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By : Matt Daniel M. Daep

 Fatty acid synthesis is the creation of fatty acids from
acetyl-CoA and NADPH through the action of enzymes
called fatty acid synthases. This process takes place in the
cytoplasm of the cell. Most of the acetyl-CoA which is
converted into fatty acids is derived from carbohydrates
via the glycolytic pathway. The glycolytic pathway also
provides the glycerol with which three fatty acids can
combine (by means of ester bonds) to form triglycerides
(also known as "triacylglycerols", to distinguish them
from fatty "acids" - or simply as "fat"), the final product of
the lipogenic process
What is fatty acid
synthesis:

The input to fatty acid synthesis is acetyl-
CoA, which is carboxylated to malonyl-CoA.
The ATP-dependent carboxylation provides
energy input. The CO2 is lost later during
condensation with the growing fatty acid.
The spontaneous decarboxylation drives the
condensation.
Acetyl-CoA Carboxylase catalyzes the 2-step
reaction by which acetyl-CoA is carboxylated
to form malonyl-CoA.
As with other carboxylation reactions (e.g.,
Pyruvate Carboxylase), the enzyme
prosthetic group is biotin.
 Phospholipids are a class of lipids that are a major component of all
cell membranes. They can form lipid bilayers because of their
amphiphilic characteristic. The structure of the phospholipid molecule
generally consists of two hydrophobic fatty acid "tails" and a
hydrophilic "head" consisting of a phosphate group. The two
components are joined together by a glycerol molecule. The phosphate
groups can be modified with simple organic molecules such as choline.
 The first phospholipid identified in 1847 as such in biological tissues
was lecithin, or phosphatidylcholine, in the egg yolk of chickens by the
French chemist and pharmacist, Theodore Nicolas Gobley. Biological
membranes in eukaryotes also contain another class of lipid, sterol,
interspersed among the phospholipids and together they provide
membrane fluidity and mechanical strength. Purified phospholipids
are produced commercially and have found applications in
nanotechnology and materials science.
What are
phospholipids?

 Phosphatidic Acid
 Biosynthesis:
 While quantitatively a minor component of
membrane phospholipids, phosphatidic acid forms
the backbone on which the synthesis of other
phospholipid species and triacylglycerol is based.
Biosynthesis


Phosphatidic acid synthesis begins with the addition of a
fatty acyl-CoA, usually saturated, to glycerol 3-
phosphate at the sn-1 position to produce
lysophosphatidic acid. This reaction is catalyzed by
glycerol 3-phosphate acyltransferase and is rate limiting
for phosphatidic acid synthesis. There are two forms of
this enzyme; one is found in the outer mitochondrial
membrane, while the other is found in the endoplasmic
reticulum. A second fatty acyl-CoA, often unsaturated, is
added to lysophosphatidic acid at the sn-2 position by
acylglycerol-3-acyltransferase to form phosphatidic acid.
This occurs primarily in the endoplasmic reticulum.

 Ketone bodies are three water-soluble molecules
(acetoacetate, beta-hydroxybutyrate, and their
spontaneous breakdown product, acetone) that are
produced by the liver from fatty acids[1] during periods
of low food intake (fasting), carbohydrate restrictive diets,
starvation, prolonged intense exercise,[2] or in untreated
(or inadequately treated) type 1 diabetes mellitus. These
ketone bodies are readily picked up by the extra-hepatic
tissues, and converted into acetyl-CoA which then enters
the citric acid cycle and is oxidized in the mitochondria
for energy.[3] In the brain, ketone bodies are also used to
make acetyl-CoA into long-chain fatty acids.
What are ketones?

Ketone bodies are produced by the liver under the
circumstances listed above (i.e. fasting, starving, low
carbohydrate diets, prolonged exercise and untreated
type 1 diabetes mellitus) as a result of intense
gluconeogenesis, which is the production of glucose
from non-carbohydrate sources (not including fatty
acids). They are therefore always released into the
blood by the liver together with newly produced
glucose, after the liver glycogen stores have been
depleted. (These glycogen stores are depleted after only
24 hours of fasting.)
Ketone synthesis

 Isoprenoid quinones are one of the most important
groups of compounds occurring in membranes of living
organisms. These compounds are composed of a
hydrophilic head group and an apolar isoprenoid side
chain, giving the molecules a lipid-soluble character.
Isoprenoid quinones function mainly as electron and
proton carriers in photosynthetic and respiratory electron
transport chains and these compounds show also
additional functions, such as antioxidant function. Most
of naturally occurring isoprenoid quinones belong to
naphthoquinones or evolutionary younger
benzoquinones.
Isoprenoids
 Isoprenoid quinones are membrane-bound compounds found in
nearly all living organisms. The only exception presently known is
some obligatory fermentative bacteria that lost the ability of synthesis
of isoprenoid quinones and methanogenic Archea, belonging to
Methanosarcinales . Isoprenoid quinones are composed of a polar
head group and a hydrophobic side chain. The apolar isoprenoid side
chain gives the molecules a lipid-soluble character and anchors them
in membrane lipid bilayers, whereas the hydrophilic head group
enables interaction with hydrophilic parts of proteins. It is generally
accepted that long-chain, isoprenoid quinones localize in the
hydrophobic mid-plane region of the lipid bilayer, whereas the polar
head can oscillate between mid-plane and polar interphase of the
membrane . The quinone ring can undergo two-step reversible
reduction leading to quinol form . The reduced form of isoprenoid
quinones is more polar and the quinol head group is thought to
preferentially localize in polar, interphase region of membranes and.
Synthesis

 Sterols are constituents of the cellular membranes that are
essential for their normal structure and function. In
mammalian cells, cholesterol is the main sterol found in
the various membranes. However, other sterols
predominate in eukaryotic microorganisms such as fungi
and protozoa. It is now well established that an important
metabolic pathway in fungi and in members of the
Trypanosomatidae family is one that produces a special
class of sterols, including ergosterol, and other 24-methyl
sterols, which are required for parasitic growth and
viability, but are absent from mammalian host cells.
What are sterols

 Figure 1: Molecular structures of cholesterol and
ergosterol. The arrows indicate the parts of the
molecules which have been shown to be essential for
the growth of mammalian cells (cholesterol), fungi,
and trypanosomatids (ergosterol and 24-methyl
sterols).

Figure 1:

 Figure 2: Schematic representation of main
morphologies found during the life cycle of some
members of the Trypanosomatidae family in the
invertebrate host (insect) and vertebrate host
(mammal).

Figure 2:

 Figure 3: The biosynthesis of ergosterol and
cholesterol showing the main steps, the enzymes
involved, and the known inhibitors.

Figure 3

 A compound of the sterol type found in most body
tissues. Cholesterol and its derivatives are important
constituents of cell membranes and precursors of
other steroid compounds, but a high proportion in
the blood of low-density lipoprotein (which
transports cholesterol to the tissues) is associated
with an increased risk of coronary heart disease.
What is cholesterol?

 The amount of cholesterol that is synthesized in the
liver is tightly regulated by dietary cholesterol
levels. When dietary intake of cholesterol is high,
synthesis is decreased and when dietary intake is
low, synthesis is increased. However, cholesterol
produced in other tissues is under no such feedback
control. Cholesterol and similar oxysterols act as
regulatory molecules to maintain healthy levels of
cholesterol.
Cholesterol regulation

 The rate of synthesis of reductase mRNA is controlled by the
sterol regulatory element binding protein (SREBP). This
transcription factor binds to a short DNA sequence called the
sterol regulatory element (SRE) on the 5′ side of the reductase
gene. In its inactive state, the SREBP is anchored to the
endoplasmic reticulum or nuclear membrane. When cholesterol
levels fall, the amino-terminal domain is released from its
association with the membrane by two specific proteolytic
cleavages. The released protein migrates to the nucleus and
binds the SRE of the HMG-CoA reductase gene, as well as
several other genes in the cholesterol biosynthetic pathway, to
enhance transcription. When cholesterol levels rise, the
proteolytic release of the SREBP is blocked, and the SREBP in
the nucleus is rapidly degraded. These two events halt the
transcription of the genes of the cholesterol biosynthetic
pathways.

 any of a group of soluble proteins that combine with
and transport fat or other lipids in the blood plasma.
What are lipoproteins?

 LPL actions within tissues are modulated at both the
transcriptional and posttranscriptional levels.
The latter might involve actions of the
glycosylphosphatidylinositol HDL binding protein
(GPIHBP) protein (19), angiopoietin-like proteins, which
reduce LPL dimer formation (20), and the recently
described lipase maturation factor (21). LPL regulation is
tissue specific. LPL is present in the liver during fetal and
early postnatal life but is then suppressed by a putative
transcriptional regulatory mechanism, perhaps involving
a novel transcription factor, termed RF-1-LPL, which
binds to an NF-1-like site in the region of the
glucocorticoid response element. This extinction of the
hepatic expression of LPL is also under the influence of
thyroid hormone and glucocorticoids.

 Amino acid synthesis is the set of biochemical
processes (metabolic pathways) by which the various
amino acids are produced from other compounds.
The substrates for these processes are various
compounds in the organism's diet or growth media.
Not all organisms are able to synthesise all amino
acids. Humans are excellent example of this, since
humans can only synthesise 11 of the 20 standard
amino acids (aka non-essential amino acid), and in
time of accelerated growth, histidine, can be
considered an essential amino acid.
What is amino acids synthesis?

 Most amino acids are synthesized from α-ketoacids,
and later transaminated from another amino acid,
usually glutamate. The enzyme involved in this
reaction is an aminotransferase.
α-ketoacid + glutamate ⇄ amino acid + α-ketoglutarate
Glutamate itself is formed by amination of α-
ketoglutarate:
α-ketoglutarate + NH+
4 ⇄ glutamate

 Porphyrins are a group of heterocyclic macrocycle organic
compounds, composed of four modified pyrrole subunits
interconnected at their α carbon atoms via methine bridges
(=CH−). The parent porphyrin is porphin, and substituted
porphines are called porphyrins. The porphyrin ring structure
is aromatic, with a total of 26 electrons in the conjugated
system. Various analyses indicate that not all atoms of the ring
are involved equally in the conjugation or that the molecule's
overall nature is substantially based on several smaller
conjugated systems. One result of the large conjugated system
is that porphyrin molecules typically have very intense
absorption bands in the visible region and may be deeply
colored; the name "porphyrin" comes from the Greek word
πορφύρα (porphyra), meaning purple.
Porphyrins

Illustration:

 Nucleotides are small organic molecules consisting of a
five ring sugar (which can be a ribose or a deoxyribose), a
nitrogen base, and one to three phosphate groups. The
most important function of nucleotides is there
polymerization in nucleic acids such as DNA or RNA. As
such they serve as the building blocks for the extremely
long molecules that make up the chromosomes in every
cell of our bodies, and that carry the genetic blueprint.
The genetic information is carried out in DNA, and is
organised in genes. Messenger RNA, is transcribed from
the DNA whenever genes are expressed and carries the
information from the cell nucleus to the cytoplasm, where
it acts as a blueprint for protein synthesis.
What are nucleotides?

Nucleotides are required for cell growth and replication
A key enzyme for the synthesis of one nucleotide is
dihydrofolate reductase. Cells grown in the presence of
methotrexate, a reductase inhibitor, respond by
increasing the number of copies of the reductase gene.
The bright yellow regions visible on three of the
chromosomes in the fluorescence micrograph (left),
which were grown in the presence of methotrexate,
contain hundreds of copies of the reductase gene
Nucleotide synthesis



