Download DNA Replication in Bacteria

UNIT 2
DNA Replication
Objectives
 Discuss experimental evidence supporting semiconservative mechanism of DNA replication
 Explain DNA replication in Prokaryotes and
Eukaryotes
 Define mutation
 Identify different types of mutations and their
effects on the protein products produced
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Eukaryotic genes have interrupted coding sequences.
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That is, there are long sequences of bases within the
protein-coding sequences of the gene that do not code
for amino acids in the final protein product.
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The nocoding regions within the gene are called introns
(intervening sequences).
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The exons (expressed sequences) which are part of the
protein-coding sequence.
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A typical eukaryotic gene may have multiple exons and
introns and the numbers are quite variable.
Eg. the β-globulin gene has 2 and the ovalbumin gene
of egg white has 7.
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In many cases the lengths of the introns are much
greater than those of the exon sequences.
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For instance the ovalbumin gene contains about 7700
base pairs, 1859 of them in exons.
DNA Replication
Three theories were suggested:
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Conservative replication
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Dispersive replication
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intact the original DNA molecule and generate a completely
new molecule.
produce two DNA molecules with sections of both old and
new DNA interspersed along each strand.
Semi-conservative replication
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produce molecules with both old and new DNA - each
molecule would be composed of one old strand and one new
one.
DNA Replication is semi-conservative
Experimental Proof
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(1957) Mathew Meselson and Franklin Stahl grew the
bacterium Escherichia coli on medium that contained 15N
in the form of ammonium chloride.
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The 15N became incorporated into DNA (nitrogenous
bases).
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The resulting heavy nitrogen-containing DNA
molecules were extracted from some of the cells.
DNA Replication is semi-conservative
Experimental Proof
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When subject to density gradient centrifugation, they
accumulated in the high-density region of the gradient.
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The rest of the bacteria were transferred to a new
growth medium in which ammonium chloride
contained the naturally abundant, lighter 14N isotope.
DNA Replication is semi-conservative
Experimental Proof
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The newly synthesized strands were expected to be less
dense since they incorporated bases containing the
lighter 14N isotope.
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The DNA from cells isolated after one generation had
an intermediate density, indicating that they contained
half as many 15N isotope as the parent DNA.
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This finding supported the semi-conservative model each double helix would contain one previously
synthesized strand and a newly synthesized strand.
DNA Replication is semi-conservative
Experimental Proof
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It is also consistent with the dispersive model which
would yield one class of molecules, all with intermediate
density.
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It was inconsistent with the conservative model which
predicted that there would be two classes of doublestranded molecules, those with two heavy strands and
those with two light strands.
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After another cycle of cell division in the medium with
the lighter 14N isotope, two types of DNA appeared in
the density gradient.
DNA Replication is semi-conservative
Experimental Proof
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One with hybrid DNA helices ( one strand 15N isotope
and the other strand 14N), whereas the other contained
only strands of the light isotope.
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This finding refuted the dispersive model, which predicted
that all stands should have intermediate density.
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It however supported the semiconservative method which
predicted that each parent strand would act as a template
for the synthesis of new strands.
Animation of DNA Replication
Experimental Proof
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http://highered.mcgrawhill.com/sites/0072437316/student_view0/chap
ter14/animations.html
Tutorial
 http://www.sumanasinc.com/webcontent/anim
ations/content/meselson.html
DNA Replication in Bacteria
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In general, DNA is replicated by:
uncoiling of the helix
 strand separation by breaking of the hydrogen
bonds between the complementary strands
 synthesis of two new strands by
complementary base pairing
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Replication begins at a specific site in the DNA called
the origin of replication (ori)
DNA Replication in Bacteria
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DNA replication is bidirectional from the
origin of replication
DNA replication occurs in both directions from
the origin of replication in the circular DNA
found in most bacteria.
DNA Replication in Bacteria
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To begin DNA replication, unwinding enzymes
called DNA helicases cause the two parent
DNA strands to unwind and separate from one
another at the origin of replication to form two
"Y"-shaped replication forks.
