell's progress through the cell cycle
exert their effects at either the initiation of the S or M phases. Both of these transitions are
regulated by a protein complex consisting of a catalytic subunit and a regulatory subunit. The
regulatory subunits are called cyclins because their concentrations cycle with the cell cycle. The
catalytic subunits are protein kinases, and their activity depends on their association with a
cyclin, leading to the name, cyclin-dependent kinase, or CdK. When these CdKs are activated
they phosphorylate many different substrates, activating some and inhibiting others, to control
the growth and division of a cell. The CdK-cyclin complexes are themselves regulated by other
protein kinases and protein phosphatases, and, in turn, these enzymes are regulated. Further
levels of regulation are exerted on the CdKs and cyclins at the level of initiation of transcription
of the genes and by regulating the degradation of the proteins.
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DNA damage induces checkpoints that prevent entry into the S and the M phases of the
cell cycle until the damage is repaired, or if the damage to the DNA is irreparable, the cell may
enter another pathway of programmed cell death termed apoptosis. In eukaryotic cells, DNA
damage leads to activation of the p53 transcription factor, which induces transcription of the
gene encoding Cki1, a small protein that binds to and inhibits G1 CdK-cyclin complexes,
blocking entry into the S phase. In humans, genetic mutations that inactivate p53 predispose
cells to develop into cancer cells. Initial research on apoptosis led researchers to hypothesize
that cancer might result from the failure of cells to die when they are supposed to. If apoptosis
can be considered an ultimate cell regulatory program, then loss or inappropriate use of this
program would have devastating effects on an organism.
Morphologically, the cell nucleus and cytoplasm of an apoptotic cell condense, and then
the nucleus breaks into fragments. Large blebs form on the cell and eventually break off to form
multiple membrane-bound cell fragments or apoptotic bodies. These bodies are rapidly
phagocytosed and degraded intracellularly. In this way, cells that are either damaged or
dangerous are rapidly removed without provoking the immune response, and the "parts"
recycled. These original morphological observations led to an intense research effort to
understand the molecular mechanisms of apoptosis. Research has delineated two separate
pathways that induce apoptosis; one depends on extracellular factors and the other depends on
intracellular factors. The extracellular pathway is activated via a family of cell surface
transmembrane receptors (death receptors) whose purpose may be to instruct cells to undergo
apoptosis during certain stages of development or during immune responses. Mitochondria play
the role of the intracellular initiator of apoptosis by releasing cytochrome c and ATP. Toxins,
free radicals, calcium ions, ultraviolet irradiation, and a host of other pro-apoptotic proteins
induce the release of cytochrome c and ATP by mitochondria.
Once initiated, apoptosis proceeds through the action of a set of enzymes called caspases.
Caspases are a family of highly specific cysteine proteases that cleave proteins after aspartic acid
residues. They exist within cells in an inactive state as procaspases, and they are converted to
their active forms by proteolysis carried out by themselves. Thus, once an initiator caspase is
activated, it can induce a cascade of proteolytic events that lead to activation of the effector
caspases, and ultimately to the proteolysis and fragmentation of the cell.
Apoptosis is regulated by a set of complex control mechanisms. One eukaryotic protein
that can oppose apoptosis is the product of the bcl-2 oncogene. The Bcl-2 protein is important in
the production of B cell lymphomas, and it is now the archetypal member of a large family of
related anti-apoptotic proteins. Conversely, a large set of proteins that promote apoptosis acts in
concert with the anti-apoptotic proteins. Currently, it is thought that cells regulate the levels of
these sets of proteins to promote either survival or suicide.
So, with all of these control mechanisms and checkpoints both within the cell cycle and
without, how do cells grow and proliferate in an abnormal way? Asked in another way, how can
cancer develop? First, it may be helpful to define cancer, or the transformation of cells, as the
loss of normal growth control regulation. More specifically, cancer arises from a single cell that
loses growth control regulation. At the molecular level, this can be detected as a series of
accumulated mutations in a specific subset of genes. Two classes of genes have been identified:
the proto-oncogenes which stimulate passage through the cell cycle (cyclins, Cdks and others)
and the tumor-suppressor genes (e.g., p53 transcription factor). For a cancer to occur, multiple
mutations must be present within these genes. Altered forms of other genes may contribute to
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the ability of a cancer cell to metastasize or migrate from its' original site with formation of a
tumor at a distal site. Tumor cells may also be able to induce anti-apoptotic proteins as in the
case of Bcl-2 to circumvent the cell's own programmed cell death mechanism.
Figure 1. Gene Expression.
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Figure 2. The Cell Cycle.
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