Da en’
DNA and
Chromosome
Structure
DNA (or deoxyribonucleic acid) is the molecule
that carries the genetic information in all cellular
forms of life and some viruses. It belongs to a
class of molecules called the nucleic acids, which
are polynucleotides - that is, long chains of
nucleotides.
Each nucleotide consists of three components:
---a nitrogenous base: cytosine (C), guanine (G),
adenine (A) or thymine (T)
---a five-carbon sugar molecule (deoxyribose in
the case of DNA)
---a phosphate molecule
The backbone of the polynucleotide is a chain of sugar
and phosphate molecules. Each of the sugar groups in
this sugar-phosphate backbone is linked to one of the four
nitrogenous bases.
Strand of polynucleotides
Strand of polynucleotides
DNA's ability to store - and transmit - information lies in the fact that
it consists of two polynucleotide strands that twist around each other
to form a double-stranded helix. The bases link across the two
strands in a specific manner using hydrogen bonds: cytosine (C) pairs
with guanine (G), and adenine (A) pairs with thymine (T).
Double strand of polynucleotides
The double helix of the complete DNA molecule resembles a spiral staircase,
with two sugar phosphate backbones and the paired bases in the centre of the
helix. This structure explains two of the most important properties of the
molecule. First, it can be copied or 'replicated', as each strand can act as a
template for the generation of the complementary strand. Second, it can store
information in the linear sequence of the nucleotides along each strand.
Chromosomes
Eukaryotic chromosomes
The label eukaryote is taken from the Greek for 'true nucleus', and
eukaryotes (all organisms except viruses, Eubacteria and Archaea) are
defined by the possession of a nucleus and other membrane-bound cell
organelles.
The nucleus of each cell in our bodies contains approximately 1.8
metres of DNA in total, although each strand is less than one millionth
of a centimetre thick. This DNA is tightly packed into structures
called chromosomes, which consist of long chains of DNA and
associated proteins.
In eukaryotes, DNA molecules are tightly wound around proteins -
called histone proteins - which provide structural support and play a role
in controlling the activities of the genes.
A strand 150 to 200 nucleotides long is wrapped twice around a core of
eight histone proteins to form a structure called a nucleosome. The
histone octamer at the centre of the nucleosome is formed from two
units each of histones H2A, H2B, H3, and H4. The chains of histones are
coiled in turn to form asolenoid, which is stabilised by the histone H1.
Further coiling of the solenoids forms the structure of the chromosome
proper.
Each chromosome has a p arm and a q arm. The p arm (from the French word
'petit', meaning small) is the short arm, and the q arm (the next letter in the
alphabet) is the long arm. In their replicated form, each chromosome consists of
two chromatids.
Chromosome unraveling to show the base pairings of the DNA
The chromosomes - and the DNA they contain - are copied as part of the cell cycle,
and passed to daughter cells through the processes of mitosis and meiosis.
Human beings have 46 chromosomes, consisting of 22 pairs of autosomes and a
pair of sex chromosomes: two X sex chromosomes for females (XX) and an X
and Y sex chromosome for males (XY). One member of each pair of
chromosomes comes from the mother (through the egg cell); one member of
each pair comes from the father (through the sperm cell).
A photograph of the chromosomes in a cell is known as a karyotype. The
autosomes are numbered 1-22 in decreasing size order.
Karyotype of a human male
The prokaryotes (Greek for 'before nucleus' - including Eubacteria and
Archaea) lack a discrete nucleus, and the chromosomes of prokaryotic cells are
not enclosed by a separate membrane.
Prokaryotic chromosomes
Most bacteria contain a single, circular chromosome. (There are exceptions:
some bacteria - for example, the genus Streptomyces - possess linear
chromosomes, and Vibrio cholerae, the causative agent of cholera, has two
circular chromosomes.) The chromosome - together with ribosomes and
proteins associated with gene expression - is located in a region of the cell
cytoplasm known as the nucleoid.
In addition to the main chromosome, bacteria are also characterised by the
presence of extra-chromosomal genetic elements called plasmids. These
relatively small circular DNA molecules usually contain genes that are not
essential to growth or reproduction.
In addition to the main chromosome, bacteria are also characterised by the
presence of extra-chromosomal genetic elements called plasmids. These
relatively small circular DNA molecules usually contain genes that are not
essential to growth or reproduction.
Retrieved from
http://www2.le.ac.uk/departments/genetics/vgec/schoolscolleges/topics/dna
-genes-chromosomes
DNA Replication
Every time a cell divides to produce new cells its DNA is copied. Each
molecule of DNA undergoes semi-conservative replication. Put very simply,
the DNA unwinds and unzips to expose nucleotide bases. DNA polymerases
catalyse the addition of activated DNA nucleotides, according to
complementary base-pairing rules, to make two new identical molecules of
DNA, each one containing one old strand and one new strand. Hence each
new molecule contains half of the original molecule
Before DNA synthesis begins the original strands are separated and the
synthesis of the daughter strands begins at the replication fork at a site
called an origin of replication where a replisome is assembled from many
proteins. The initiation complex that is formed attracts DNA polymerases.
Synthesis of the new strands is called elongation and is aided by the proteins
in the replisome.
Lastly the termination site replicates
Figure 1. The DNA replication fork. Because both daughter strands are
synthesised in the 5’ to 3’ direction, the DNA complementary to the lagging
strand is synthesised in small fragments called Okazaki fragments. These
fragments are then joined together.
Figure 2. Enzymes involved in DNA replication.
The replisome
The replisome consists of many proteins, including helicase, gyrase/
topoisomerase, primase, DNA polymerases, RNAse H and ligase. One DNA
polymerase complex synthesises the lagging strand and another synthesises
the leading strand. There are also factors, called replication proteins, that
protect both the unstable single-stranded unwound leading and lagging
strands from making hydrogen bonds with themselves and forming hairpins.
Helicase causes the hydrogen bonds between complementary base pairs to
break and so catalyses the separation of the two parental strands that will
act as templates for synthesis of the daughter molecules. Helicase moves
along the DNA in a 3’ to 5’ direction.
Helicase
Gyrase (a form of topoisomerase) unwinds the resulting supercoil that forms
upstream of the section of unwound DNA.
Gyrase
DNA polymerases catalyse the elongation phase of replication.
DNA polymerases
Clamp proteins help keep the DNA polymerases attached to the leading and
lagging strands and make sure the process proceeds at a suitably fast rate.
Clamp proteins
Priming
In eukaryotic cells a DNA-dependent RNA polymerase creates an RNA primer,
of about 10 bases long, on both the newly separated leading and lagging
strands, once for the leading strand and once per Okazaki fragment (about
1000 base pairs long) on the lagging strand. The RNA primer attached to its DNA
template is called A-form DNA. (Normal DNA is called B-form DNA.)
In prokaryotes primase creates an RNA primer at the beginning of the newly
separated leading and lagging strands. DNA polymerase enzymes cannot bind
directly to single-stranded DNA and these primers provide a short chain of
nucleotides that give the correct configuration to allow the active site of DNA
polymerase to fit on and begin elongation.
Elongation
The leading and lagging strands are anti-parallel. In the leading strand
nucleotide synthesis (catalysed by DNA polymerase epsilon in eukaryotes and
by DNA polymerase III in prokaryotes) proceeds in the 5’ to 3’ direction (3’ to
5’ direction on the template strand) and makes a continuous complementary
strand. Synthesis of the other strand in the opposite direction cannot occur at
the same time so replication of the lagging strand is discontinuous. It involves
making short discrete nucleotide chains, called Okazaki fragments, that are
then joined by DNA repair enzymes, such as DNA polymerase I and ligase, so it
is not made in one continuous strand.
This can only happen once a sufficient length of DNA has been unwound so
replication of this strand lags behind that of the leading strand.
RNAse H enzymes remove the unstable RNA primers from the newly
synthesised fragments and replace them with DNA fragments.
DNA ligase (aided by polymerase I in prokaryotes) enzyme connects the
Okazaki fragments, closing the gaps between their sugar-phosphate backbones
by catalysing the formation of phosphodiester bonds.
Proofreading enzymes correct any mistakes due to insertion of incorrect
bases.
Retrieved from
http://www.contentextra.com/lifesciences/files/topicguides/Topic-guide-7.3-
DNA-replication.pdf
Mutagenesis and DNA
Repair Mechanisms
This rare albino alligator must have the specific
"instructions," or DNA, to have this quality. The cause of
albinism is a mutation in a gene for melanin, a protein
found in skin and eyes. Such a mutation may result in no
melanin production at all or a significant decline in the
amount of melanin.
What causes
albinism?
A change in the sequence of bases in DNA or RNA is called a
mutation. Does the word mutation make you think of
science fiction and bug-eyed monsters? Think again.
Everyone has mutations. In fact, most people have dozens
or even hundreds of mutations in their DNA.
Mutations are essential for evolution to occur. They are the
ultimate source of all new genetic material—new alleles in
a species. Although most mutations have no effect on the
organisms in which they occur, some mutations are
beneficial. Even harmful mutations rarely cause drastic
changes in organisms.
Causes of Mutation
Mutations have many possible causes. Some mutations
seem to happen spontaneously without any outside
influence. They occur when mistakes are made during DNA
replication or transcription.
Other mutations are caused by environmental factors.
Anything in the environment that can cause a mutation is
known as a mutagen. Examples of mutagens are pictured
in Figure 1.
Figure 1 Examples of Mutagens. Types of mutagens include radiation,
chemicals, and infectious agents. Do you know of other examples of each
type of mutagen shown here?
Types of Mutations
There are a variety of types of mutations. Two major
categories of mutations are germline mutations and
somatic mutations.
• Germline mutations occur in gametes. These
mutations are especially significant because they can be
transmitted to offspring and every cell in the offspring
will have the mutation.
• Somatic mutations occur in other cells of the body.
These mutations may have little effect on the organism
because they are confined to just one cell and its
daughter cells. Somatic mutations cannot be passed on
to offspring. Mutations also differ in the way that the
genetic material is changed. Mutations may change the
structure of a chromosome or just change a single
nucleotide.
What does radiation contamination do?
It mutates DNA. The Chernobyl disaster was a nuclear
accident that occurred on April 26, 1986. It is considered
the worst nuclear power plant accident in history. A
Russian publication concludes that 985,000 excess
cancers occurred between 1986 and 2004 as a result of
radioactive contamination. The 2011 report of the
European Committee on Radiation Risk calculates a total
of 1.4 million excess cancers occurred as a result of this
contamination.
Chromosomal Alterations
Chromosomal alterations are mutations that change
chromosome structure. They occur when a section of a
chromosome breaks off and rejoins incorrectly or does
not rejoin at all. Possible ways these mutations can occur
are illustrated in Figure 2.
Figure 2 Chromosomal Alterations. Chromosomal alterations are
major changes in the genetic material.
Point Mutations
Mutations A point mutation is a change in a single
nucleotide in DNA. This type of mutation is usually less
serious than a chromosomal alteration. An example of a
point mutation is a mutation that changes the codon UUU
to the codon UCU. Point mutations can be silent,
missense, or nonsense mutations, as shown in Table 1. The
effects of point mutations depend on how they change the
genetic code.
Table 1: Point Mutations and Their Effects
Frameshift Mutations
A frameshift mutation is a deletion or insertion of one or
more nucleotides that changes the reading frame of the
base sequence. Deletions remove nucleotides, and
insertions add nucleotides. Consider the following
sequence of bases in RNA: AUG-AAU-ACG-GCU = start-
asparagine-threoninealanine
Now, assume an insertion occurs in this sequence. Let’s
say an A nucleotide is inserted after the start codon AUG:
AUG-AAA-UAC-GGC-U = start-lysine-tyrosine-glycine
Even though the rest of the sequence is unchanged, this
insertion changes the reading frame and thus all of the
codons that follow it. As this example shows, a frameshift
mutation can dramatically change how the codons in
mRNA are read. This can have a drastic effect on the
protein product.
Spontaneous Mutations
There are five common types of spontaneous mutations. These are described
in the Table 2 below.
Table 7.6: Spontaneous Mutations Described
Effects of Mutations
The majority of mutations have neither negative nor
positive effects on the organism in which they occur.
These mutations are called neutral mutations. Examples
include silent point mutations. They are neutral because
they do not change the amino acids in the proteins they
encode. Many other mutations have no effect on the
organism because they are repaired before protein
synthesis occurs.
Cells have multiple repair mechanisms to fix mutations in
DNA. One way DNA can be repaired is illustrated in Figure
3. If a cell’s DNA is permanently damaged and cannot be
repaired, the cell is likely to be prevented from dividing.
Figure 3: DNA Repair Pathway. This flow chart shows one way
that damaged DNA is repaired in E. coli bacteria.
Is this rat hairless?
Yes. Why? The result of a
mutation, a change in the DNA
sequence. The effects of
mutations can vary widely, from
being beneficial, to having no
effect, to having lethal
consequences, and every
possibility in between.
Beneficial Mutations
Some mutations have a positive effect on the organism
in which they occur. They are called beneficial
mutations. They lead to new versions of proteins that
help organisms adapt to changes in their environment.
Beneficial mutations are essential for evolution to
occur. They increase an organism’s changes of surviving
or reproducing, so they are likely to become more
common over time. There are several well-known
examples of beneficial mutations. Here are just two:
1. Mutations in many bacteria that allow them to survive in the
presence of antibiotic drugs. The mutations lead to antibiotic-
resistant strains of bacteria. 2. A unique mutation is found in people
in a small town in Italy. The mutation protects them from developing
atherosclerosis, which is the dangerous buildup of fatty materials in
blood vessels. The individual in which the mutation first appeared has
even been identified.
Harmful Mutations
Imagine making a random change in a complicated
machine such as a car engine. The chance that the random
change would improve the functioning of the car is very
small. The change is far more likely to result in a car that
does not run well or perhaps does not run at all. By the
same token, any random change in a gene’s DNA is likely to
result in a protein that does not function normally or may
not function at all. Such mutations are likely to be
harmful. Harmful mutations may cause genetic disorders or
cancer.
• Cancer is a disease in which cells grow out of control
and form abnormal masses of cells. It is generally caused
by mutations in genes that regulate the cell cycle.
Because of the mutations, cells with damaged DNA are
allowed to divide without limits. Cancer genes can be
inherited.
• A genetic disorder is a disease caused by a mutation in
one or a few genes. A human example is cystic fibrosis. A
mutation in a single gene causes the body to produce
thick, sticky mucus that clogs the lungs and blocks ducts
in digestive organs.
Genetic Disorders
Many genetic disorders are caused by mutations in one or
a few genes. Other genetic disorders are caused by
abnormal numbers of chromosomes.
Genetic Disorders Caused by Mutations
Table 3 lists several genetic disorders caused by
mutations in just one gene. Some of the disorders are
caused by mutations in autosomal genes, others by
mutations in X-linked genes. Which disorder would you
expect to be more common in males than females?
Table 3: Genetic Disorders Caused by Mutations in One Gene
Few genetic disorders are controlled by dominant alleles.
A mutant dominant allele is expressed in every individual
who inherits even one copy of it. If it causes a serious
disorder, affected people may die young and fail to
reproduce. Therefore, the mutant dominant allele is likely
to die out of the population.
A mutant recessive allele, such as the allele that causes
sickle cell anemia (see Figure 7.43), is not expressed in
people who inherit just one copy of it. These people are
called carriers. They do not have the disorder themselves,
but they carry the mutant allele and can pass it to their
offspring. Thus, the allele is likely to pass on to the next
generation rather than die out.
Figure 5 Sickle-Shaped and Normal Red Blood Cells. Sickle
cell anemia is an autosomal recessive disorder. The
mutation that causes the disorder affects just one amino
acid in a single protein, but it has serious consequences for
the affected person. This photo shows the sickle shape of
red blood cells in people with sickle cell anemia.
Chromosomal Disorders
Mistakes may occur during meiosis that result in
nondisjunction. This is the failure of replicated
chromosomes to separate during meiosis (the animation at
the link below shows how this happens). Some of the
resulting gametes will be missing a chromosome, while
others will have an extra copy of the chromosome. If such
gametes are fertilized and form zygotes, they usually do
not survive. If they do survive, the individuals are likely to
have serious genetic disorders.
Table 4 lists several genetic disorders that are caused by
abnormal numbers of chromosomes. Figure 7.44 shows a
karyotype for trisomy 21 or Down’s Syndrome. Most
chromosomal disorders involve the X chromosome. Look back
at the X and Y chromosomes and you will see why. The X and
Y chromosomes are very different in size, so nondisjunction
of the sex chromosomes occurs relatively often.
Table 4: Genetic Disorders Caused by Abnormal Number of Chromosomes
Figure 6 Trisomy 21 (Down Syndrome) Karyotype. A karyotype is a picture
of a cell's chromosomes. Note the extra chromosome 21. (right) Child with
Down syndrome, exhibiting characteristic facial appearance.
Diagnosing Genetic Disorders
A genetic disorder that is caused by a mutation can be
inherited. Therefore, people with a genetic disorder in
their family may be concerned about having children with
the disorder. Professionals known as genetic counselors can
help them understand the risks of their children being
affected. If they decide to have children, they may be
advised to have prenatal (“before birth”) testing to see if
the fetus has any genetic abnormalities. One method of
prenatal testing is amniocentesis. In this procedure, a few
fetal cells are extracted from the fluid surrounding the
fetus, and the fetal chromosomes are examined.
Treating Genetic Disorders
The symptoms of genetic disorders can sometimes be
treated, but cures for genetic disorders are still in the early
stages of development. One potential cure that has already
been used with some success is gene therapy. This involves
inserting normal genes into cells with mutant genes.
• Mutations are caused by environmental factors known as mutagens. Types of
mutagens include radiation, chemicals, and infectious agents.
• Germline mutations occur in gametes. Somatic mutations occur in other
body cells. Chromosomal alterations are mutations that change chromosome
structure. Point mutations change a single nucleotide. Frameshift mutations
are additions or deletions of nucleotides that cause a shift in the reading
frame.
• Mutations are essential for evolution to occur because they increase
genetic variation and the potential for individuals to differ. The majority of
mutations are neutral in their effects on the organisms in which they occur.
Beneficial mutations may become more common through natural selection.
Harmful mutations may cause genetic disorders or cancer.
• Many genetic disorders are caused by mutations in one or a few genes.
• Other genetic disorders are caused by abnormal numbers of chromosomes.
Summary
Retrieved from:
www.ck-12.org
http://www.boyertownasd.org/cms/lib07/PA01916192/Centricity/Domain/743/D
.%20Chapter%207%20Lesson%204-Mutations.pdf
http://www.pubinfo.vcu.edu/secretsofthesequence/playlist_frame.asp
http://www.dnalc.org/resources/3d/17-sicklecell.html
http://www.kqed.org/quest/television/genetic-testing-through-the-web.
http://genetics.wustl.edu/bio5491/files/2016/01/DNA-mutagenesis-lecture-.pdf
http://www.biostudio.com/d_%20Nonsense%20Suppression%20I%20Nonsense%20M
utation.htm
Transcription
To transcribe means "to paraphrase or summarize in writing." The
information in DNA is transcribed - or summarized - into a smaller version -
RNA - that can be used by the cell. This process is called transcription.
Transcription is the first part of the central dogma of molecular biology:
DNA → RNA. It is the transfer of genetic instructions in DNA to mRNA.
During transcription, a strand of mRNA is made that is complementary to a
strand of DNA. Figure 1 shows how this occurs.
Figure 1 Overview of Transcription. Transcription uses the sequence of bases
in a strand of DNA to make a complementary strand of mRNA. Triplets are
groups of three successive nucleotide bases in DNA. Codons are
complementary groups of bases in mRNA.
Steps of Transcription
Transcription takes place in three steps: initiation, elongation, and
termination. The steps are illustrated in Figure 2.
1. Initiation is the beginning of transcription. It occurs when the enzyme
RNA polymerase binds to a region of a gene called the promoter. This
signals the DNA to unwind so the enzyme can ‘‘read” the bases in one of
the DNA strands. The enzyme is ready to make a strand of mRNA with a
complementary sequence of bases.
2. Elongation is the addition of nucleotides to the mRNA strand.
3. Termination is the ending of transcription, and occurs when RNA
polymerase crosses a stop (termination) sequence in the gene. The mRNA
strand is complete, and it detaches from DNA.
Figure 2. Steps of Transcription. Transcription occurs in the three steps -
initiation, elongation, and termination - shown here.
Source:
www.ck-12.org
*Processing RNA
• In prokaryotes, no RNA processing is necessary:
– the nascent RNA is usually the mRNA.
• In eukaryotes, the nascent RNA is called primary
transcript-RNA
– needs to be processed
– and transported to the cytoplasm for translation to
occur.
Fig. 1 Processes for synthesis of functional mRNA in prokaryotes and eukaryotes
Splicing removes introns from mRNA (see Figure 2).
Introns are regions that do not code for proteins. The
remaining mRNA consists only of regions that do code for
proteins, which are called exons. Ribonucleoproteins are
nucleoproteins that contain RNA. Small nuclear
ribonuclearproteins are involved in pre-mRNA splicing.
Figure 2 Splicing. Splicing removes introns from mRNA. UTR is an
untranslated region of the mRNA.
Editing changes some of the nucleotides in mRNA. For
example, the human protein called APOB, which helps
transport lipids in the blood, has two different forms
because of editing. One form is smaller than the other
because editing adds a premature stop signal in mRNA.
Polyadenylation adds a “tail” to the mRNA. The tail
consists of a string of As (adenine bases). It signals the end
of mRNA. It is also involved in exporting mRNA from the
nucleus. In addition, the tail protects mRNA from enzymes
that might break it down.
Figure 3 The ends of eukaryotic mRNAs
Questions
• Why is the mRNA not equal in length to the DNA it was transcribed from?
– 1) the mRNA was longer because it has a Poly A tail
– 2) The mRNA was longer because it contains only introns
– 3) The DNA was shorter because it does not have the Methylated cap
– 4) The mRNA was shorter because of Intron splicing
• Which nucleotides signal the 5’ end of an intron splice site?
– 1. AT
– 2. GU
– 3. AG
– 4. GG
Retrieved from:
http://vcell.ndsu.edu/animations/mrnasplicing/movie-flash.htm.
http://www.boyertownasd.org/cms/lib07/PA01916192/Centricity/Domain/743/
A.%20Chapter%207%20Lesson%201-From%20DNA%20to%20Proteins.pdf
http://www.csun.edu/~cmalone/pdf360/Ch13-2RNAprocess.pdf
Translation
Translation is the second part of the central dogma of
molecular biology: RNA → Protein. It is the process in
which the genetic code in mRNA is read to make a protein.
Figure 1 shows how this happens. After mRNA leaves the
nucleus, it moves to a ribosome, which consists of rRNA
and proteins. The ribosome reads the sequence of codons
in mRNA. Molecules of tRNA bring amino acids to the
ribosome in the correct sequence.
To understand the role of tRNA, you need to know more
about its structure. Each tRNA molecule has an anticodon
for the amino acid it carries. An anticodon is complementary
to the codon for an amino acid. For example, the amino
acid lysine has the codon AAG, so the anticodon is UUC.
Therefore, lysine would be carried by a tRNA molecule with
the anticodon UUC.
Wherever the codon AAG appears in mRNA, a UUC
anticodon of tRNA temporarily binds. While bound to mRNA,
tRNA gives up its amino acid. Bonds form between the
amino acids as they are brought one by one to the
ribosome, forming a polypeptide chain. The chain of amino
acids keeps growing until a stop codon is reached. To see
how this happens, go the link below.
Figure 1: Translation. Translation of the codons in mRNA to a chain of
amino acids occurs at a ribosome. Find the different types of RNA in
the diagram. What are their roles in translation?
After a polypeptide chain is synthesized, it may undergo
additional processes. For example, it may assume a
folded shape due to interactions among its amino acids.
It may also bind with other polypeptides or with
different types of molecules, such as lipids or
carbohydrates. Many proteins travel to the Golgi
apparatus to be modified for the specific job they will
do.
Source:
www.ck-12.org
GENE
REGULATION
Gene regulation is a label for the cellular
processes that control the rate and manner of
gene expression. A complex set of interactions