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These replication forks are the actual site of
DNA copying
Replication Fork
Animation of Replication Fork
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http://highered.mcgrawhill.com/sites/0072437316/student_view0/chap
ter14/animations.html#
DNA Replication in Bacteria
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Helix destabilizing proteins bind to the
single-stranded regions so the two strands do
not rejoin
Enzymes called topoisimerases produce breaks
in the DNA and then rejoin them in order to
relieve the stress in the helical molecule during
replication.
DNA Replication in Bacteria
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As the strands continue to unwind in both directions
around the entire DNA molecule, new
complementary strands are produced by the
hydrogen bonding of free DNA nucleotides with
those on each parent strand
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As the new nucleotides line up opposite each parent
strand by hydrogen bonding, enzymes called DNA
polymerases join the nucleotides by way of
phosphodiester bonds.
DNA Replication in Bacteria
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The nucleotides lining up by complementary
base pairing are deoxynucleoside triphosphates
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As the phosphodiester bond forms between the
5' phosphate group of the new nucleotide and
the 3' OH of the last nucleotide in the DNA
strand, two of the phosphates are removed
providing energy for bonding
DNA Replication by Complementary
Base Pairing
Animation of How Nucleotides are
added
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http://highered.mcgrawhill.com/sites/0072437316/student_view0/chap
ter14/animations.html#
DNA replication in a 5' to 3'
direction
DNA Replication in Bacteria
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DNA replication is more complicated than this because
of the nature of the DNA polymerases.
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DNA polymerase enzymes are only able to join the
phosphate group at the 5' carbon of a new
nucleotide to the hydroxyl (OH) group of the 3'
carbon of a nucleotide already in the chain.
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As a result, DNA can only be synthesized in a 5' to 3'
direction while copying a parent strand running in a 3'
to 5' direction.
DNA Replication in Bacteria
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The two strands are antiparallel –
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one parent strand - the one running 3' to 5' is called
the leading strand can be copied directly down its
entire length
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the other parent strand - the one running 5' to 3' is
called the lagging strand must be copied
discontinuously in short fragments –
Okazaki fragments of around 100-1000 nucleotides
each as the DNA unwinds.
DNA Replication in Bacteria
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DNA polymerase enzymes cannot begin a new
DNA chain from scratch.
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can only attach new nucleotides onto 3' OH group of a
nucleotide in a preexisting strand.
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To start the synthesis of the leading strand and each DNA
fragment of the lagging strand, an RNA polymerase
complex called a primosome or primase is required.
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The primase is capable of joining RNA nucleotides without
requiring a preexisting strand of nucleic acid - forms what is
called an RNA primer
RNA primer
DNA Replication in Bacteria
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After a few nucleotides are added, primase is replaced
by DNA polymerase.
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DNA polymerase can now add nucleotides to the 3'
end of the short RNA primer.
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The primer is later degraded and filled in with DNA.
DNA Replication in Bacteria
Bacteria have 5 known DNA polymerases:
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Pol I:
DNA repair
 has 5'→3' (Polymerase) activity
 both 3' → 5' (proof reading) and 5' → 3' exonuclease
activity (in removing RNA primers).
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DNA polymerase I is not the replicative polymerase:
1. The enzyme is too slow!
adds dNTPs at a rate of 20 nt/sec. So it would require
460,000 sec (= 7667 min = 128 hr = 5.3 days) to replicate
the E. coli chromosome! Too slow for an organism which
can divide every 20 mins.
2. The enzyme is too abundant
There are 400 molecules per E. coli cell. This is excessive
given that there are generally only 2 replication forks per
cell.
3.The enzyme is not processive enough
DNA polymerase I dissociates after catalysing the
incorporation of 20-50 nucleotides.
DNA Replication in Bacteria
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Pol II:
involved in repair of damaged DNA
 has 3' → 5' exonuclease activity.
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Proof that this is not the main polymerase:
1. Strains lacking the gene show no defect in growth or
replication.