between genes, RNA molecules, proteins
(including transcription factors) and other
components of the expression system determine
when and where specific genes are activated
and the amount of protein or RNA product
produced. Often, one gene regulator controls
another, and so on, in a gene regulatory
network. Gene regulation is essential
for viruses, prokaryotes and eukaryotes as it
increases the versatility and adaptability of
WHAT IS GENE REGULATION?
Although as early as 1951, Barbara
McClintock showed interaction between
two genetic loci, Activator (Ac) and
Dissociator (Ds), in the color formation of
maize seeds, the first discovery of a gene
regulation system is widely considered to
be the identification in 1961 of
the lac operon, discovered by Jacques
Monod, in which some enzymes involved
in lactose metabolism are expressed by E.
coli only in the presence of lactose and
absence of glucose.
Any step of gene expression may be
modulated, from the DNA-
RNA transcription step to post-translational
modification of a protein. The following is a
list of stages where gene expression is
regulated, the most extensively utilized
point is Transcription Initiation:
• Chromatin domains
• Transcription
• Post-transcriptional modification
• RNA transport
• Translation
• mRNA degradation
WHY IS GENE EXPRESSION REGULATED?
Genes can't control an organism on their own;
rather, they must interact with and respond to
the organism's environment. Some genes are
constitutive, or always "on," regardless of
environmental conditions. Such genes are
among the most important elements of a cell's
genome, and they control the ability of DNA to
replicate, express itself, and repair itself.
These genes also control protein synthesis
and much of an organism's central
metabolism. It turns out that the regulation of
such genes differs between PROKARYOTES
and EUKARYOTES. For prokaryotes,
most regulatory proteins are negative and
PROKARYOTIC GENE REGULATION
For prokaryotes, most regulatory proteins are
negative and therefore turn genes off. Here,
the cells rely on protein–small molecule
binding, in which a ligand or small molecule
signals the state of the cell and whether gene
expression is needed.
The repressor or activator protein binds near
its regulatory target: the gene. Some
regulatory proteins must have a ligand
attached to them to be able to bind, whereas
others are unable to bind when attached to a
ligand. In prokaryotes, most regulatory
proteins are specific to one gene, although
there are a few proteins that act more widely.
Furthermore, some repressors have a fine-
tuning system known as attenuation, which
uses mRNA structure to stop both
transcription and translation depending on the
concentration of an operon's end-product
enzymes. (In eukaryotes, there is no exact
equivalent of attenuation, because
transcription occurs in the nucleus and
translation occurs in the cytoplasm, making
this sort of coordinated effect impossible.)
Yet another layer of prokaryotic regulation
affects the structure of RNA polymerase,
which turns on large groups of genes. Here,
the sigma factor of RNA polymerase changes
several times to produce heat- and
PROKARYOTIC GENE REGULATION
(TRANSCRIPTION REGULATION)
DIAGRAM
EUKARYOTIC GENE REGULATION
Unlike prokaryotes, multiple gene-regulating
mechanisms operate in the nucleus before
and after RNA transcription, and in the
cytoplasm both before and after translation.
Histones are small proteins packed inside
the molecular structure of the DNA double
helix. Tight histone packing prevents RNA
polymerase from contacting and transcribing
the DNA. This type of overall control of
protein synthesis is regulated by genes that
control the packing density of histones. X-
chromosome inactivation occurs when
dense packing of the X chromosome in
females totally prevents its function even in
interphase. This type of inactivation is
Activator-enhancer complex is unique in
eukaryotes because they normally have to be
activated to begin protein synthesis, which
requires the use of transcription factors and
RNA polymerase. In general, the process of
eukaryotic protein synthesis involves four
steps:
1. Activators, a special type of transcription
factor, bind to enhancers, which are discrete
DNA units located at varying points along the
chromosome.
2. The activator-enhancer complex bends the
DNA molecule so that additional transcription
factors have better access to bonding sites on
the operator.
3. The bonding of additional transcription
factors to the operator allows greater access by
Eukaryotic gene expression involves many steps,
and almost all of them can be regulated.
Different genes are regulated at different points,
and it’s not uncommon for a gene (particularly an
important or powerful one) to be regulated at
multiple steps.
• Chromatin accessibility. The structure of
chromatin (DNA and its organizing proteins)
can be regulated. More open or “relaxed”
chromatin makes a gene more available for
transcription.
• Transcription. Transcription is a key regulatory
point for many genes. Sets of transcription
factor proteins bind to specific DNA sequences
in or near a gene and promote or repress its
transcription into an RNA.
• RNA processing. Splicing, capping, and
• RNA stability. The lifetime of an mRNA
molecule in the cytosol affects how many
proteins can be made from it. Small
regulatory RNAs called miRNAs can bind
to target mRNAs and cause them to be
chopped up.
• Translation. Translation of an mRNA may
be increased or inhibited by regulators.
For instance, miRNAs sometimes block
translation of their target mRNAs (rather
than causing them to be chopped up).
• Protein activity. Proteins can undergo a
variety of modifications, such as being
chopped up or tagged with chemical
groups. These modifications can be
The processing and packaging of RNA both in the
nucleus and cytoplasm provides two more
opportunities for gene regulation to occur after
transcription but before translation.
Adding extra nucleotides as a protective cap and
tail to the RNA identifies the RNA as an mRNA by
the ribosomes, and prevents degradation by cell
enzymes as it moves from the nucleus into the
cytoplasm.
RNA splicing occurs when “gaps” of nonprotein-
code-carrying nucleotides called interons are
removed from the code-carrying nucleotides,
called exons, which are then connected to
shorten the RNA molecule for conversion into
tRNA and rRNA. The number of interons
regulates the speed at which the RNA can be
processed.
The longevity of the individual mRNA molecule
determines how many times it can be used and
reused to create proteins. In eukaryotes, the
mRNA tends to be stable, which means it can
be used multiple times; which is efficient, but
it prevents eukaryotes from making rapid
response changes to environmental
disruptions. The mRNA of prokaryotes is
unstable, allowing for the creation of new
mRNA, which has more opportunities to adjust
for changing environmental conditions.
Inhibitory proteins prevent the translation of
mRNA. They are made inactive when bonded
with the substance for which they are trying to
block production.
Post-translation control involves the selective
cutting and breakdown of proteins that prevent
EXAMPLES OF GENE REGULATION
• Enzyme induction is a process in which a
molecule (e.g., a drug) induces (i.e., initiates
or enhances) the expression of an enzyme.
• The Lac operon is an interesting example of
how gene expression can be regulated.
Viruses, despite having only a few genes,
possess mechanisms to regulate their gene
expression, typically into an early and late
phase, using collinear systems regulated by
anti-terminators (lambda phage) or splicing
modulators (HIV).
• GAL4 is a transcriptional activator that
controls the expression of GAL1, GAL7, and
GAL10 (all of which code for the metabolic of
galactose in yeast). The GAL4/UAS
ENZYME INDUCTION
• Enzyme induction is a process in which a
molecule (e.g. a drug) induces (i.e. initiates or
enhances) the expression of an enzyme. An
enzyme inducer is a type of drug which binds to
an enzyme and increases its metabolic activity.
Many of the enzymes involved in drug
metabolism may be up-regulated by exposure to
drugs and environmental chemicals leading to
increased rates of metabolism. This
phenomenon is known
as enzyme induction. Enzyme induction is a
process where production of an enzyme is
triggered or increased in response to changes in
the environment that surrounds an individual
You do not have access to view this node. The
ENZYME
INDUCTION
DIAGRAM
WHAT IS ENZYME REPRESSION?
Induction and repression are linked in that
they both focus on the binding of a molecule
known as RNA polymerase to DNA.
Particularly, the RNA polymerase binds to a
region that is immediately "upstream" from
the region of DNA that code for a protein.
The binding region is termed the ‘operator’.
The operator acts to position the
polymerase correctly, so that the molecule
can then begin to move along the DNA,
interpreting the genetic information as it
moves along. The three-dimensional shape
of the operator region manipulates the
WHAT IS THE PROCESS OF ENZYME
INDUCTION?
Enzyme induction is a process where an enzyme
is contrived in response to the presence of a
specific molecule. This molecule is termed an
inducer. Basically, an inducer molecule is a
compound that the enzyme acts upon. In
the induction process, the inducer molecule
merges with another molecule, which is called
the ‘repressor’ (a chemical compound that is
designed to limit or prevent enzyme production,
so there are no obstacles to enzyme
production). The binding of the inducer to the
repressor obstructs the function of the
repressor, which is to bind to a specific region
called an ‘operator’. The operator is the site to
which another molecule, known as ribonucleic
acid (RNA) polymerase, binds and begins the
transcription (transfer of genetic information
Thus, the binding of the inducer to
the repressor keeps the repressor
from averting transcription, and so
the gene coding for the inducible
enzyme is transcribed. Repression of
transcription is basically the default
behavior, which is dominated once
the inducing molecule is present. In
bacteria, the lactose (lac) operon is a
very well characterized system that
operates on the basis of induction. An
operon is a single unit of physically
adjacent genes that function together
under the control of a single operator
gene.
LAC OPERON
The lac operon (lactose operon) is
an operon required for the transport and
metabolism of lactose in Escherichia coli and
many other enteric bacteria. Although glucose is
the preferred carbon source for most bacteria,
the lac operon allows for the effective digestion
of lactose when glucose is not available. Bacterial
operons are polycistronic transcripts that are able
to produce multiple proteins from one mRNA
transcript. In this case, when lactose is required
as a sugar source for the bacterium, the three
genes of the lac operon can be expressed and
their subsequent proteins translated: lacZ, lacY,
and lacA. The gene product of lacZ is β-
galactosidase which cleaves lactose, a
It would be wasteful to produce the enzymes
when there is no lactose available or if there is
a more preferable energy source available,
such as glucose. The lac operon uses a two-
part control mechanism to ensure that the cell
expends energy producing the enzymes
encoded by the lac operon only when
necessary. In the absence of lactose,
the lac repressor halts production of the
enzymes encoded by the lac operon. In the
presence of glucose, the catabolite activator
protein (CAP), required for production of the
enzymes, remains inactive, and EIIAGlc shuts
down lactose permease to prevent transport of
lactose into the cell. This dual control
mechanism causes the sequential utilization of
glucose and lactose in two distinct growth
phases, known as diauxie.
STRUCTURE OF LAC OPERON
The lac operon contains three
genes: lacZ, lacY, and lacA. These genes are
transcribed as a single mRNA, under control of
one promoter.
Genes in the lac operon specify proteins that
help the cell utilize lactose. lacZ encodes an
enzyme that splits lactose into
monosaccharides (single-unit sugars) that can
be fed into glycolysis. Similarly, lacY encodes a
membrane-embedded transporter that helps
bring lactose into the cell.
In addition to the three genes, the lac operon
also contains a number of regulatory DNA
sequences. These are regions of DNA to which
• The promoter is the binding site for RNA
polymerase, the enzyme that performs
transcription.
• The operator is a negative regulatory site
bound by the lac repressor protein. The
operator overlaps with the promoter, and
when the lac repressor is bound, RNA
polymerase cannot bind to the promoter
and start transcription.
• The CAP binding site is a positive
regulatory site that is bound by catabolite
activator protein (CAP). When CAP is
bound to this site, it promotes
transcription by helping RNA polymerase
bind to the promoter.
STRUCTURE OF LAC OPERON
THE LAC REPRESSOR
• The lac repressor is a protein that
represses (inhibits) transcription of
the lac operon. It does this by binding to
the operator, which partially overlaps with
the promoter. When bound,
the lac repressor gets in RNA
polymerase's way and keeps it from
transcribing the operon.
• When lactose is not available,
the lac repressor binds tightly to the
operator, preventing transcription by RNA
polymerase. However, when lactose is
present, the lac repressor loses its ability
to bind DNA. It floats off the operator,
clearing the way for RNA polymerase to
This change in
the lac repressor is
caused by the small
molecule allolactose, an
isomer (rearranged
version) of lactose. When
lactose is available, some
molecules will be
converted to allolactose
inside the cell.
Allolactose binds to
the lac repressor and
makes it change shape so
it can no longer bind DNA.
Allolactose is an example
of an inducer, a small
molecule that triggers
expression of a gene or
operon. The lac operon is
considered an inducible
CATABOLITE ACTIVATOR PROTEIN(CAP)
When lactose is present, the lac repressor
loses its DNA-binding ability. This clears the
way for RNA polymerase to bind to the
promoter and transcribe the lac operon. As it
turns out, RNA polymerase alone does not
bind very well to the lac operon promoter. It
might make a few transcripts, but it won't do
much more unless it gets extra help
from catabolite activator protein (CAP). CAP
binds to a region of DNA just before
the lac operon promoter and helps RNA
polymerase attach to the promoter, driving
high levels of transcription.
CAP isn't always active
(able to bind DNA).
Instead, it's regulated by
a small molecule
called cyclic AMP (cAMP).
cAMP is a "hunger signal"
made by E. coli when
glucose levels are low.
cAMP binds to CAP,
changing its shape and
making it able to bind
DNA and promote
transcription. Without
cAMP, CAP cannot bind
DNA and is inactive.
CAP is only active when
glucose levels are low
(cAMP levels are high).
Thus, the lac operon can
only be transcribed at
high levels when glucose
is absent. This strategy
Glucose present,
lactose absent: No
transcription of
the lac operon
occurs. That's
because
the lac repressor
remains bound to
the operator and
prevents
transcription by
RNA polymerase.
Also, cAMP levels
are low because
glucose levels are
high, so CAP is
inactive and
cannot bind DNA.
Glucose present,
lactose present:
Low-level
transcription of
the lac operon
occurs.
The lac repressor is
released from the
operator because
the inducer
(allolactose) is
present. cAMP
levels, however, are
low because
glucose is present.
Thus, CAP remains
inactive and cannot
Glucose absent,
lactose absent: No
transcription of
the lac operon
occurs. cAMP
levels are high
because glucose
levels are low, so
CAP is active and
will be bound to
the DNA. However,
the lac repressor
will also be bound
to the operator
(due to the
absence of
Glucose absent,
lactose present:
Strong
transcription of
the lac operon
occurs.
The lac repressor
is released from
the operator
because the
inducer
(allolactose) is
present. cAMP
levels are high
because glucose
is absent, so CAP
is active and
bound to the DNA.
VIRAL REPLICATIONReplication of viruses primarily involves
the multiplication of the viral genome.
Replication also involves synthesis of
viral messenger RNA (mRNA) from
"early" genes (with exceptions for positive
sense RNA viruses),
viral protein synthesis, possible assembly
of viral proteins, then viral genome
replication mediated by early or regulatory
protein expression. This may be followed,
for complex viruses with larger genomes,
by one or more further rounds of mRNA
synthesis: "late" gene expression is, in
general, necessary for structural
or virion proteins. Viral replication usually
takes place in the cytoplasm.
SCHEMATIC SHOWING ANTISENSE DNA STRANDS CAN INTERFERE WITH
PROTEIN TRANSLATION
Viruses that replicate via RNA intermediates
need an RNA-dependent RNA-polymerase to
replicate their RNA, but animal cells do not
seem to possess a suitable enzyme. Therefore,
this type of animal RNA virus needs to code for
an RNA-dependent RNA polymerase. No viral
proteins can be made until viral messenger RNA
is available; thus, the nature of the RNA in the
virion affects the strategy of the virus: In plus-
stranded RNA viruses, the virion (genomic) RNA
is the same sense as mRNA and so functions as
mRNA. This mRNA can be translated
immediately upon infection of the host cell .
Examples: poliovirus (picornavirus),
togaviruses, and flaviviruses.
Viral gene expression regulation refers to any
of the processes by which cytoplasmic
factors influence the differential control of
gene action in viruses. The interplay of the
viral genome with the host metabolic
machinery involves modifications in both
gene expression and regulation. Retroviruses
have adapted themselves to use this
machinery while maintaining the cell
integrity, which is essential to preserve their
survival. Consequently, there can be variable
host pathogenicity associated with several
diseases such as malignancies,
immunodeficiencies, and neurological
disorders. This book describes current
research in the field, and gives a better
GAL4 SYSTEM
The GAL4-UAS system is
a biochemical method used to study gene
expression and function in organisms such as
the fruit fly. It has also been adapted to
study receptor chemical-binding functions in
vitro in cell culture. It was developed
by Andrea Brand and Norbert Perrimon in
1993 and is considered a powerful technique
for studying the expression of genes. The
system has two parts: the GAL4 gene,
encoding the yeast transcription
activator protein GAL4, and the UAS
(Upstream Activation Sequence), an enhancer
The GAL4 system allows separation of the
problems of defining which cells express a
gene or protein and what the experimenter
wants to do with this knowledge. Geneticists
have created genetic varieties of model
organisms (typically fruit flies), called GAL4
lines, each of which expresses GAL4 in some
subset of the animal's tissues. For example,
some lines might express GAL4 only in muscle
cells, or only in nerves, or only in the
antennae, and so on. For fruit flies in
particular, there are tens of thousands of such
lines, with the most useful expressing GAL4 in
only a very specific subset of the animal—
perhaps, for example, only those neurons that
connect two specific compartments of the
fly's brain. The presence of GAL4, by itself, in
Since GAL4 by itself is not visible, and has little
effect on cells, the other necessary part of this
system are the "reporter lines". These are
strains of flies with the special UAS region next
to a desired gene. These genetic instructions
occur in every cell of the animal, but in most
cells nothing happens since that cell is not
producing GAL4. In the cells that are producing
GAL4, however, the UAS is activated, the gene
next to it is turned on, and it starts producing
its resulting protein. This may report to the
investigator which cells are expressing GAL4,
hence the term "reporter line", but genes
intended to manipulate the cell behavior are
often used as well.
Typical reporter genes include:
• Fluorescent proteins like green (GFP) or red
fluorescent proteins (RFP), which allow scientists
to see which cells express GAL4
• Channelrhodopsin, which allows light-sensitive
triggering of nerve cells
• Halorhodopsin, which conversely allows light to
suppress the firing of neurons
• Shibire, which shuts neurons off, but only at higher
temperatures (30 °C and above). Flies with this gene
can be raised and tested at lower temperatures
where their neurons will behave normally. Then the
body temperature of the flies can be raised (since
they are cold-blooded), and these neurons turn
off.[3] If the fly's behavior changes, this gives a
strong clue to what those neurons do.
• GECI (Genetically Encoded Calcium Indicator), often
a member of the GCaMP family of proteins. These
proteins glow when exposed to calcium, which, in
RNA PROCESSING
RNA serves a multitude of functions within
cells. These functions are primarily involved
in converting the genetic information
contained in a cell's DNA into the proteins
that determine the cell's structure and
function. All RNAs are originally transcribed
from DNA by RNA polymerases, which are
specialized enzyme complexes, but most
RNAs must be further modified or
processed before they can carry out their
roles. Thus, RNA processing refers to any
modification made to RNA between its
transcription and its final function in the
cell. These processing steps include the
removal of extra sections of RNA, specific
modifications of RNA bases, and
TYPES OF RNA
• There are different types of RNA, each of which plays a
specific role, including specifying the amino acid
sequence of proteins (performed by messenger RNAs, or
mRNAs), organizing and catalyzing the synthesis of
proteins (ribosomal RNAs or rRNAs), translating codons in
the mRNA into amino acids (transfer RNAs or tRNAs) and
directing many of the RNA processing steps (performed
by small RNAs in the nucleus, called snRNAs and
snoRNAs).
All of these types of RNAs begin as primary transcripts
copied from DNA by one of the RNA polymerases. One of
the features that separates eukaryotes and prokaryotes is
that eukaryotes isolate their DNA inside a nucleus while
protein synthesis takes place in the cytoplasm. This
separates the processes of transcription and translation in
space and time. Prokaryotes, which lack a nucleus, can
translate an mRNA as soon as it is transcribed by RNA
polymerase. As a consequence, there is very little
processing of prokaryotic mRNAs. By contrast, in eukaryotic
cells many processing steps occur between mRNA
transcription and translation. Unlike the case of mRNAs,
both eukaryotes and prokaryotes process their rRNAs and
tRNAs in broadly similar ways.
TYPES OF RNA PROCESSING
There are three main types of RNA processing events: trimming one or both of the ends of the primary
transcript to the mature RNA length; removing internal RNA sequences by a process called RNA splicing;
and modifying RNA nucleotides either at the ends of an RNA or within the body of the RNA. We will briefly
examine each of these and then discuss how they are applied to the various types of cellular RNAs.
Almost all RNAs have extra sequences at one or both ends of the primary transcripts that must be removed.
The removal of individual nucleotides from the ends of the RNA strand is carried out by any of several
ribonucleases (enzymes that cut RNA), called exoribonucleases. An entire section of RNA sequence can be
removed by cleavage in the middle of an RNA strand. The enzymes responsible for the cleavage in this
location are called endoribonucleases. Each of these ribonucleases is targeted so that it only cleaves
particular RNAs at particular places.
RNA splicing is similar to trimming in that it removes extra RNA sequences, but it is different because the
sequence is removed from the middle of an RNA and the two flanking pieces are joined together again (see
figure). The part of the RNA that is removed is called an intron, whereas the two pieces that are joined
together, or spliced, are called exons. Just as with the cleavage enzymes, the splicing machinery recognizes
particular sites within the RNA, in this case the junctions between exons and introns, and cleaves and
rejoins the RNA at those positions.
Modification of RNA nucleotides can occur at the ends of an RNA molecule or at internal positions.
Modification of the ends can protect the RNA from degradation by exoribonucleases and can also act as a
signal to guide the transport of the molecule to a particular subcellular compartment. Some internal
modifications, particularly of tRNAs and rRNAs, are necessary for these RNAs to carry out their functions in
protein synthesis. Some internal modifications of mRNAs change the sequence of the message and so
change the amino acid sequence of the protein coded for by the mRNA. This process is called RNA editing.
As with the other types of RNA processing, the enzymes that modify RNAs are directed to specific sites on
the RNA.
PROCESSING OF VARIOUS CLASSES OF RNASRibosomal RNAs are synthesized as long primary transcripts that contain several different
rRNAs separated by spacer regions .The individual rRNAs are cut apart by endoribonucleases
that cleave within the spacer regions. Other enzymes then trim the ends to their final length.
Ribosomal RNAs are also modified at many specific sites within the RNA. Ribosomal RNA
synthesis and processing occurs in a special structure within the nucleus called
the nucleolus . The mature rRNAs bind to ribosomal proteins within the nucleolus and the
assembled ribosomes are then transported to the cytoplasm to carry out protein synthesis.
Transfer RNAs are transcribed individually from tRNA genes. The primary transcripts are
trimmed at both the 5′ and 3′ ("five prime," or "upstream" and "three prime," or
"downstream") ends, and several modifications are made to internal bases. Many eukaryotic
tRNAs also contain an intron, which must be removed by RNA splicing. The finished tRNAs are
then transported from the nucleus to the cytoplasm.
Messenger RNAs are transcribed individually from their genes as very long primary
transcripts. This is because most eukaryotic genes are divided into many exons separated by
introns. Genes may contain from zero to more than sixty introns, with a typical gene having
around ten. Introns are spliced out of primary RNA transcripts by a large structure called
the spliceosome . The spliceosome does not move along the RNA but is assembled around
each intron where it cuts and joins the RNA to remove the intron and connect the exons. This
must be done many times on a typical primary transcript to produce the mature mRNA.
In addition to removal of the introns, the mRNA is modified at
the 5′ end by the addition of a special "cap" structure that is
later recognized by the translation machinery. The mRNA is
also trimmed at the 3′ end and several hundred adenosine
nucleotides are added. This modification, which is called either
polyadenylation or poly (A) addition, helps stabilize the 3′ end
against degradation and is also recognized by the translation
machinery. Finally, the processed mature mRNA is transported
from the nucleus to the cytoplasm.
Some RNAs, called small nuclear RNAs (snRNAs) and small
nucleolar RNAs (snoRNAs), are processed in the nucleus and
are themselves part of the RNA processing systems in the
nucleus. Most snRNAs are involved in mRNA splicing, while
most snoRNAs are involved in rRNA cleavage and modification.
RNA PROCESSING AND THE HUMAN
GENOME
The fact that most human genes are composed of many exons has some
important consequences for the expression of genetic information. First, we
now know that many genes are spliced in more than one way, a phenomenon
known as alternative splicing. For example, some types of cells might leave
out an exon from the final mRNA that is left in by other types of cells, giving it
a slightly different function. This means that a single gene can code for more
than one protein. Some complicated genes appear to be spliced to give
hundreds of alternative forms. Alternative splicing, therefore, can increase
the coding capacity of the genome without increasing the number of genes.
A second consequence of the exon/intron gene structure is that many human
gene mutations affect the splicing pattern of that gene. For example, a
mutation in the sequence at an intron/exon junction that is recognized by the
spliceosome can cause the junction to be ignored. This causes splicing to
occur to the next exon in line, leaving out the exon next to the mutation. This
is called exon skipping and it usually results in an mRNA that codes for a
nonfunctional protein. Exon skipping and other errors in splicing are seen in
many human genetic diseases.
POST-TRANSCRIPTIONAL MODIFICATION
Post-transcriptional modification or Co-transcriptional
modification is the process in eukaryotic cells where primary
transcript RNA is converted into mature RNA. A notable
example is the conversion of precursor messenger
RNA into maturemessenger RNA (mRNA) that occurs prior to
protein translation. The process includes three major steps:
addition of a 5' cap, addition of a 3' poly-adenylation tail,
and splicing. This process is vital for the correct translation of
the genomes of eukaryotes because the initial precursor
mRNA produced during transcription contains
both exons (coding or important sequences involved in
translation), and introns (non-coding sequences)
Example of a signal that directs post-transcriptional processing: the
conserved eukaryotic polyadenylation signal directs cleavage at the cleavage
signal and addition of a poly-A tail to the mRNA transcript
5’ PROCESSING
Capping
Capping of the pre-mRNA involves the addition of 7-methylguanosine (m7G) to the
5' end. To achieve this, the terminal 5' phosphate requires removal, which is done
with the aid of a phosphatase enzyme. The enzyme guanosyl transferase then
catalyses the reaction, which produces the diphosphate 5' end. The diphosphate 5'
end then attacks the alpha phosphorus atom of a GTP molecule in order to add
the guanine residue in a 5'5' triphosphate link. The enzyme (guanine-N7-)-
methyltransferase ("cap MTase") transfers a methyl group from S-adenosyl
methionine to the guanine ring.[3] This type of cap, with just the (m7G) in position is
called a cap 0 structure. The ribose of the adjacent nucleotide may also be
methylated to give a cap 1. Methylation of nucleotides downstream of the RNA
molecule produce cap 2, cap 3 structures and so on. In these cases the methyl
groups are added to the 2' OH groups of the ribose sugar. The cap protects the 5'
end of the primary RNA transcript from attack by ribonucleases that have specificity
to the 3'5' phosphodiester bonds.
3’ PROCESSING
Cleavage and polyadenylation The pre-mRNA processing at the 3' end of the RNA
molecule involves cleavage of its 3' end and then the addition of about
250 adenine residues to form a poly(A) tail. The cleavage and adenylation reactions
occur if a polyadenylation signal sequence (5'- AAUAAA-3') is located near the 3' end
of the pre-mRNA molecule, which is followed by another sequence, which is
usually (5'-CA-3') and is the site of cleavage. A GU-rich sequence is also usually
present further downstream on the pre-mRNA molecule. After the synthesis of the
sequence elements, two multisubunit proteins called cleavage and polyadenylation
specificity factor (CPSF) and cleavage stimulation factor (CStF) are transferred
from RNA Polymerase II to the RNA molecule. The two factors bind to the sequence
elements. A protein complex forms that contains additional cleavage factors and the
enzyme Polyadenylate Polymerase (PAP). This complex cleaves the RNA between the
polyadenylation sequence and the GU-rich sequence at the cleavage site marked by
the (5'-CA-3') sequences. Poly(A) polymerase then adds about 200 adenine units to
the new 3' end of the RNA molecule using ATP as a precursor. As the poly(A) tail is
synthesised, it binds multiple copies of poly(A) binding protein, which protects the
3'end from ribonuclease digestion
SPLICING
Splicing
RNA splicing is the process by which introns, regions of RNA that do not
code for protein, are removed from the pre-mRNA and the
remaining exons connected to re-form a single continuous molecule. Exons
are sections of mRNA which become "expressed" or translated into a
protein. They are the coding portions of a mRNA molecule.[5] Although most
RNA splicing occurs after the complete synthesis and end-capping of the
pre-mRNA, transcripts with many exons can be spliced co-
transcriptionally.[6] The splicing reaction is catalyzed by a large protein
complex called the spliceosome assembled from proteins and small nuclear
RNA molecules that recognize splice sites in the pre-mRNA sequence. Many
pre-mRNAs, including those encoding antibodies, can be spliced in multiple
ways to produce different mature mRNAs that encode different protein
sequences. This process is known as alternative splicing, and allows
production of a large variety of proteins from a limited amount of DNA.
TRANSLATION
Translation is the process in which ribosomes in a cell's cytoplasm create proteins,
following transcription of DNA to RNA in the cell's nucleus. The entire process is a
part of gene expression.
In translation, messenger RNA (mRNA) is decoded by a ribosome, outside the
nucleus, to produce a specific amino acid chain, or polypeptide. The polypeptide
later folds into an active protein and performs its functions in
the cell. The ribosome facilitates decoding by inducing the binding
of complementary tRNA anticodon sequences to mRNA codons. The tRNAs carry
specific amino acids that are chained together into a polypeptide as the mRNA
passes through and is "read" by the ribosome.
Translation proceeds in three phases:
Initiation: The ribosome assembles around the target mRNA. The first tRNA is
attached at the start codon.
Elongation: The tRNA transfers an amino acid to the tRNA corresponding to the
next codon. The ribosome then moves (translocates) to the next mRNA codon to
continue the process, creating an amino acid chain.
Termination: When a stop codon is reached, the ribosome releases the
polypeptide.
In bacteria, translation occurs in the cytoplasm, where the large and small subunits
of the ribosome bind to the mRNA. In eukaryotes, translation occurs in
the cytosol or across the membrane of the endoplasmic reticulum in a process
THE GENETIC CODE
During translation, a cell “reads” the information in a messenger RNA (mRNA)
and uses it to build a protein. Actually, to be a little more techical, an mRNA
doesn’t always encode—provide instructions for—a whole protein. Instead, what
we can confidently say is that it always encodes a polypeptide, or chain of amino
acids. In an mRNA, the instructions for building a polypeptide are RNA
nucleotides (As, Us, Cs, and Gs) read in groups of three. These groups of three
are called codons.
There are 616161 codons for amino acids, and each of them is "read" to specify a
certain amino acid out of the 202020 commonly found in proteins. One codon,
AUG, specifies the amino acid methionine and also acts as a start codon to signal
the start of protein construction.
There are three more codons that do not specify amino acids. These stop
codons, UAA, UAG, and UGA, tell the cell when a polypeptide is complete. All
together, this collection of codon-amino acid relationships is called the genetic
code, because it lets cells “decode” an mRNA into a chain of amino acids.
TRANSFER RNAS (TRNAS)
Transfer RNAs, or tRNAs, are molecular
"bridges" that connect mRNA codons to
the amino amino acids they encode. One
end of each tRNA has a sequence of three
nucleotides called an anticodon, which
can bind to specific mRNA codons. The
other end of the tRNA carries the amino
acid specified by the codons.
There are many different types of tRNAs.
Each type reads one or a few codons and
brings the right amino acid matching
those codons.
RIBOSOMAL RNA
Ribosomes are the structures where polypeptides (proteins) are built. They are
made up of protein and RNA (ribosomal RNA, or rRNA). Each ribosome has two
subunits, a large one and a small one, which come together around an mRNA—
kind of like the two halves of a hamburger bun coming together around the
patty.
The ribosome provides a set of handy slots where tRNAs can find their matching
codons on the mRNA template and deliver their amino acids. These slots are
called the A, P, and E sites. Not only that, but the ribosome also acts as an
enzyme, catalyzing the chemical reaction that links amino acids together to make
a chain.
GETTING STARTED: INITIATION
In initiation, the ribosome assembles around the mRNA to be
read and the first tRNA (carrying the amino acid methionine,
which matches the start codon, AUG). This setup, called the
initiation complex, is needed in order for translation to get
started.
EXTENDING THE CHAIN: ELONGATION
Elongation is the stage where the amino acid chain gets longer.
In elongation, the mRNA is read one codon at a time, and the
amino acid matching each codon is added to a growing protein
chain.
Each time a new codon is exposed:
• A matching tRNA binds to the codon
• The existing amino acid chain (polypeptide) is linked onto the
amino acid of the tRNA via a chemical reaction
• The mRNA is shifted one codon over in the ribosome,
exposing a new codon for reading
During
elongation,
tRNAs move
through the A,
P, and E sites of
the ribosome,
as shown
above. This
process repeats
many times as
new codons are
read and new
amino acids are
added to the
chain.
FINISHING UP: TERMINATION
Termination is the stage in which the finished polypeptide
chain is released. It begins when a stop codon (UAG, UAA, or
UGA) enters the ribosome, triggering a series of events that
separate the chain from its tRNA and allow it to drift out of
the ribosome.
After termination, the polypeptide may still need to fold into
the right 3D shape, undergo processing (such as the removal
of amino acids), get shipped to the right place in the cell, or
combine with other polypeptides before it can do its job as a
functional protein.
EPIGENETICS
Epigenetics is the study of potentially heritable changes in gene expression (active versus
inactive genes) that does not involve changes to the underlying DNA sequence — a
change in phenotype without a change in genotype — which in turn affects how cells
read the genes. Epigenetic change is a regular and natural occurrence but can also be
influenced by several factors including age, the environment/lifestyle, and disease state.
Epigenetic modifications can manifest as commonly as the manner in which cells
terminally differentiate to end up as skin cells, liver cells, brain cells, etc. Or, epigenetic
change can have more damaging effects that can result in diseases like cancer. At least
three systems including DNA methylation, histone modification and non-coding
RNA (ncRNA)-associated gene silencing are currently considered to initiate and sustain
epigenetic change.1 New and ongoing research is continuously uncovering the role
of epigenetics in a variety of human disorders and fatal diseases.
EPIGENETIC MECHANISM
One example of an epigenetic change in eukaryotic biology
is the process of cellular differentiation.
During morphogenesis, totipotent stem cells become the
various pluripotentcell lines of the embryo, which in turn
become fully differentiated cells. In other words, as a
single fertilized egg cell – the zygote – continues to divide,
the resulting daughter cells change into all the different
cell types in an organism, including neurons, muscle
cells, epithelium, endothelium of blood vessels, etc., by
activating some genes while inhibiting the expression of
others.Historically, some phenomena not necessarily
heritable have also been described as epigenetic. For
example, epigenetic has been used to describe any
modification of chromosomal regions, especially histone
modifications, whether or not these changes are heritable
or associated with a phenotype. The consensus definition
HISTORY
What began as broad research focused on combining genetics and developmental
biology by well-respected scientists including Conrad H. Waddington and Ernst Hadorn
during the mid-twentieth century has evolved into the field we currently refer to
as epigenetics. The term epigenetics, which was coined by Waddington in 1942, was
derived from the Greek word “epigenesis” which originally described the influence of
genetic processes on development.2 During the 1990s there became a renewed
interest in genetic assimilation. This lead to elucidation of the molecular basis of
Conrad Waddington’s observations in which environmental stress caused genetic
assimilation of certain phenotypic characteristics in Drosophila fruit flies. Since then,
research efforts have been focused on unraveling the epigenetic mechanisms related
to these types of changes.3
Currently, DNA methylation is one of the most broadly studied and well-characterized
epigenetic modifications dating back to studies done by Griffith and Mahler in 1969
which suggested that DNA methylation may be important in long term memory
function.4 Other major modifications include chromatin remodeling, histone
modifications, and non-coding RNA mechanisms. The renewed interest in epigenetics
has led to new findings about the relationship between epigenetic changes and a host
of disorders including various cancers, mental retardation associated disorders,
EPIGENETICS: FUNDAMENTALS
Cancer. Cancer was the first human disease to be linked to epigenetics. Studies
performed by Feinberg and Vogelstein in 1983, using primary human tumor tissues,
found that genes of colorectal cancer cells were substantially hypomethylated compared
with normal tissues.1 DNA hypomethylation can activate oncogenes and
initiate chromosome instability, whereas DNA hypermethylation initiates silencing of
tumor suppressor genes. An accumulation of genetic and epigenetic errors can
transform a normal cell into an invasive or metastatic tumor cell. Additionally,
DNA methylation patterns may cause abnormal expression of cancer-associated genes.
Global histone modification patterns are also found to correlate with cancers such as
prostate, breast, and pancreatic cancer. Subsequently, epigenetic changes can be used as
biomarkers for the molecular diagnosis of early cancer.
Mental Retardation Disorders. Epigenetic changes are also linked to several
disorders that result in intellectual disabilities such as ATR-X, Fragile X, Rett,
Beckwith-Weidman (BWS), Prader-Willi and Angelman syndromes..2 For example,
the imprint disorders Prader-Willi syndrome and Angelman syndrome, display an
abnormal phenotype as a result of the absence of the paternal or maternal copy of
a gene, respectively. In these imprint disorders, there is a genetic deletion in
chromosome 15 in a majority of patients. The same gene on the corresponding
chromosome cannot compensate for the deletion because it has been turned off
by methylation, an epigenetic modification. Genetic deletions inherited from the
father result in Prader-Willi syndrome, and those inherited from the mother,
Angelman syndrome.
Immunity & Related Disorders. There are several pieces of evidence showing
that loss of epigenetic control over complex immune processes contributes to
autoimmune disease. Abnormal DNA methylation has been observed in patients
with lupus whose T cells exhibit decreased DNA methyltransferase activity and
hypomethylated DNA. Disregulation of this pathway apparently leads to
overexpression of methylation-sensitive genes such as the leukocyte function-
associated factor (LFA1), which causes lupus-like autoimmunity. Interestingly, LFA1
expression is also required for the development of arthritis, which raises the
possibility that altered DNA methylation patterns may contribute to other diseases
displaying idiopathic autoimmunity.
Neuropsychiatric Disorders. Epigenetic errors also play a role in the
causation of complex adult psychiatric, autistic, and neurodegenerative
disorders. Several reports have associated schizophrenia and mood disorders
with DNA rearrangements that include the DNMT genes. DNMT1 is selectively
overexpressed in gamma-aminobutyric acid (GABA)-ergic interneurons of
schizophrenic brains, whereas hypermethylation has been shown to repress
expression of Reelin (a protein required for normal neurotransmission,
memory formation and synaptic plasticity) in brain tissue from patients with
schizophrenia and patients with bipolar illness and psychosis. A role for
aberrant methylation mediated by folate levels has been suggested as a factor
in Alzheimer’s disease; also some preliminary evidence supports a model that
incorporates both genetic and epigenetic contributions in the causation of
autism. Autism has been linked to the region on chromosome 15 that is
responsible for Prader-Willi syndrome and Angelman syndrome. Findings at
autopsy of brain tissue from patients with autism have revealed a deficiency in
MECP2 expression that appears to account for reduced expression of several
relevant genes.
Pediatric Syndromes. In addition to epigenetic alterations, specific mutations
affecting components of the epigenetic pathway have been identified that are
responsible for several syndromes: DNMT3B in ICF (immunodeficiency, centromeric
instability and facial anomalies) syndrome, MECP2 in Rett syndrome, ATRX in ATR-X
syndrome (a-thalassemia/mental retardation syndrome, X-linked), and DNA repeats
in facioscapulohumeral muscular dystrophy. In Rett syndrome, for example, MECP2
encodes a protein that binds to methylated DNA; mutations in this protein cause
abnormal gene expression patterns within the first year of life. Girls with Rett
syndrome display reduced brain growth, loss of developmental milestones and
profound mental disabilities. Similarly, the ATR-X syndrome also includes severe
developmental deficiencies due to loss of ATRX, a protein involved in maintaining
the condensed, inactive state of DNA. Together, this constellation of clinical pediatric
syndromes is associated with alterations in genes and chromosomal regions
necessary for proper neurologic and physical development.
The increased knowledge and technologies in epigenetics over the last ten years
allow us to better understand the interplay between epigenetic change, gene
regulation, and human diseases, and will lead to the development of new
approaches for molecular diagnosis and targeted treatments across the clinical
spectrum.
MECHANISMS
DNA methylation
is an epigenetic mechanism that occurs by the addition of a methyl (CH3)
group to DNA, thereby often modifying the function of the genes. The most
widely characterized DNA methylation process is the covalent addition of the
methyl group at the 5-carbon of the cytosine ring resulting in 5-methylcytosine
(5-mC), also informally known as the “fifth base” of DNA. These methyl groups
project into the major groove of DNA and inhibit transcription. In human DNA,
5-methylcytosine is found in approximately 1.5% of genomic DNA.1 In somatic
cells, 5-mC occurs almost exclusively in the context of paired symmetrical
methylation of a CpG site, in which a cytosine nucleotide is located next to a
guanidine nucleotide. An exception to this is seen in embryonic stem (ES) cells,
where a substantial amount of 5-mC is also observed in non-CpG contexts. In
the bulk of genomic DNA, most CpG sites are heavily methylated while CpG
islands (sites of CpG clusters) in germ-line tissues and located near promoters
of normal somatic cells, remain unmethylated, thus allowing gene expression
to occur. When a CpG island in the promoter region of a gene is methylated,
In human DNA, 5-methylcytosine is found in approximately 1.5% of
genomic DNA.1 In somatic cells, 5-mC occurs almost exclusively in the
context of paired symmetrical methylation of a CpG site, in which a
cytosine nucleotide is located next to a guanidine nucleotide. An
exception to this is seen in embryonic stem (ES) cells, where a
substantial amount of 5-mC is also observed in non-CpG contexts. In
the bulk of genomic DNA, most CpG sites are heavily methylated while
CpG islands (sites of CpG clusters) in germ-line tissues and located near
promoters of normal somatic cells, remain unmethylated, thus allowing
gene expression to occur. When a CpG island in the promoter region of
a gene is methylated, expression of the gene is repressed (it is turned
off).
RNA TRANSCRIPTS
Sometimes a gene, after being turned on, transcribes a product that (directly or
indirectly) maintains the activity of that gene. For example, Hnf4 and MyoD enhance
the transcription of many liver- and muscle-specific genes, respectively, including
their own, through the transcription factor activity of the proteins they encode. RNA
signalling includes differential recruitment of a hierarchy of generic chromatin
modifying complexes and DNA methyltransferases to specific loci by RNAs during
differentiation and development.[60] Other epigenetic changes are mediated by the
production of different splice forms of RNA, or by formation of double-stranded RNA
(RNAi). Descendants of the cell in which the gene was turned on will inherit this
activity, even if the original stimulus for gene-activation is no longer present. These
genes are often turned on or off by signal transduction, although in some systems
where syncytia or gap junctions are important, RNA may spread directly to other
cells or nuclei by diffusion. A large amount of RNA and protein is contributed to
the zygote by the mother during oogenesis or via nurse cells, resulting in maternal
effect phenotypes. A smaller quantity of sperm RNA is transmitted from the father,
but there is recent evidence that this epigenetic information can lead to visible
changes in several generations of offspring
MICRORNAS
MicroRNAs (miRNAs) are members of non-coding RNAs that range in size from
17 to 25 nucleotides. miRNAs regulate a large variety of biological functions in
plants and animals.[62] So far, in 2013, about 2000 miRNAs have been
discovered in humans and these can be found online in a miRNA
database.[63] Each miRNA expressed in a cell may target about 100 to 200
messenger RNAs that it downregulates.[64] Most of the downregulation of
mRNAs occurs by causing the decay of the targeted mRNA, while some
downregulation occurs at the level of translation into protein.[65]
It appears that about 60% of human protein coding genes are regulated by
miRNAs.[66] Many miRNAs are epigenetically regulated. About 50% of miRNA
genes are associated with CpG islands,[62] that may be repressed by epigenetic
methylation. Transcription from methylated CpG islands is strongly and
heritably repressed.[67] Other miRNAs are epigenetically regulated by either
histone modifications or by combined DNA methylation and histone
modification.[
MRNA
In 2011, it was demonstrated that the methylation of mRNA plays a critical role
in human energy homeostasis. The obesity-associated FTO gene is shown to be
able to demethylate N6-methyladenosine in RNA
SRNA
sRNAs are small (50–250 nucleotides), highly structured, non-coding RNA fragments
found in bacteria. They control gene expression including virulence genes in
pathogens and are viewed as new targets in the fight against drug-resistant
bacteria.[70] They play an important role in many biological processes, binding to
mRNA and protein targets in prokaryotes. Their phylogenetic analyses, for example
through sRNA–mRNA target interactions or protein binding properties, are used to
build comprehensive databases.[71]sRNA-gene maps based on their targets in
microbial genomes are also constructed.
PRIONS
Prions are infectious forms of proteins. In general, proteins fold into discrete units that perform
distinct cellular functions, but some proteins are also capable of forming an infectious
conformational state known as a prion. Although often viewed in the context of infectious
disease, prions are more loosely defined by their ability to catalytically convert other native
state versions of the same protein to an infectious conformational state. It is in this latter sense
that they can be viewed as epigenetic agents capable of inducing a phenotypic change without
a modification of the genome.[73]
Fungal prions are considered by some to be epigenetic because the infectious phenotype
caused by the prion can be inherited without modification of the genome. PSI+ and URE3,
discovered in yeast in 1965 and 1971, are the two best studied of this type of prion.[74][75] Prions
can have a phenotypic effect through the sequestration of protein in aggregates, thereby
reducing that protein's activity. In PSI+ cells, the loss of the Sup35 protein (which is involved in
termination of translation) causes ribosomes to have a higher rate of read-through of
stop codons, an effect that results in suppression of nonsense mutations in other genes.[76] The
ability of Sup35 to form prions may be a conserved trait. It could confer an adaptive advantage
by giving cells the ability to switch into a PSI+ state and express dormant genetic features
normally terminated by stop codon mutations
STRUCTURAL INHERITANCE
In ciliates such as Tetrahymena and Paramecium, genetically
identical cells show heritable differences in the patterns of ciliary
rows on their cell surface. Experimentally altered patterns can be
transmitted to daughter cells. It seems existing structures act as
templates for new structures. The mechanisms of such
inheritance are unclear, but reasons exist to assume that
multicellular organisms also use existing cell structures to
assemble new ones
NUCLEOSOME POSITIONING
Eukaryotic genomes have numerous nucleosomes. Nucleosome position is not
random, and determine the accessibility of DNA to regulatory proteins. This
determines differences in gene expression and cell differentiation. It has been
shown that at least some nucleosomes are retained in sperm cells (where most
but not all histones are replaced by protamines). Thus nucleosome positioning
is to some degree inheritable. Recent studies have uncovered connections
between nucleosome positioning and other epigenetic factors, such as DNA
methylation and hydroxymethylation
MOLECULAR
TECHNIQUES
Biochem pt2
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Biochem pt2

  • 1. By : Matt Daniel M. Daep
  • 2.   Fatty acid synthesis is the creation of fatty acids from acetyl-CoA and NADPH through the action of enzymes called fatty acid synthases. This process takes place in the cytoplasm of the cell. Most of the acetyl-CoA which is converted into fatty acids is derived from carbohydrates via the glycolytic pathway. The glycolytic pathway also provides the glycerol with which three fatty acids can combine (by means of ester bonds) to form triglycerides (also known as "triacylglycerols", to distinguish them from fatty "acids" - or simply as "fat"), the final product of the lipogenic process What is fatty acid synthesis:
  • 3.  The input to fatty acid synthesis is acetyl- CoA, which is carboxylated to malonyl-CoA. The ATP-dependent carboxylation provides energy input. The CO2 is lost later during condensation with the growing fatty acid. The spontaneous decarboxylation drives the condensation. Acetyl-CoA Carboxylase catalyzes the 2-step reaction by which acetyl-CoA is carboxylated to form malonyl-CoA. As with other carboxylation reactions (e.g., Pyruvate Carboxylase), the enzyme prosthetic group is biotin.
  • 4.  Phospholipids are a class of lipids that are a major component of all cell membranes. They can form lipid bilayers because of their amphiphilic characteristic. The structure of the phospholipid molecule generally consists of two hydrophobic fatty acid "tails" and a hydrophilic "head" consisting of a phosphate group. The two components are joined together by a glycerol molecule. The phosphate groups can be modified with simple organic molecules such as choline.  The first phospholipid identified in 1847 as such in biological tissues was lecithin, or phosphatidylcholine, in the egg yolk of chickens by the French chemist and pharmacist, Theodore Nicolas Gobley. Biological membranes in eukaryotes also contain another class of lipid, sterol, interspersed among the phospholipids and together they provide membrane fluidity and mechanical strength. Purified phospholipids are produced commercially and have found applications in nanotechnology and materials science. What are phospholipids?
  • 5.   Phosphatidic Acid  Biosynthesis:  While quantitatively a minor component of membrane phospholipids, phosphatidic acid forms the backbone on which the synthesis of other phospholipid species and triacylglycerol is based. Biosynthesis
  • 6.
  • 7.  Phosphatidic acid synthesis begins with the addition of a fatty acyl-CoA, usually saturated, to glycerol 3- phosphate at the sn-1 position to produce lysophosphatidic acid. This reaction is catalyzed by glycerol 3-phosphate acyltransferase and is rate limiting for phosphatidic acid synthesis. There are two forms of this enzyme; one is found in the outer mitochondrial membrane, while the other is found in the endoplasmic reticulum. A second fatty acyl-CoA, often unsaturated, is added to lysophosphatidic acid at the sn-2 position by acylglycerol-3-acyltransferase to form phosphatidic acid. This occurs primarily in the endoplasmic reticulum.
  • 8.
  • 9.   Ketone bodies are three water-soluble molecules (acetoacetate, beta-hydroxybutyrate, and their spontaneous breakdown product, acetone) that are produced by the liver from fatty acids[1] during periods of low food intake (fasting), carbohydrate restrictive diets, starvation, prolonged intense exercise,[2] or in untreated (or inadequately treated) type 1 diabetes mellitus. These ketone bodies are readily picked up by the extra-hepatic tissues, and converted into acetyl-CoA which then enters the citric acid cycle and is oxidized in the mitochondria for energy.[3] In the brain, ketone bodies are also used to make acetyl-CoA into long-chain fatty acids. What are ketones?
  • 10.  Ketone bodies are produced by the liver under the circumstances listed above (i.e. fasting, starving, low carbohydrate diets, prolonged exercise and untreated type 1 diabetes mellitus) as a result of intense gluconeogenesis, which is the production of glucose from non-carbohydrate sources (not including fatty acids). They are therefore always released into the blood by the liver together with newly produced glucose, after the liver glycogen stores have been depleted. (These glycogen stores are depleted after only 24 hours of fasting.) Ketone synthesis
  • 11.   Isoprenoid quinones are one of the most important groups of compounds occurring in membranes of living organisms. These compounds are composed of a hydrophilic head group and an apolar isoprenoid side chain, giving the molecules a lipid-soluble character. Isoprenoid quinones function mainly as electron and proton carriers in photosynthetic and respiratory electron transport chains and these compounds show also additional functions, such as antioxidant function. Most of naturally occurring isoprenoid quinones belong to naphthoquinones or evolutionary younger benzoquinones. Isoprenoids
  • 12.  Isoprenoid quinones are membrane-bound compounds found in nearly all living organisms. The only exception presently known is some obligatory fermentative bacteria that lost the ability of synthesis of isoprenoid quinones and methanogenic Archea, belonging to Methanosarcinales . Isoprenoid quinones are composed of a polar head group and a hydrophobic side chain. The apolar isoprenoid side chain gives the molecules a lipid-soluble character and anchors them in membrane lipid bilayers, whereas the hydrophilic head group enables interaction with hydrophilic parts of proteins. It is generally accepted that long-chain, isoprenoid quinones localize in the hydrophobic mid-plane region of the lipid bilayer, whereas the polar head can oscillate between mid-plane and polar interphase of the membrane . The quinone ring can undergo two-step reversible reduction leading to quinol form . The reduced form of isoprenoid quinones is more polar and the quinol head group is thought to preferentially localize in polar, interphase region of membranes and. Synthesis
  • 13.   Sterols are constituents of the cellular membranes that are essential for their normal structure and function. In mammalian cells, cholesterol is the main sterol found in the various membranes. However, other sterols predominate in eukaryotic microorganisms such as fungi and protozoa. It is now well established that an important metabolic pathway in fungi and in members of the Trypanosomatidae family is one that produces a special class of sterols, including ergosterol, and other 24-methyl sterols, which are required for parasitic growth and viability, but are absent from mammalian host cells. What are sterols
  • 14.   Figure 1: Molecular structures of cholesterol and ergosterol. The arrows indicate the parts of the molecules which have been shown to be essential for the growth of mammalian cells (cholesterol), fungi, and trypanosomatids (ergosterol and 24-methyl sterols).
  • 16.   Figure 2: Schematic representation of main morphologies found during the life cycle of some members of the Trypanosomatidae family in the invertebrate host (insect) and vertebrate host (mammal).
  • 18.   Figure 3: The biosynthesis of ergosterol and cholesterol showing the main steps, the enzymes involved, and the known inhibitors.
  • 20.
  • 21.   A compound of the sterol type found in most body tissues. Cholesterol and its derivatives are important constituents of cell membranes and precursors of other steroid compounds, but a high proportion in the blood of low-density lipoprotein (which transports cholesterol to the tissues) is associated with an increased risk of coronary heart disease. What is cholesterol?
  • 22.   The amount of cholesterol that is synthesized in the liver is tightly regulated by dietary cholesterol levels. When dietary intake of cholesterol is high, synthesis is decreased and when dietary intake is low, synthesis is increased. However, cholesterol produced in other tissues is under no such feedback control. Cholesterol and similar oxysterols act as regulatory molecules to maintain healthy levels of cholesterol. Cholesterol regulation
  • 23.   The rate of synthesis of reductase mRNA is controlled by the sterol regulatory element binding protein (SREBP). This transcription factor binds to a short DNA sequence called the sterol regulatory element (SRE) on the 5′ side of the reductase gene. In its inactive state, the SREBP is anchored to the endoplasmic reticulum or nuclear membrane. When cholesterol levels fall, the amino-terminal domain is released from its association with the membrane by two specific proteolytic cleavages. The released protein migrates to the nucleus and binds the SRE of the HMG-CoA reductase gene, as well as several other genes in the cholesterol biosynthetic pathway, to enhance transcription. When cholesterol levels rise, the proteolytic release of the SREBP is blocked, and the SREBP in the nucleus is rapidly degraded. These two events halt the transcription of the genes of the cholesterol biosynthetic pathways.
  • 24.   any of a group of soluble proteins that combine with and transport fat or other lipids in the blood plasma. What are lipoproteins?
  • 25.   LPL actions within tissues are modulated at both the transcriptional and posttranscriptional levels. The latter might involve actions of the glycosylphosphatidylinositol HDL binding protein (GPIHBP) protein (19), angiopoietin-like proteins, which reduce LPL dimer formation (20), and the recently described lipase maturation factor (21). LPL regulation is tissue specific. LPL is present in the liver during fetal and early postnatal life but is then suppressed by a putative transcriptional regulatory mechanism, perhaps involving a novel transcription factor, termed RF-1-LPL, which binds to an NF-1-like site in the region of the glucocorticoid response element. This extinction of the hepatic expression of LPL is also under the influence of thyroid hormone and glucocorticoids.
  • 26.   Amino acid synthesis is the set of biochemical processes (metabolic pathways) by which the various amino acids are produced from other compounds. The substrates for these processes are various compounds in the organism's diet or growth media. Not all organisms are able to synthesise all amino acids. Humans are excellent example of this, since humans can only synthesise 11 of the 20 standard amino acids (aka non-essential amino acid), and in time of accelerated growth, histidine, can be considered an essential amino acid. What is amino acids synthesis?
  • 27.   Most amino acids are synthesized from α-ketoacids, and later transaminated from another amino acid, usually glutamate. The enzyme involved in this reaction is an aminotransferase. α-ketoacid + glutamate ⇄ amino acid + α-ketoglutarate Glutamate itself is formed by amination of α- ketoglutarate: α-ketoglutarate + NH+ 4 ⇄ glutamate
  • 28.   Porphyrins are a group of heterocyclic macrocycle organic compounds, composed of four modified pyrrole subunits interconnected at their α carbon atoms via methine bridges (=CH−). The parent porphyrin is porphin, and substituted porphines are called porphyrins. The porphyrin ring structure is aromatic, with a total of 26 electrons in the conjugated system. Various analyses indicate that not all atoms of the ring are involved equally in the conjugation or that the molecule's overall nature is substantially based on several smaller conjugated systems. One result of the large conjugated system is that porphyrin molecules typically have very intense absorption bands in the visible region and may be deeply colored; the name "porphyrin" comes from the Greek word πορφύρα (porphyra), meaning purple. Porphyrins
  • 30.
  • 31.   Nucleotides are small organic molecules consisting of a five ring sugar (which can be a ribose or a deoxyribose), a nitrogen base, and one to three phosphate groups. The most important function of nucleotides is there polymerization in nucleic acids such as DNA or RNA. As such they serve as the building blocks for the extremely long molecules that make up the chromosomes in every cell of our bodies, and that carry the genetic blueprint. The genetic information is carried out in DNA, and is organised in genes. Messenger RNA, is transcribed from the DNA whenever genes are expressed and carries the information from the cell nucleus to the cytoplasm, where it acts as a blueprint for protein synthesis. What are nucleotides?
  • 32.  Nucleotides are required for cell growth and replication A key enzyme for the synthesis of one nucleotide is dihydrofolate reductase. Cells grown in the presence of methotrexate, a reductase inhibitor, respond by increasing the number of copies of the reductase gene. The bright yellow regions visible on three of the chromosomes in the fluorescence micrograph (left), which were grown in the presence of methotrexate, contain hundreds of copies of the reductase gene Nucleotide synthesis
  • 33.
  • 34.
  • 37. DNA (or deoxyribonucleic acid) is the molecule that carries the genetic information in all cellular forms of life and some viruses. It belongs to a class of molecules called the nucleic acids, which are polynucleotides - that is, long chains of nucleotides. Each nucleotide consists of three components: ---a nitrogenous base: cytosine (C), guanine (G), adenine (A) or thymine (T) ---a five-carbon sugar molecule (deoxyribose in the case of DNA) ---a phosphate molecule
  • 38. The backbone of the polynucleotide is a chain of sugar and phosphate molecules. Each of the sugar groups in this sugar-phosphate backbone is linked to one of the four nitrogenous bases. Strand of polynucleotides Strand of polynucleotides
  • 39. DNA's ability to store - and transmit - information lies in the fact that it consists of two polynucleotide strands that twist around each other to form a double-stranded helix. The bases link across the two strands in a specific manner using hydrogen bonds: cytosine (C) pairs with guanine (G), and adenine (A) pairs with thymine (T). Double strand of polynucleotides
  • 40. The double helix of the complete DNA molecule resembles a spiral staircase, with two sugar phosphate backbones and the paired bases in the centre of the helix. This structure explains two of the most important properties of the molecule. First, it can be copied or 'replicated', as each strand can act as a template for the generation of the complementary strand. Second, it can store information in the linear sequence of the nucleotides along each strand.
  • 41. Chromosomes Eukaryotic chromosomes The label eukaryote is taken from the Greek for 'true nucleus', and eukaryotes (all organisms except viruses, Eubacteria and Archaea) are defined by the possession of a nucleus and other membrane-bound cell organelles. The nucleus of each cell in our bodies contains approximately 1.8 metres of DNA in total, although each strand is less than one millionth of a centimetre thick. This DNA is tightly packed into structures called chromosomes, which consist of long chains of DNA and associated proteins.
  • 42. In eukaryotes, DNA molecules are tightly wound around proteins - called histone proteins - which provide structural support and play a role in controlling the activities of the genes. A strand 150 to 200 nucleotides long is wrapped twice around a core of eight histone proteins to form a structure called a nucleosome. The histone octamer at the centre of the nucleosome is formed from two units each of histones H2A, H2B, H3, and H4. The chains of histones are coiled in turn to form asolenoid, which is stabilised by the histone H1. Further coiling of the solenoids forms the structure of the chromosome proper.
  • 43. Each chromosome has a p arm and a q arm. The p arm (from the French word 'petit', meaning small) is the short arm, and the q arm (the next letter in the alphabet) is the long arm. In their replicated form, each chromosome consists of two chromatids. Chromosome unraveling to show the base pairings of the DNA The chromosomes - and the DNA they contain - are copied as part of the cell cycle, and passed to daughter cells through the processes of mitosis and meiosis.
  • 44. Human beings have 46 chromosomes, consisting of 22 pairs of autosomes and a pair of sex chromosomes: two X sex chromosomes for females (XX) and an X and Y sex chromosome for males (XY). One member of each pair of chromosomes comes from the mother (through the egg cell); one member of each pair comes from the father (through the sperm cell). A photograph of the chromosomes in a cell is known as a karyotype. The autosomes are numbered 1-22 in decreasing size order. Karyotype of a human male
  • 45. The prokaryotes (Greek for 'before nucleus' - including Eubacteria and Archaea) lack a discrete nucleus, and the chromosomes of prokaryotic cells are not enclosed by a separate membrane. Prokaryotic chromosomes Most bacteria contain a single, circular chromosome. (There are exceptions: some bacteria - for example, the genus Streptomyces - possess linear chromosomes, and Vibrio cholerae, the causative agent of cholera, has two circular chromosomes.) The chromosome - together with ribosomes and proteins associated with gene expression - is located in a region of the cell cytoplasm known as the nucleoid.
  • 46. In addition to the main chromosome, bacteria are also characterised by the presence of extra-chromosomal genetic elements called plasmids. These relatively small circular DNA molecules usually contain genes that are not essential to growth or reproduction. In addition to the main chromosome, bacteria are also characterised by the presence of extra-chromosomal genetic elements called plasmids. These relatively small circular DNA molecules usually contain genes that are not essential to growth or reproduction. Retrieved from http://www2.le.ac.uk/departments/genetics/vgec/schoolscolleges/topics/dna -genes-chromosomes
  • 48.
  • 49. Every time a cell divides to produce new cells its DNA is copied. Each molecule of DNA undergoes semi-conservative replication. Put very simply, the DNA unwinds and unzips to expose nucleotide bases. DNA polymerases catalyse the addition of activated DNA nucleotides, according to complementary base-pairing rules, to make two new identical molecules of DNA, each one containing one old strand and one new strand. Hence each new molecule contains half of the original molecule Before DNA synthesis begins the original strands are separated and the synthesis of the daughter strands begins at the replication fork at a site called an origin of replication where a replisome is assembled from many proteins. The initiation complex that is formed attracts DNA polymerases. Synthesis of the new strands is called elongation and is aided by the proteins in the replisome.
  • 50. Lastly the termination site replicates Figure 1. The DNA replication fork. Because both daughter strands are synthesised in the 5’ to 3’ direction, the DNA complementary to the lagging strand is synthesised in small fragments called Okazaki fragments. These fragments are then joined together.
  • 51. Figure 2. Enzymes involved in DNA replication.
  • 52. The replisome The replisome consists of many proteins, including helicase, gyrase/ topoisomerase, primase, DNA polymerases, RNAse H and ligase. One DNA polymerase complex synthesises the lagging strand and another synthesises the leading strand. There are also factors, called replication proteins, that protect both the unstable single-stranded unwound leading and lagging strands from making hydrogen bonds with themselves and forming hairpins. Helicase causes the hydrogen bonds between complementary base pairs to break and so catalyses the separation of the two parental strands that will act as templates for synthesis of the daughter molecules. Helicase moves along the DNA in a 3’ to 5’ direction. Helicase
  • 53. Gyrase (a form of topoisomerase) unwinds the resulting supercoil that forms upstream of the section of unwound DNA. Gyrase DNA polymerases catalyse the elongation phase of replication. DNA polymerases Clamp proteins help keep the DNA polymerases attached to the leading and lagging strands and make sure the process proceeds at a suitably fast rate. Clamp proteins
  • 54. Priming In eukaryotic cells a DNA-dependent RNA polymerase creates an RNA primer, of about 10 bases long, on both the newly separated leading and lagging strands, once for the leading strand and once per Okazaki fragment (about 1000 base pairs long) on the lagging strand. The RNA primer attached to its DNA template is called A-form DNA. (Normal DNA is called B-form DNA.) In prokaryotes primase creates an RNA primer at the beginning of the newly separated leading and lagging strands. DNA polymerase enzymes cannot bind directly to single-stranded DNA and these primers provide a short chain of nucleotides that give the correct configuration to allow the active site of DNA polymerase to fit on and begin elongation.
  • 55. Elongation The leading and lagging strands are anti-parallel. In the leading strand nucleotide synthesis (catalysed by DNA polymerase epsilon in eukaryotes and by DNA polymerase III in prokaryotes) proceeds in the 5’ to 3’ direction (3’ to 5’ direction on the template strand) and makes a continuous complementary strand. Synthesis of the other strand in the opposite direction cannot occur at the same time so replication of the lagging strand is discontinuous. It involves making short discrete nucleotide chains, called Okazaki fragments, that are then joined by DNA repair enzymes, such as DNA polymerase I and ligase, so it is not made in one continuous strand. This can only happen once a sufficient length of DNA has been unwound so replication of this strand lags behind that of the leading strand.
  • 56. RNAse H enzymes remove the unstable RNA primers from the newly synthesised fragments and replace them with DNA fragments. DNA ligase (aided by polymerase I in prokaryotes) enzyme connects the Okazaki fragments, closing the gaps between their sugar-phosphate backbones by catalysing the formation of phosphodiester bonds. Proofreading enzymes correct any mistakes due to insertion of incorrect bases. Retrieved from http://www.contentextra.com/lifesciences/files/topicguides/Topic-guide-7.3- DNA-replication.pdf
  • 58.
  • 59. This rare albino alligator must have the specific "instructions," or DNA, to have this quality. The cause of albinism is a mutation in a gene for melanin, a protein found in skin and eyes. Such a mutation may result in no melanin production at all or a significant decline in the amount of melanin. What causes albinism?
  • 60. A change in the sequence of bases in DNA or RNA is called a mutation. Does the word mutation make you think of science fiction and bug-eyed monsters? Think again. Everyone has mutations. In fact, most people have dozens or even hundreds of mutations in their DNA. Mutations are essential for evolution to occur. They are the ultimate source of all new genetic material—new alleles in a species. Although most mutations have no effect on the organisms in which they occur, some mutations are beneficial. Even harmful mutations rarely cause drastic changes in organisms.
  • 61. Causes of Mutation Mutations have many possible causes. Some mutations seem to happen spontaneously without any outside influence. They occur when mistakes are made during DNA replication or transcription. Other mutations are caused by environmental factors. Anything in the environment that can cause a mutation is known as a mutagen. Examples of mutagens are pictured in Figure 1.
  • 62. Figure 1 Examples of Mutagens. Types of mutagens include radiation, chemicals, and infectious agents. Do you know of other examples of each type of mutagen shown here?
  • 63. Types of Mutations There are a variety of types of mutations. Two major categories of mutations are germline mutations and somatic mutations. • Germline mutations occur in gametes. These mutations are especially significant because they can be transmitted to offspring and every cell in the offspring will have the mutation. • Somatic mutations occur in other cells of the body. These mutations may have little effect on the organism because they are confined to just one cell and its daughter cells. Somatic mutations cannot be passed on to offspring. Mutations also differ in the way that the genetic material is changed. Mutations may change the structure of a chromosome or just change a single nucleotide.
  • 64. What does radiation contamination do? It mutates DNA. The Chernobyl disaster was a nuclear accident that occurred on April 26, 1986. It is considered the worst nuclear power plant accident in history. A Russian publication concludes that 985,000 excess cancers occurred between 1986 and 2004 as a result of radioactive contamination. The 2011 report of the European Committee on Radiation Risk calculates a total of 1.4 million excess cancers occurred as a result of this contamination.
  • 65. Chromosomal Alterations Chromosomal alterations are mutations that change chromosome structure. They occur when a section of a chromosome breaks off and rejoins incorrectly or does not rejoin at all. Possible ways these mutations can occur are illustrated in Figure 2.
  • 66. Figure 2 Chromosomal Alterations. Chromosomal alterations are major changes in the genetic material.
  • 67. Point Mutations Mutations A point mutation is a change in a single nucleotide in DNA. This type of mutation is usually less serious than a chromosomal alteration. An example of a point mutation is a mutation that changes the codon UUU to the codon UCU. Point mutations can be silent, missense, or nonsense mutations, as shown in Table 1. The effects of point mutations depend on how they change the genetic code. Table 1: Point Mutations and Their Effects
  • 68. Frameshift Mutations A frameshift mutation is a deletion or insertion of one or more nucleotides that changes the reading frame of the base sequence. Deletions remove nucleotides, and insertions add nucleotides. Consider the following sequence of bases in RNA: AUG-AAU-ACG-GCU = start- asparagine-threoninealanine Now, assume an insertion occurs in this sequence. Let’s say an A nucleotide is inserted after the start codon AUG: AUG-AAA-UAC-GGC-U = start-lysine-tyrosine-glycine Even though the rest of the sequence is unchanged, this insertion changes the reading frame and thus all of the codons that follow it. As this example shows, a frameshift mutation can dramatically change how the codons in mRNA are read. This can have a drastic effect on the protein product.
  • 69. Spontaneous Mutations There are five common types of spontaneous mutations. These are described in the Table 2 below. Table 7.6: Spontaneous Mutations Described
  • 70. Effects of Mutations The majority of mutations have neither negative nor positive effects on the organism in which they occur. These mutations are called neutral mutations. Examples include silent point mutations. They are neutral because they do not change the amino acids in the proteins they encode. Many other mutations have no effect on the organism because they are repaired before protein synthesis occurs. Cells have multiple repair mechanisms to fix mutations in DNA. One way DNA can be repaired is illustrated in Figure 3. If a cell’s DNA is permanently damaged and cannot be repaired, the cell is likely to be prevented from dividing.
  • 71. Figure 3: DNA Repair Pathway. This flow chart shows one way that damaged DNA is repaired in E. coli bacteria.
  • 72. Is this rat hairless? Yes. Why? The result of a mutation, a change in the DNA sequence. The effects of mutations can vary widely, from being beneficial, to having no effect, to having lethal consequences, and every possibility in between.
  • 73. Beneficial Mutations Some mutations have a positive effect on the organism in which they occur. They are called beneficial mutations. They lead to new versions of proteins that help organisms adapt to changes in their environment. Beneficial mutations are essential for evolution to occur. They increase an organism’s changes of surviving or reproducing, so they are likely to become more common over time. There are several well-known examples of beneficial mutations. Here are just two: 1. Mutations in many bacteria that allow them to survive in the presence of antibiotic drugs. The mutations lead to antibiotic- resistant strains of bacteria. 2. A unique mutation is found in people in a small town in Italy. The mutation protects them from developing atherosclerosis, which is the dangerous buildup of fatty materials in blood vessels. The individual in which the mutation first appeared has even been identified.
  • 74. Harmful Mutations Imagine making a random change in a complicated machine such as a car engine. The chance that the random change would improve the functioning of the car is very small. The change is far more likely to result in a car that does not run well or perhaps does not run at all. By the same token, any random change in a gene’s DNA is likely to result in a protein that does not function normally or may not function at all. Such mutations are likely to be harmful. Harmful mutations may cause genetic disorders or cancer.
  • 75. • Cancer is a disease in which cells grow out of control and form abnormal masses of cells. It is generally caused by mutations in genes that regulate the cell cycle. Because of the mutations, cells with damaged DNA are allowed to divide without limits. Cancer genes can be inherited. • A genetic disorder is a disease caused by a mutation in one or a few genes. A human example is cystic fibrosis. A mutation in a single gene causes the body to produce thick, sticky mucus that clogs the lungs and blocks ducts in digestive organs.
  • 76. Genetic Disorders Many genetic disorders are caused by mutations in one or a few genes. Other genetic disorders are caused by abnormal numbers of chromosomes. Genetic Disorders Caused by Mutations Table 3 lists several genetic disorders caused by mutations in just one gene. Some of the disorders are caused by mutations in autosomal genes, others by mutations in X-linked genes. Which disorder would you expect to be more common in males than females?
  • 77. Table 3: Genetic Disorders Caused by Mutations in One Gene
  • 78. Few genetic disorders are controlled by dominant alleles. A mutant dominant allele is expressed in every individual who inherits even one copy of it. If it causes a serious disorder, affected people may die young and fail to reproduce. Therefore, the mutant dominant allele is likely to die out of the population. A mutant recessive allele, such as the allele that causes sickle cell anemia (see Figure 7.43), is not expressed in people who inherit just one copy of it. These people are called carriers. They do not have the disorder themselves, but they carry the mutant allele and can pass it to their offspring. Thus, the allele is likely to pass on to the next generation rather than die out. Figure 5 Sickle-Shaped and Normal Red Blood Cells. Sickle cell anemia is an autosomal recessive disorder. The mutation that causes the disorder affects just one amino acid in a single protein, but it has serious consequences for the affected person. This photo shows the sickle shape of red blood cells in people with sickle cell anemia.
  • 79. Chromosomal Disorders Mistakes may occur during meiosis that result in nondisjunction. This is the failure of replicated chromosomes to separate during meiosis (the animation at the link below shows how this happens). Some of the resulting gametes will be missing a chromosome, while others will have an extra copy of the chromosome. If such gametes are fertilized and form zygotes, they usually do not survive. If they do survive, the individuals are likely to have serious genetic disorders. Table 4 lists several genetic disorders that are caused by abnormal numbers of chromosomes. Figure 7.44 shows a karyotype for trisomy 21 or Down’s Syndrome. Most chromosomal disorders involve the X chromosome. Look back at the X and Y chromosomes and you will see why. The X and Y chromosomes are very different in size, so nondisjunction of the sex chromosomes occurs relatively often.
  • 80. Table 4: Genetic Disorders Caused by Abnormal Number of Chromosomes
  • 81. Figure 6 Trisomy 21 (Down Syndrome) Karyotype. A karyotype is a picture of a cell's chromosomes. Note the extra chromosome 21. (right) Child with Down syndrome, exhibiting characteristic facial appearance.
  • 82. Diagnosing Genetic Disorders A genetic disorder that is caused by a mutation can be inherited. Therefore, people with a genetic disorder in their family may be concerned about having children with the disorder. Professionals known as genetic counselors can help them understand the risks of their children being affected. If they decide to have children, they may be advised to have prenatal (“before birth”) testing to see if the fetus has any genetic abnormalities. One method of prenatal testing is amniocentesis. In this procedure, a few fetal cells are extracted from the fluid surrounding the fetus, and the fetal chromosomes are examined.
  • 83. Treating Genetic Disorders The symptoms of genetic disorders can sometimes be treated, but cures for genetic disorders are still in the early stages of development. One potential cure that has already been used with some success is gene therapy. This involves inserting normal genes into cells with mutant genes.
  • 84. • Mutations are caused by environmental factors known as mutagens. Types of mutagens include radiation, chemicals, and infectious agents. • Germline mutations occur in gametes. Somatic mutations occur in other body cells. Chromosomal alterations are mutations that change chromosome structure. Point mutations change a single nucleotide. Frameshift mutations are additions or deletions of nucleotides that cause a shift in the reading frame. • Mutations are essential for evolution to occur because they increase genetic variation and the potential for individuals to differ. The majority of mutations are neutral in their effects on the organisms in which they occur. Beneficial mutations may become more common through natural selection. Harmful mutations may cause genetic disorders or cancer. • Many genetic disorders are caused by mutations in one or a few genes. • Other genetic disorders are caused by abnormal numbers of chromosomes. Summary
  • 87.
  • 88. To transcribe means "to paraphrase or summarize in writing." The information in DNA is transcribed - or summarized - into a smaller version - RNA - that can be used by the cell. This process is called transcription. Transcription is the first part of the central dogma of molecular biology: DNA → RNA. It is the transfer of genetic instructions in DNA to mRNA. During transcription, a strand of mRNA is made that is complementary to a strand of DNA. Figure 1 shows how this occurs. Figure 1 Overview of Transcription. Transcription uses the sequence of bases in a strand of DNA to make a complementary strand of mRNA. Triplets are groups of three successive nucleotide bases in DNA. Codons are complementary groups of bases in mRNA.
  • 89. Steps of Transcription Transcription takes place in three steps: initiation, elongation, and termination. The steps are illustrated in Figure 2. 1. Initiation is the beginning of transcription. It occurs when the enzyme RNA polymerase binds to a region of a gene called the promoter. This signals the DNA to unwind so the enzyme can ‘‘read” the bases in one of the DNA strands. The enzyme is ready to make a strand of mRNA with a complementary sequence of bases. 2. Elongation is the addition of nucleotides to the mRNA strand. 3. Termination is the ending of transcription, and occurs when RNA polymerase crosses a stop (termination) sequence in the gene. The mRNA strand is complete, and it detaches from DNA.
  • 90. Figure 2. Steps of Transcription. Transcription occurs in the three steps - initiation, elongation, and termination - shown here.
  • 93. • In prokaryotes, no RNA processing is necessary: – the nascent RNA is usually the mRNA. • In eukaryotes, the nascent RNA is called primary transcript-RNA – needs to be processed – and transported to the cytoplasm for translation to occur.
  • 94. Fig. 1 Processes for synthesis of functional mRNA in prokaryotes and eukaryotes
  • 95. Splicing removes introns from mRNA (see Figure 2). Introns are regions that do not code for proteins. The remaining mRNA consists only of regions that do code for proteins, which are called exons. Ribonucleoproteins are nucleoproteins that contain RNA. Small nuclear ribonuclearproteins are involved in pre-mRNA splicing. Figure 2 Splicing. Splicing removes introns from mRNA. UTR is an untranslated region of the mRNA.
  • 96. Editing changes some of the nucleotides in mRNA. For example, the human protein called APOB, which helps transport lipids in the blood, has two different forms because of editing. One form is smaller than the other because editing adds a premature stop signal in mRNA. Polyadenylation adds a “tail” to the mRNA. The tail consists of a string of As (adenine bases). It signals the end of mRNA. It is also involved in exporting mRNA from the nucleus. In addition, the tail protects mRNA from enzymes that might break it down.
  • 97. Figure 3 The ends of eukaryotic mRNAs
  • 98. Questions • Why is the mRNA not equal in length to the DNA it was transcribed from? – 1) the mRNA was longer because it has a Poly A tail – 2) The mRNA was longer because it contains only introns – 3) The DNA was shorter because it does not have the Methylated cap – 4) The mRNA was shorter because of Intron splicing • Which nucleotides signal the 5’ end of an intron splice site? – 1. AT – 2. GU – 3. AG – 4. GG
  • 100. Translation Translation is the second part of the central dogma of molecular biology: RNA → Protein. It is the process in which the genetic code in mRNA is read to make a protein. Figure 1 shows how this happens. After mRNA leaves the nucleus, it moves to a ribosome, which consists of rRNA and proteins. The ribosome reads the sequence of codons in mRNA. Molecules of tRNA bring amino acids to the ribosome in the correct sequence.
  • 101. To understand the role of tRNA, you need to know more about its structure. Each tRNA molecule has an anticodon for the amino acid it carries. An anticodon is complementary to the codon for an amino acid. For example, the amino acid lysine has the codon AAG, so the anticodon is UUC. Therefore, lysine would be carried by a tRNA molecule with the anticodon UUC. Wherever the codon AAG appears in mRNA, a UUC anticodon of tRNA temporarily binds. While bound to mRNA, tRNA gives up its amino acid. Bonds form between the amino acids as they are brought one by one to the ribosome, forming a polypeptide chain. The chain of amino acids keeps growing until a stop codon is reached. To see how this happens, go the link below.
  • 102.
  • 103. Figure 1: Translation. Translation of the codons in mRNA to a chain of amino acids occurs at a ribosome. Find the different types of RNA in the diagram. What are their roles in translation?
  • 104. After a polypeptide chain is synthesized, it may undergo additional processes. For example, it may assume a folded shape due to interactions among its amino acids. It may also bind with other polypeptides or with different types of molecules, such as lipids or carbohydrates. Many proteins travel to the Golgi apparatus to be modified for the specific job they will do. Source: www.ck-12.org
  • 106. Gene regulation is a label for the cellular processes that control the rate and manner of gene expression. A complex set of interactions between genes, RNA molecules, proteins (including transcription factors) and other components of the expression system determine when and where specific genes are activated and the amount of protein or RNA product produced. Often, one gene regulator controls another, and so on, in a gene regulatory network. Gene regulation is essential for viruses, prokaryotes and eukaryotes as it increases the versatility and adaptability of WHAT IS GENE REGULATION?
  • 107. Although as early as 1951, Barbara McClintock showed interaction between two genetic loci, Activator (Ac) and Dissociator (Ds), in the color formation of maize seeds, the first discovery of a gene regulation system is widely considered to be the identification in 1961 of the lac operon, discovered by Jacques Monod, in which some enzymes involved in lactose metabolism are expressed by E. coli only in the presence of lactose and absence of glucose.
  • 108. Any step of gene expression may be modulated, from the DNA- RNA transcription step to post-translational modification of a protein. The following is a list of stages where gene expression is regulated, the most extensively utilized point is Transcription Initiation: • Chromatin domains • Transcription • Post-transcriptional modification • RNA transport • Translation • mRNA degradation
  • 109. WHY IS GENE EXPRESSION REGULATED? Genes can't control an organism on their own; rather, they must interact with and respond to the organism's environment. Some genes are constitutive, or always "on," regardless of environmental conditions. Such genes are among the most important elements of a cell's genome, and they control the ability of DNA to replicate, express itself, and repair itself. These genes also control protein synthesis and much of an organism's central metabolism. It turns out that the regulation of such genes differs between PROKARYOTES and EUKARYOTES. For prokaryotes, most regulatory proteins are negative and
  • 110.
  • 111. PROKARYOTIC GENE REGULATION For prokaryotes, most regulatory proteins are negative and therefore turn genes off. Here, the cells rely on protein–small molecule binding, in which a ligand or small molecule signals the state of the cell and whether gene expression is needed. The repressor or activator protein binds near its regulatory target: the gene. Some regulatory proteins must have a ligand attached to them to be able to bind, whereas others are unable to bind when attached to a ligand. In prokaryotes, most regulatory proteins are specific to one gene, although there are a few proteins that act more widely.
  • 112. Furthermore, some repressors have a fine- tuning system known as attenuation, which uses mRNA structure to stop both transcription and translation depending on the concentration of an operon's end-product enzymes. (In eukaryotes, there is no exact equivalent of attenuation, because transcription occurs in the nucleus and translation occurs in the cytoplasm, making this sort of coordinated effect impossible.) Yet another layer of prokaryotic regulation affects the structure of RNA polymerase, which turns on large groups of genes. Here, the sigma factor of RNA polymerase changes several times to produce heat- and
  • 114. EUKARYOTIC GENE REGULATION Unlike prokaryotes, multiple gene-regulating mechanisms operate in the nucleus before and after RNA transcription, and in the cytoplasm both before and after translation. Histones are small proteins packed inside the molecular structure of the DNA double helix. Tight histone packing prevents RNA polymerase from contacting and transcribing the DNA. This type of overall control of protein synthesis is regulated by genes that control the packing density of histones. X- chromosome inactivation occurs when dense packing of the X chromosome in females totally prevents its function even in interphase. This type of inactivation is
  • 115.
  • 116. Activator-enhancer complex is unique in eukaryotes because they normally have to be activated to begin protein synthesis, which requires the use of transcription factors and RNA polymerase. In general, the process of eukaryotic protein synthesis involves four steps: 1. Activators, a special type of transcription factor, bind to enhancers, which are discrete DNA units located at varying points along the chromosome. 2. The activator-enhancer complex bends the DNA molecule so that additional transcription factors have better access to bonding sites on the operator. 3. The bonding of additional transcription factors to the operator allows greater access by
  • 117.
  • 118. Eukaryotic gene expression involves many steps, and almost all of them can be regulated. Different genes are regulated at different points, and it’s not uncommon for a gene (particularly an important or powerful one) to be regulated at multiple steps. • Chromatin accessibility. The structure of chromatin (DNA and its organizing proteins) can be regulated. More open or “relaxed” chromatin makes a gene more available for transcription. • Transcription. Transcription is a key regulatory point for many genes. Sets of transcription factor proteins bind to specific DNA sequences in or near a gene and promote or repress its transcription into an RNA. • RNA processing. Splicing, capping, and
  • 119. • RNA stability. The lifetime of an mRNA molecule in the cytosol affects how many proteins can be made from it. Small regulatory RNAs called miRNAs can bind to target mRNAs and cause them to be chopped up. • Translation. Translation of an mRNA may be increased or inhibited by regulators. For instance, miRNAs sometimes block translation of their target mRNAs (rather than causing them to be chopped up). • Protein activity. Proteins can undergo a variety of modifications, such as being chopped up or tagged with chemical groups. These modifications can be
  • 120.
  • 121. The processing and packaging of RNA both in the nucleus and cytoplasm provides two more opportunities for gene regulation to occur after transcription but before translation. Adding extra nucleotides as a protective cap and tail to the RNA identifies the RNA as an mRNA by the ribosomes, and prevents degradation by cell enzymes as it moves from the nucleus into the cytoplasm. RNA splicing occurs when “gaps” of nonprotein- code-carrying nucleotides called interons are removed from the code-carrying nucleotides, called exons, which are then connected to shorten the RNA molecule for conversion into tRNA and rRNA. The number of interons regulates the speed at which the RNA can be processed.
  • 122. The longevity of the individual mRNA molecule determines how many times it can be used and reused to create proteins. In eukaryotes, the mRNA tends to be stable, which means it can be used multiple times; which is efficient, but it prevents eukaryotes from making rapid response changes to environmental disruptions. The mRNA of prokaryotes is unstable, allowing for the creation of new mRNA, which has more opportunities to adjust for changing environmental conditions. Inhibitory proteins prevent the translation of mRNA. They are made inactive when bonded with the substance for which they are trying to block production. Post-translation control involves the selective cutting and breakdown of proteins that prevent
  • 123. EXAMPLES OF GENE REGULATION • Enzyme induction is a process in which a molecule (e.g., a drug) induces (i.e., initiates or enhances) the expression of an enzyme. • The Lac operon is an interesting example of how gene expression can be regulated. Viruses, despite having only a few genes, possess mechanisms to regulate their gene expression, typically into an early and late phase, using collinear systems regulated by anti-terminators (lambda phage) or splicing modulators (HIV). • GAL4 is a transcriptional activator that controls the expression of GAL1, GAL7, and GAL10 (all of which code for the metabolic of galactose in yeast). The GAL4/UAS
  • 124. ENZYME INDUCTION • Enzyme induction is a process in which a molecule (e.g. a drug) induces (i.e. initiates or enhances) the expression of an enzyme. An enzyme inducer is a type of drug which binds to an enzyme and increases its metabolic activity. Many of the enzymes involved in drug metabolism may be up-regulated by exposure to drugs and environmental chemicals leading to increased rates of metabolism. This phenomenon is known as enzyme induction. Enzyme induction is a process where production of an enzyme is triggered or increased in response to changes in the environment that surrounds an individual You do not have access to view this node. The
  • 126. WHAT IS ENZYME REPRESSION? Induction and repression are linked in that they both focus on the binding of a molecule known as RNA polymerase to DNA. Particularly, the RNA polymerase binds to a region that is immediately "upstream" from the region of DNA that code for a protein. The binding region is termed the ‘operator’. The operator acts to position the polymerase correctly, so that the molecule can then begin to move along the DNA, interpreting the genetic information as it moves along. The three-dimensional shape of the operator region manipulates the
  • 127. WHAT IS THE PROCESS OF ENZYME INDUCTION? Enzyme induction is a process where an enzyme is contrived in response to the presence of a specific molecule. This molecule is termed an inducer. Basically, an inducer molecule is a compound that the enzyme acts upon. In the induction process, the inducer molecule merges with another molecule, which is called the ‘repressor’ (a chemical compound that is designed to limit or prevent enzyme production, so there are no obstacles to enzyme production). The binding of the inducer to the repressor obstructs the function of the repressor, which is to bind to a specific region called an ‘operator’. The operator is the site to which another molecule, known as ribonucleic acid (RNA) polymerase, binds and begins the transcription (transfer of genetic information
  • 128. Thus, the binding of the inducer to the repressor keeps the repressor from averting transcription, and so the gene coding for the inducible enzyme is transcribed. Repression of transcription is basically the default behavior, which is dominated once the inducing molecule is present. In bacteria, the lactose (lac) operon is a very well characterized system that operates on the basis of induction. An operon is a single unit of physically adjacent genes that function together under the control of a single operator gene.
  • 129.
  • 130. LAC OPERON The lac operon (lactose operon) is an operon required for the transport and metabolism of lactose in Escherichia coli and many other enteric bacteria. Although glucose is the preferred carbon source for most bacteria, the lac operon allows for the effective digestion of lactose when glucose is not available. Bacterial operons are polycistronic transcripts that are able to produce multiple proteins from one mRNA transcript. In this case, when lactose is required as a sugar source for the bacterium, the three genes of the lac operon can be expressed and their subsequent proteins translated: lacZ, lacY, and lacA. The gene product of lacZ is β- galactosidase which cleaves lactose, a
  • 131. It would be wasteful to produce the enzymes when there is no lactose available or if there is a more preferable energy source available, such as glucose. The lac operon uses a two- part control mechanism to ensure that the cell expends energy producing the enzymes encoded by the lac operon only when necessary. In the absence of lactose, the lac repressor halts production of the enzymes encoded by the lac operon. In the presence of glucose, the catabolite activator protein (CAP), required for production of the enzymes, remains inactive, and EIIAGlc shuts down lactose permease to prevent transport of lactose into the cell. This dual control mechanism causes the sequential utilization of glucose and lactose in two distinct growth phases, known as diauxie.
  • 132.
  • 133. STRUCTURE OF LAC OPERON The lac operon contains three genes: lacZ, lacY, and lacA. These genes are transcribed as a single mRNA, under control of one promoter. Genes in the lac operon specify proteins that help the cell utilize lactose. lacZ encodes an enzyme that splits lactose into monosaccharides (single-unit sugars) that can be fed into glycolysis. Similarly, lacY encodes a membrane-embedded transporter that helps bring lactose into the cell. In addition to the three genes, the lac operon also contains a number of regulatory DNA sequences. These are regions of DNA to which
  • 134. • The promoter is the binding site for RNA polymerase, the enzyme that performs transcription. • The operator is a negative regulatory site bound by the lac repressor protein. The operator overlaps with the promoter, and when the lac repressor is bound, RNA polymerase cannot bind to the promoter and start transcription. • The CAP binding site is a positive regulatory site that is bound by catabolite activator protein (CAP). When CAP is bound to this site, it promotes transcription by helping RNA polymerase bind to the promoter.
  • 135. STRUCTURE OF LAC OPERON
  • 136. THE LAC REPRESSOR • The lac repressor is a protein that represses (inhibits) transcription of the lac operon. It does this by binding to the operator, which partially overlaps with the promoter. When bound, the lac repressor gets in RNA polymerase's way and keeps it from transcribing the operon. • When lactose is not available, the lac repressor binds tightly to the operator, preventing transcription by RNA polymerase. However, when lactose is present, the lac repressor loses its ability to bind DNA. It floats off the operator, clearing the way for RNA polymerase to
  • 137. This change in the lac repressor is caused by the small molecule allolactose, an isomer (rearranged version) of lactose. When lactose is available, some molecules will be converted to allolactose inside the cell. Allolactose binds to the lac repressor and makes it change shape so it can no longer bind DNA. Allolactose is an example of an inducer, a small molecule that triggers expression of a gene or operon. The lac operon is considered an inducible
  • 138. CATABOLITE ACTIVATOR PROTEIN(CAP) When lactose is present, the lac repressor loses its DNA-binding ability. This clears the way for RNA polymerase to bind to the promoter and transcribe the lac operon. As it turns out, RNA polymerase alone does not bind very well to the lac operon promoter. It might make a few transcripts, but it won't do much more unless it gets extra help from catabolite activator protein (CAP). CAP binds to a region of DNA just before the lac operon promoter and helps RNA polymerase attach to the promoter, driving high levels of transcription.
  • 139. CAP isn't always active (able to bind DNA). Instead, it's regulated by a small molecule called cyclic AMP (cAMP). cAMP is a "hunger signal" made by E. coli when glucose levels are low. cAMP binds to CAP, changing its shape and making it able to bind DNA and promote transcription. Without cAMP, CAP cannot bind DNA and is inactive. CAP is only active when glucose levels are low (cAMP levels are high). Thus, the lac operon can only be transcribed at high levels when glucose is absent. This strategy
  • 140. Glucose present, lactose absent: No transcription of the lac operon occurs. That's because the lac repressor remains bound to the operator and prevents transcription by RNA polymerase. Also, cAMP levels are low because glucose levels are high, so CAP is inactive and cannot bind DNA.
  • 141. Glucose present, lactose present: Low-level transcription of the lac operon occurs. The lac repressor is released from the operator because the inducer (allolactose) is present. cAMP levels, however, are low because glucose is present. Thus, CAP remains inactive and cannot
  • 142. Glucose absent, lactose absent: No transcription of the lac operon occurs. cAMP levels are high because glucose levels are low, so CAP is active and will be bound to the DNA. However, the lac repressor will also be bound to the operator (due to the absence of
  • 143. Glucose absent, lactose present: Strong transcription of the lac operon occurs. The lac repressor is released from the operator because the inducer (allolactose) is present. cAMP levels are high because glucose is absent, so CAP is active and bound to the DNA.
  • 144. VIRAL REPLICATIONReplication of viruses primarily involves the multiplication of the viral genome. Replication also involves synthesis of viral messenger RNA (mRNA) from "early" genes (with exceptions for positive sense RNA viruses), viral protein synthesis, possible assembly of viral proteins, then viral genome replication mediated by early or regulatory protein expression. This may be followed, for complex viruses with larger genomes, by one or more further rounds of mRNA synthesis: "late" gene expression is, in general, necessary for structural or virion proteins. Viral replication usually takes place in the cytoplasm.
  • 145. SCHEMATIC SHOWING ANTISENSE DNA STRANDS CAN INTERFERE WITH PROTEIN TRANSLATION
  • 146. Viruses that replicate via RNA intermediates need an RNA-dependent RNA-polymerase to replicate their RNA, but animal cells do not seem to possess a suitable enzyme. Therefore, this type of animal RNA virus needs to code for an RNA-dependent RNA polymerase. No viral proteins can be made until viral messenger RNA is available; thus, the nature of the RNA in the virion affects the strategy of the virus: In plus- stranded RNA viruses, the virion (genomic) RNA is the same sense as mRNA and so functions as mRNA. This mRNA can be translated immediately upon infection of the host cell . Examples: poliovirus (picornavirus), togaviruses, and flaviviruses.
  • 147. Viral gene expression regulation refers to any of the processes by which cytoplasmic factors influence the differential control of gene action in viruses. The interplay of the viral genome with the host metabolic machinery involves modifications in both gene expression and regulation. Retroviruses have adapted themselves to use this machinery while maintaining the cell integrity, which is essential to preserve their survival. Consequently, there can be variable host pathogenicity associated with several diseases such as malignancies, immunodeficiencies, and neurological disorders. This book describes current research in the field, and gives a better
  • 148. GAL4 SYSTEM The GAL4-UAS system is a biochemical method used to study gene expression and function in organisms such as the fruit fly. It has also been adapted to study receptor chemical-binding functions in vitro in cell culture. It was developed by Andrea Brand and Norbert Perrimon in 1993 and is considered a powerful technique for studying the expression of genes. The system has two parts: the GAL4 gene, encoding the yeast transcription activator protein GAL4, and the UAS (Upstream Activation Sequence), an enhancer
  • 149. The GAL4 system allows separation of the problems of defining which cells express a gene or protein and what the experimenter wants to do with this knowledge. Geneticists have created genetic varieties of model organisms (typically fruit flies), called GAL4 lines, each of which expresses GAL4 in some subset of the animal's tissues. For example, some lines might express GAL4 only in muscle cells, or only in nerves, or only in the antennae, and so on. For fruit flies in particular, there are tens of thousands of such lines, with the most useful expressing GAL4 in only a very specific subset of the animal— perhaps, for example, only those neurons that connect two specific compartments of the fly's brain. The presence of GAL4, by itself, in
  • 150. Since GAL4 by itself is not visible, and has little effect on cells, the other necessary part of this system are the "reporter lines". These are strains of flies with the special UAS region next to a desired gene. These genetic instructions occur in every cell of the animal, but in most cells nothing happens since that cell is not producing GAL4. In the cells that are producing GAL4, however, the UAS is activated, the gene next to it is turned on, and it starts producing its resulting protein. This may report to the investigator which cells are expressing GAL4, hence the term "reporter line", but genes intended to manipulate the cell behavior are often used as well.
  • 151. Typical reporter genes include: • Fluorescent proteins like green (GFP) or red fluorescent proteins (RFP), which allow scientists to see which cells express GAL4 • Channelrhodopsin, which allows light-sensitive triggering of nerve cells • Halorhodopsin, which conversely allows light to suppress the firing of neurons • Shibire, which shuts neurons off, but only at higher temperatures (30 °C and above). Flies with this gene can be raised and tested at lower temperatures where their neurons will behave normally. Then the body temperature of the flies can be raised (since they are cold-blooded), and these neurons turn off.[3] If the fly's behavior changes, this gives a strong clue to what those neurons do. • GECI (Genetically Encoded Calcium Indicator), often a member of the GCaMP family of proteins. These proteins glow when exposed to calcium, which, in
  • 152.
  • 154. RNA serves a multitude of functions within cells. These functions are primarily involved in converting the genetic information contained in a cell's DNA into the proteins that determine the cell's structure and function. All RNAs are originally transcribed from DNA by RNA polymerases, which are specialized enzyme complexes, but most RNAs must be further modified or processed before they can carry out their roles. Thus, RNA processing refers to any modification made to RNA between its transcription and its final function in the cell. These processing steps include the removal of extra sections of RNA, specific modifications of RNA bases, and
  • 155. TYPES OF RNA • There are different types of RNA, each of which plays a specific role, including specifying the amino acid sequence of proteins (performed by messenger RNAs, or mRNAs), organizing and catalyzing the synthesis of proteins (ribosomal RNAs or rRNAs), translating codons in the mRNA into amino acids (transfer RNAs or tRNAs) and directing many of the RNA processing steps (performed by small RNAs in the nucleus, called snRNAs and snoRNAs).
  • 156. All of these types of RNAs begin as primary transcripts copied from DNA by one of the RNA polymerases. One of the features that separates eukaryotes and prokaryotes is that eukaryotes isolate their DNA inside a nucleus while protein synthesis takes place in the cytoplasm. This separates the processes of transcription and translation in space and time. Prokaryotes, which lack a nucleus, can translate an mRNA as soon as it is transcribed by RNA polymerase. As a consequence, there is very little processing of prokaryotic mRNAs. By contrast, in eukaryotic cells many processing steps occur between mRNA transcription and translation. Unlike the case of mRNAs, both eukaryotes and prokaryotes process their rRNAs and tRNAs in broadly similar ways.
  • 157. TYPES OF RNA PROCESSING There are three main types of RNA processing events: trimming one or both of the ends of the primary transcript to the mature RNA length; removing internal RNA sequences by a process called RNA splicing; and modifying RNA nucleotides either at the ends of an RNA or within the body of the RNA. We will briefly examine each of these and then discuss how they are applied to the various types of cellular RNAs. Almost all RNAs have extra sequences at one or both ends of the primary transcripts that must be removed. The removal of individual nucleotides from the ends of the RNA strand is carried out by any of several ribonucleases (enzymes that cut RNA), called exoribonucleases. An entire section of RNA sequence can be removed by cleavage in the middle of an RNA strand. The enzymes responsible for the cleavage in this location are called endoribonucleases. Each of these ribonucleases is targeted so that it only cleaves particular RNAs at particular places. RNA splicing is similar to trimming in that it removes extra RNA sequences, but it is different because the sequence is removed from the middle of an RNA and the two flanking pieces are joined together again (see figure). The part of the RNA that is removed is called an intron, whereas the two pieces that are joined together, or spliced, are called exons. Just as with the cleavage enzymes, the splicing machinery recognizes particular sites within the RNA, in this case the junctions between exons and introns, and cleaves and rejoins the RNA at those positions. Modification of RNA nucleotides can occur at the ends of an RNA molecule or at internal positions. Modification of the ends can protect the RNA from degradation by exoribonucleases and can also act as a signal to guide the transport of the molecule to a particular subcellular compartment. Some internal modifications, particularly of tRNAs and rRNAs, are necessary for these RNAs to carry out their functions in protein synthesis. Some internal modifications of mRNAs change the sequence of the message and so change the amino acid sequence of the protein coded for by the mRNA. This process is called RNA editing. As with the other types of RNA processing, the enzymes that modify RNAs are directed to specific sites on the RNA.
  • 158. PROCESSING OF VARIOUS CLASSES OF RNASRibosomal RNAs are synthesized as long primary transcripts that contain several different rRNAs separated by spacer regions .The individual rRNAs are cut apart by endoribonucleases that cleave within the spacer regions. Other enzymes then trim the ends to their final length. Ribosomal RNAs are also modified at many specific sites within the RNA. Ribosomal RNA synthesis and processing occurs in a special structure within the nucleus called the nucleolus . The mature rRNAs bind to ribosomal proteins within the nucleolus and the assembled ribosomes are then transported to the cytoplasm to carry out protein synthesis. Transfer RNAs are transcribed individually from tRNA genes. The primary transcripts are trimmed at both the 5′ and 3′ ("five prime," or "upstream" and "three prime," or "downstream") ends, and several modifications are made to internal bases. Many eukaryotic tRNAs also contain an intron, which must be removed by RNA splicing. The finished tRNAs are then transported from the nucleus to the cytoplasm. Messenger RNAs are transcribed individually from their genes as very long primary transcripts. This is because most eukaryotic genes are divided into many exons separated by introns. Genes may contain from zero to more than sixty introns, with a typical gene having around ten. Introns are spliced out of primary RNA transcripts by a large structure called the spliceosome . The spliceosome does not move along the RNA but is assembled around each intron where it cuts and joins the RNA to remove the intron and connect the exons. This must be done many times on a typical primary transcript to produce the mature mRNA.
  • 159. In addition to removal of the introns, the mRNA is modified at the 5′ end by the addition of a special "cap" structure that is later recognized by the translation machinery. The mRNA is also trimmed at the 3′ end and several hundred adenosine nucleotides are added. This modification, which is called either polyadenylation or poly (A) addition, helps stabilize the 3′ end against degradation and is also recognized by the translation machinery. Finally, the processed mature mRNA is transported from the nucleus to the cytoplasm. Some RNAs, called small nuclear RNAs (snRNAs) and small nucleolar RNAs (snoRNAs), are processed in the nucleus and are themselves part of the RNA processing systems in the nucleus. Most snRNAs are involved in mRNA splicing, while most snoRNAs are involved in rRNA cleavage and modification.
  • 160. RNA PROCESSING AND THE HUMAN GENOME The fact that most human genes are composed of many exons has some important consequences for the expression of genetic information. First, we now know that many genes are spliced in more than one way, a phenomenon known as alternative splicing. For example, some types of cells might leave out an exon from the final mRNA that is left in by other types of cells, giving it a slightly different function. This means that a single gene can code for more than one protein. Some complicated genes appear to be spliced to give hundreds of alternative forms. Alternative splicing, therefore, can increase the coding capacity of the genome without increasing the number of genes. A second consequence of the exon/intron gene structure is that many human gene mutations affect the splicing pattern of that gene. For example, a mutation in the sequence at an intron/exon junction that is recognized by the spliceosome can cause the junction to be ignored. This causes splicing to occur to the next exon in line, leaving out the exon next to the mutation. This is called exon skipping and it usually results in an mRNA that codes for a nonfunctional protein. Exon skipping and other errors in splicing are seen in many human genetic diseases.
  • 161. POST-TRANSCRIPTIONAL MODIFICATION Post-transcriptional modification or Co-transcriptional modification is the process in eukaryotic cells where primary transcript RNA is converted into mature RNA. A notable example is the conversion of precursor messenger RNA into maturemessenger RNA (mRNA) that occurs prior to protein translation. The process includes three major steps: addition of a 5' cap, addition of a 3' poly-adenylation tail, and splicing. This process is vital for the correct translation of the genomes of eukaryotes because the initial precursor mRNA produced during transcription contains both exons (coding or important sequences involved in translation), and introns (non-coding sequences)
  • 162. Example of a signal that directs post-transcriptional processing: the conserved eukaryotic polyadenylation signal directs cleavage at the cleavage signal and addition of a poly-A tail to the mRNA transcript
  • 163. 5’ PROCESSING Capping Capping of the pre-mRNA involves the addition of 7-methylguanosine (m7G) to the 5' end. To achieve this, the terminal 5' phosphate requires removal, which is done with the aid of a phosphatase enzyme. The enzyme guanosyl transferase then catalyses the reaction, which produces the diphosphate 5' end. The diphosphate 5' end then attacks the alpha phosphorus atom of a GTP molecule in order to add the guanine residue in a 5'5' triphosphate link. The enzyme (guanine-N7-)- methyltransferase ("cap MTase") transfers a methyl group from S-adenosyl methionine to the guanine ring.[3] This type of cap, with just the (m7G) in position is called a cap 0 structure. The ribose of the adjacent nucleotide may also be methylated to give a cap 1. Methylation of nucleotides downstream of the RNA molecule produce cap 2, cap 3 structures and so on. In these cases the methyl groups are added to the 2' OH groups of the ribose sugar. The cap protects the 5' end of the primary RNA transcript from attack by ribonucleases that have specificity to the 3'5' phosphodiester bonds.
  • 164.
  • 165. 3’ PROCESSING Cleavage and polyadenylation The pre-mRNA processing at the 3' end of the RNA molecule involves cleavage of its 3' end and then the addition of about 250 adenine residues to form a poly(A) tail. The cleavage and adenylation reactions occur if a polyadenylation signal sequence (5'- AAUAAA-3') is located near the 3' end of the pre-mRNA molecule, which is followed by another sequence, which is usually (5'-CA-3') and is the site of cleavage. A GU-rich sequence is also usually present further downstream on the pre-mRNA molecule. After the synthesis of the sequence elements, two multisubunit proteins called cleavage and polyadenylation specificity factor (CPSF) and cleavage stimulation factor (CStF) are transferred from RNA Polymerase II to the RNA molecule. The two factors bind to the sequence elements. A protein complex forms that contains additional cleavage factors and the enzyme Polyadenylate Polymerase (PAP). This complex cleaves the RNA between the polyadenylation sequence and the GU-rich sequence at the cleavage site marked by the (5'-CA-3') sequences. Poly(A) polymerase then adds about 200 adenine units to the new 3' end of the RNA molecule using ATP as a precursor. As the poly(A) tail is synthesised, it binds multiple copies of poly(A) binding protein, which protects the 3'end from ribonuclease digestion
  • 166.
  • 167. SPLICING Splicing RNA splicing is the process by which introns, regions of RNA that do not code for protein, are removed from the pre-mRNA and the remaining exons connected to re-form a single continuous molecule. Exons are sections of mRNA which become "expressed" or translated into a protein. They are the coding portions of a mRNA molecule.[5] Although most RNA splicing occurs after the complete synthesis and end-capping of the pre-mRNA, transcripts with many exons can be spliced co- transcriptionally.[6] The splicing reaction is catalyzed by a large protein complex called the spliceosome assembled from proteins and small nuclear RNA molecules that recognize splice sites in the pre-mRNA sequence. Many pre-mRNAs, including those encoding antibodies, can be spliced in multiple ways to produce different mature mRNAs that encode different protein sequences. This process is known as alternative splicing, and allows production of a large variety of proteins from a limited amount of DNA.
  • 168.
  • 170. Translation is the process in which ribosomes in a cell's cytoplasm create proteins, following transcription of DNA to RNA in the cell's nucleus. The entire process is a part of gene expression. In translation, messenger RNA (mRNA) is decoded by a ribosome, outside the nucleus, to produce a specific amino acid chain, or polypeptide. The polypeptide later folds into an active protein and performs its functions in the cell. The ribosome facilitates decoding by inducing the binding of complementary tRNA anticodon sequences to mRNA codons. The tRNAs carry specific amino acids that are chained together into a polypeptide as the mRNA passes through and is "read" by the ribosome. Translation proceeds in three phases: Initiation: The ribosome assembles around the target mRNA. The first tRNA is attached at the start codon. Elongation: The tRNA transfers an amino acid to the tRNA corresponding to the next codon. The ribosome then moves (translocates) to the next mRNA codon to continue the process, creating an amino acid chain. Termination: When a stop codon is reached, the ribosome releases the polypeptide. In bacteria, translation occurs in the cytoplasm, where the large and small subunits of the ribosome bind to the mRNA. In eukaryotes, translation occurs in the cytosol or across the membrane of the endoplasmic reticulum in a process
  • 172. During translation, a cell “reads” the information in a messenger RNA (mRNA) and uses it to build a protein. Actually, to be a little more techical, an mRNA doesn’t always encode—provide instructions for—a whole protein. Instead, what we can confidently say is that it always encodes a polypeptide, or chain of amino acids. In an mRNA, the instructions for building a polypeptide are RNA nucleotides (As, Us, Cs, and Gs) read in groups of three. These groups of three are called codons. There are 616161 codons for amino acids, and each of them is "read" to specify a certain amino acid out of the 202020 commonly found in proteins. One codon, AUG, specifies the amino acid methionine and also acts as a start codon to signal the start of protein construction. There are three more codons that do not specify amino acids. These stop codons, UAA, UAG, and UGA, tell the cell when a polypeptide is complete. All together, this collection of codon-amino acid relationships is called the genetic code, because it lets cells “decode” an mRNA into a chain of amino acids.
  • 173. TRANSFER RNAS (TRNAS) Transfer RNAs, or tRNAs, are molecular "bridges" that connect mRNA codons to the amino amino acids they encode. One end of each tRNA has a sequence of three nucleotides called an anticodon, which can bind to specific mRNA codons. The other end of the tRNA carries the amino acid specified by the codons. There are many different types of tRNAs. Each type reads one or a few codons and brings the right amino acid matching those codons.
  • 174. RIBOSOMAL RNA Ribosomes are the structures where polypeptides (proteins) are built. They are made up of protein and RNA (ribosomal RNA, or rRNA). Each ribosome has two subunits, a large one and a small one, which come together around an mRNA— kind of like the two halves of a hamburger bun coming together around the patty. The ribosome provides a set of handy slots where tRNAs can find their matching codons on the mRNA template and deliver their amino acids. These slots are called the A, P, and E sites. Not only that, but the ribosome also acts as an enzyme, catalyzing the chemical reaction that links amino acids together to make a chain.
  • 175. GETTING STARTED: INITIATION In initiation, the ribosome assembles around the mRNA to be read and the first tRNA (carrying the amino acid methionine, which matches the start codon, AUG). This setup, called the initiation complex, is needed in order for translation to get started.
  • 176. EXTENDING THE CHAIN: ELONGATION Elongation is the stage where the amino acid chain gets longer. In elongation, the mRNA is read one codon at a time, and the amino acid matching each codon is added to a growing protein chain. Each time a new codon is exposed: • A matching tRNA binds to the codon • The existing amino acid chain (polypeptide) is linked onto the amino acid of the tRNA via a chemical reaction • The mRNA is shifted one codon over in the ribosome, exposing a new codon for reading
  • 177. During elongation, tRNAs move through the A, P, and E sites of the ribosome, as shown above. This process repeats many times as new codons are read and new amino acids are added to the chain.
  • 178. FINISHING UP: TERMINATION Termination is the stage in which the finished polypeptide chain is released. It begins when a stop codon (UAG, UAA, or UGA) enters the ribosome, triggering a series of events that separate the chain from its tRNA and allow it to drift out of the ribosome. After termination, the polypeptide may still need to fold into the right 3D shape, undergo processing (such as the removal of amino acids), get shipped to the right place in the cell, or combine with other polypeptides before it can do its job as a functional protein.
  • 179.
  • 181.
  • 182. Epigenetics is the study of potentially heritable changes in gene expression (active versus inactive genes) that does not involve changes to the underlying DNA sequence — a change in phenotype without a change in genotype — which in turn affects how cells read the genes. Epigenetic change is a regular and natural occurrence but can also be influenced by several factors including age, the environment/lifestyle, and disease state. Epigenetic modifications can manifest as commonly as the manner in which cells terminally differentiate to end up as skin cells, liver cells, brain cells, etc. Or, epigenetic change can have more damaging effects that can result in diseases like cancer. At least three systems including DNA methylation, histone modification and non-coding RNA (ncRNA)-associated gene silencing are currently considered to initiate and sustain epigenetic change.1 New and ongoing research is continuously uncovering the role of epigenetics in a variety of human disorders and fatal diseases.
  • 184. One example of an epigenetic change in eukaryotic biology is the process of cellular differentiation. During morphogenesis, totipotent stem cells become the various pluripotentcell lines of the embryo, which in turn become fully differentiated cells. In other words, as a single fertilized egg cell – the zygote – continues to divide, the resulting daughter cells change into all the different cell types in an organism, including neurons, muscle cells, epithelium, endothelium of blood vessels, etc., by activating some genes while inhibiting the expression of others.Historically, some phenomena not necessarily heritable have also been described as epigenetic. For example, epigenetic has been used to describe any modification of chromosomal regions, especially histone modifications, whether or not these changes are heritable or associated with a phenotype. The consensus definition
  • 185. HISTORY What began as broad research focused on combining genetics and developmental biology by well-respected scientists including Conrad H. Waddington and Ernst Hadorn during the mid-twentieth century has evolved into the field we currently refer to as epigenetics. The term epigenetics, which was coined by Waddington in 1942, was derived from the Greek word “epigenesis” which originally described the influence of genetic processes on development.2 During the 1990s there became a renewed interest in genetic assimilation. This lead to elucidation of the molecular basis of Conrad Waddington’s observations in which environmental stress caused genetic assimilation of certain phenotypic characteristics in Drosophila fruit flies. Since then, research efforts have been focused on unraveling the epigenetic mechanisms related to these types of changes.3 Currently, DNA methylation is one of the most broadly studied and well-characterized epigenetic modifications dating back to studies done by Griffith and Mahler in 1969 which suggested that DNA methylation may be important in long term memory function.4 Other major modifications include chromatin remodeling, histone modifications, and non-coding RNA mechanisms. The renewed interest in epigenetics has led to new findings about the relationship between epigenetic changes and a host of disorders including various cancers, mental retardation associated disorders,
  • 186. EPIGENETICS: FUNDAMENTALS Cancer. Cancer was the first human disease to be linked to epigenetics. Studies performed by Feinberg and Vogelstein in 1983, using primary human tumor tissues, found that genes of colorectal cancer cells were substantially hypomethylated compared with normal tissues.1 DNA hypomethylation can activate oncogenes and initiate chromosome instability, whereas DNA hypermethylation initiates silencing of tumor suppressor genes. An accumulation of genetic and epigenetic errors can transform a normal cell into an invasive or metastatic tumor cell. Additionally, DNA methylation patterns may cause abnormal expression of cancer-associated genes. Global histone modification patterns are also found to correlate with cancers such as prostate, breast, and pancreatic cancer. Subsequently, epigenetic changes can be used as biomarkers for the molecular diagnosis of early cancer.
  • 187. Mental Retardation Disorders. Epigenetic changes are also linked to several disorders that result in intellectual disabilities such as ATR-X, Fragile X, Rett, Beckwith-Weidman (BWS), Prader-Willi and Angelman syndromes..2 For example, the imprint disorders Prader-Willi syndrome and Angelman syndrome, display an abnormal phenotype as a result of the absence of the paternal or maternal copy of a gene, respectively. In these imprint disorders, there is a genetic deletion in chromosome 15 in a majority of patients. The same gene on the corresponding chromosome cannot compensate for the deletion because it has been turned off by methylation, an epigenetic modification. Genetic deletions inherited from the father result in Prader-Willi syndrome, and those inherited from the mother, Angelman syndrome. Immunity & Related Disorders. There are several pieces of evidence showing that loss of epigenetic control over complex immune processes contributes to autoimmune disease. Abnormal DNA methylation has been observed in patients with lupus whose T cells exhibit decreased DNA methyltransferase activity and hypomethylated DNA. Disregulation of this pathway apparently leads to overexpression of methylation-sensitive genes such as the leukocyte function- associated factor (LFA1), which causes lupus-like autoimmunity. Interestingly, LFA1 expression is also required for the development of arthritis, which raises the possibility that altered DNA methylation patterns may contribute to other diseases displaying idiopathic autoimmunity.
  • 188. Neuropsychiatric Disorders. Epigenetic errors also play a role in the causation of complex adult psychiatric, autistic, and neurodegenerative disorders. Several reports have associated schizophrenia and mood disorders with DNA rearrangements that include the DNMT genes. DNMT1 is selectively overexpressed in gamma-aminobutyric acid (GABA)-ergic interneurons of schizophrenic brains, whereas hypermethylation has been shown to repress expression of Reelin (a protein required for normal neurotransmission, memory formation and synaptic plasticity) in brain tissue from patients with schizophrenia and patients with bipolar illness and psychosis. A role for aberrant methylation mediated by folate levels has been suggested as a factor in Alzheimer’s disease; also some preliminary evidence supports a model that incorporates both genetic and epigenetic contributions in the causation of autism. Autism has been linked to the region on chromosome 15 that is responsible for Prader-Willi syndrome and Angelman syndrome. Findings at autopsy of brain tissue from patients with autism have revealed a deficiency in MECP2 expression that appears to account for reduced expression of several relevant genes.
  • 189. Pediatric Syndromes. In addition to epigenetic alterations, specific mutations affecting components of the epigenetic pathway have been identified that are responsible for several syndromes: DNMT3B in ICF (immunodeficiency, centromeric instability and facial anomalies) syndrome, MECP2 in Rett syndrome, ATRX in ATR-X syndrome (a-thalassemia/mental retardation syndrome, X-linked), and DNA repeats in facioscapulohumeral muscular dystrophy. In Rett syndrome, for example, MECP2 encodes a protein that binds to methylated DNA; mutations in this protein cause abnormal gene expression patterns within the first year of life. Girls with Rett syndrome display reduced brain growth, loss of developmental milestones and profound mental disabilities. Similarly, the ATR-X syndrome also includes severe developmental deficiencies due to loss of ATRX, a protein involved in maintaining the condensed, inactive state of DNA. Together, this constellation of clinical pediatric syndromes is associated with alterations in genes and chromosomal regions necessary for proper neurologic and physical development. The increased knowledge and technologies in epigenetics over the last ten years allow us to better understand the interplay between epigenetic change, gene regulation, and human diseases, and will lead to the development of new approaches for molecular diagnosis and targeted treatments across the clinical spectrum.
  • 190. MECHANISMS DNA methylation is an epigenetic mechanism that occurs by the addition of a methyl (CH3) group to DNA, thereby often modifying the function of the genes. The most widely characterized DNA methylation process is the covalent addition of the methyl group at the 5-carbon of the cytosine ring resulting in 5-methylcytosine (5-mC), also informally known as the “fifth base” of DNA. These methyl groups project into the major groove of DNA and inhibit transcription. In human DNA, 5-methylcytosine is found in approximately 1.5% of genomic DNA.1 In somatic cells, 5-mC occurs almost exclusively in the context of paired symmetrical methylation of a CpG site, in which a cytosine nucleotide is located next to a guanidine nucleotide. An exception to this is seen in embryonic stem (ES) cells, where a substantial amount of 5-mC is also observed in non-CpG contexts. In the bulk of genomic DNA, most CpG sites are heavily methylated while CpG islands (sites of CpG clusters) in germ-line tissues and located near promoters of normal somatic cells, remain unmethylated, thus allowing gene expression to occur. When a CpG island in the promoter region of a gene is methylated,
  • 191. In human DNA, 5-methylcytosine is found in approximately 1.5% of genomic DNA.1 In somatic cells, 5-mC occurs almost exclusively in the context of paired symmetrical methylation of a CpG site, in which a cytosine nucleotide is located next to a guanidine nucleotide. An exception to this is seen in embryonic stem (ES) cells, where a substantial amount of 5-mC is also observed in non-CpG contexts. In the bulk of genomic DNA, most CpG sites are heavily methylated while CpG islands (sites of CpG clusters) in germ-line tissues and located near promoters of normal somatic cells, remain unmethylated, thus allowing gene expression to occur. When a CpG island in the promoter region of a gene is methylated, expression of the gene is repressed (it is turned off).
  • 192. RNA TRANSCRIPTS Sometimes a gene, after being turned on, transcribes a product that (directly or indirectly) maintains the activity of that gene. For example, Hnf4 and MyoD enhance the transcription of many liver- and muscle-specific genes, respectively, including their own, through the transcription factor activity of the proteins they encode. RNA signalling includes differential recruitment of a hierarchy of generic chromatin modifying complexes and DNA methyltransferases to specific loci by RNAs during differentiation and development.[60] Other epigenetic changes are mediated by the production of different splice forms of RNA, or by formation of double-stranded RNA (RNAi). Descendants of the cell in which the gene was turned on will inherit this activity, even if the original stimulus for gene-activation is no longer present. These genes are often turned on or off by signal transduction, although in some systems where syncytia or gap junctions are important, RNA may spread directly to other cells or nuclei by diffusion. A large amount of RNA and protein is contributed to the zygote by the mother during oogenesis or via nurse cells, resulting in maternal effect phenotypes. A smaller quantity of sperm RNA is transmitted from the father, but there is recent evidence that this epigenetic information can lead to visible changes in several generations of offspring
  • 193. MICRORNAS MicroRNAs (miRNAs) are members of non-coding RNAs that range in size from 17 to 25 nucleotides. miRNAs regulate a large variety of biological functions in plants and animals.[62] So far, in 2013, about 2000 miRNAs have been discovered in humans and these can be found online in a miRNA database.[63] Each miRNA expressed in a cell may target about 100 to 200 messenger RNAs that it downregulates.[64] Most of the downregulation of mRNAs occurs by causing the decay of the targeted mRNA, while some downregulation occurs at the level of translation into protein.[65] It appears that about 60% of human protein coding genes are regulated by miRNAs.[66] Many miRNAs are epigenetically regulated. About 50% of miRNA genes are associated with CpG islands,[62] that may be repressed by epigenetic methylation. Transcription from methylated CpG islands is strongly and heritably repressed.[67] Other miRNAs are epigenetically regulated by either histone modifications or by combined DNA methylation and histone modification.[
  • 194. MRNA In 2011, it was demonstrated that the methylation of mRNA plays a critical role in human energy homeostasis. The obesity-associated FTO gene is shown to be able to demethylate N6-methyladenosine in RNA SRNA sRNAs are small (50–250 nucleotides), highly structured, non-coding RNA fragments found in bacteria. They control gene expression including virulence genes in pathogens and are viewed as new targets in the fight against drug-resistant bacteria.[70] They play an important role in many biological processes, binding to mRNA and protein targets in prokaryotes. Their phylogenetic analyses, for example through sRNA–mRNA target interactions or protein binding properties, are used to build comprehensive databases.[71]sRNA-gene maps based on their targets in microbial genomes are also constructed.
  • 195. PRIONS Prions are infectious forms of proteins. In general, proteins fold into discrete units that perform distinct cellular functions, but some proteins are also capable of forming an infectious conformational state known as a prion. Although often viewed in the context of infectious disease, prions are more loosely defined by their ability to catalytically convert other native state versions of the same protein to an infectious conformational state. It is in this latter sense that they can be viewed as epigenetic agents capable of inducing a phenotypic change without a modification of the genome.[73] Fungal prions are considered by some to be epigenetic because the infectious phenotype caused by the prion can be inherited without modification of the genome. PSI+ and URE3, discovered in yeast in 1965 and 1971, are the two best studied of this type of prion.[74][75] Prions can have a phenotypic effect through the sequestration of protein in aggregates, thereby reducing that protein's activity. In PSI+ cells, the loss of the Sup35 protein (which is involved in termination of translation) causes ribosomes to have a higher rate of read-through of stop codons, an effect that results in suppression of nonsense mutations in other genes.[76] The ability of Sup35 to form prions may be a conserved trait. It could confer an adaptive advantage by giving cells the ability to switch into a PSI+ state and express dormant genetic features normally terminated by stop codon mutations
  • 196. STRUCTURAL INHERITANCE In ciliates such as Tetrahymena and Paramecium, genetically identical cells show heritable differences in the patterns of ciliary rows on their cell surface. Experimentally altered patterns can be transmitted to daughter cells. It seems existing structures act as templates for new structures. The mechanisms of such inheritance are unclear, but reasons exist to assume that multicellular organisms also use existing cell structures to assemble new ones
  • 197. NUCLEOSOME POSITIONING Eukaryotic genomes have numerous nucleosomes. Nucleosome position is not random, and determine the accessibility of DNA to regulatory proteins. This determines differences in gene expression and cell differentiation. It has been shown that at least some nucleosomes are retained in sperm cells (where most but not all histones are replaced by protamines). Thus nucleosome positioning is to some degree inheritable. Recent studies have uncovered connections between nucleosome positioning and other epigenetic factors, such as DNA methylation and hydroxymethylation
  • 198.