2. Synthesis of Pol II is induced during the stationary
phase of cell growth - a phase in which little growth and
DNA synthesis occurs. But DNA can accumulate
damage such as short gaps
3. Pol II has a low error rate but it is much too slow to be
of any use in normal DNA synthesis.
DNA Replication in Bacteria
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Pol III:
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the main polymerase in bacteria (elongates in
DNA replication)
has 3' → 5' exonuclease proofreading ability.
is the principal replicative enzyme
Proof of function:
1. is highly processive
2. catalyses polymerization at a high rate.
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There are two forms of the enzyme.
Core enzyme - consists of only those subunits
that are required for the basic underlying enzymatic
activity: alpha (a), epsilon (e) and theta (q).
Holoenzyme- the fully functional form of an
enzyme, complete with all of its necessary
accessory subunits.
The DNA polymerase III holoenzyme consists of
the core enzyme, the b sliding clamp and the
clamp-loading complex.
DNA Replication in Bacteria
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Pol IV and Pol V:
Are Y-family DNA polymerases
 participates in bypassing DNA damage
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DNA Replication in Bacteria
Animation of bidirectional
replication of DNA
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http://highered.mcgrawhill.com/sites/0072437316/student_view0/chap
ter11/animations.html#
DNA Replication in Eukaryotes
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multiple origins of replication in eukaryotes
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human genome about 30,000 origins
each origin produces two replication forks
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moving in opposite direction
DNA Replication in Eukaryotes
DNA Replication in Eukaryotes
Eukaryotes have at least 15 DNA Polymerases:
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Pol α : act as a primase (synthesizing an RNA primer),
elongates the primer
Pol β : repairs DNA, (excision repair and gap-filling).
Pol γ: Replicates and repairs mitochondrial DNA and has
proofreading 3' → 5' exonuclease activity.
Pol δ: Highly processive and has proofreading 3' → 5'
exonuclease activity, reposible for replication of lagging
strand.
Pol ε: Highly processive and has proofreading 3' → 5'
exonuclease activity, reponsible for replication of leading
strand.
η, ι, κ, Rev1 and Pol ζ are involved in the bypass of DNA
damage.
θ, λ, φ, σ, and μ are not as well characterized:
There are also others, but the nomenclature has become
quite jumbled.
DNA Replication in Eukaryotes
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the polymerases that deal with the elongation are
Pol α, Pol ε,Polδ.
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Pol α : forms a complex to act as a primase
(synthesizing an RNA primer), and then elongates that
primer with DNA nucleotides.
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After around 20 nucleotides elongation by Pol α is taken
over by Pol ε (on the leading strand) and δ (on the
lagging strand).
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Other enzymes are responsible for primer remover in
Eukaryotes as none of their polymerases have 5′→3′
exonuclease activity
DNA Replication in Eukaryotes
DNA damage bypass
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All organisms need to deal with the problems that arise
when a moving replication fork encounters damage in the
template strand.
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The best way to deal with this situation is to repair the
damage by an excision mechanisms.
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In some cases, however, the damage may not be repairable,
or the advancing replication fork may already have
unwound the parental strands, thus preventing excision
mechanisms from using the complementary strand as
template for repair, or excision repair may not yet have had
an opportunity to repair the damage.
DNA damage bypass
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It is important for the cell to be able to move
replication forks past unrepaired damage:
 Long-term blockage of replication forks leads
to cell death.
 Replication of damaged DNA provides a sister
chromatid that can be used as template for
subsequent repair by homologous
recombination.
Replication fork bypass mechanisms cannot,
strictly speaking, be considered examples of DNA
repair, because the damage is left in the DNA, at
least temporarily.
Rate of Replication
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In prokaryotes replication proceeds at about 1000
nucleotides per second, and thus is done in no more
than 40 minutes.
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In Eukaryotes replication takes proceeds at 50
nucleotides per second, and is completed in 60
minutes.
Mutations
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changes in the nucleotide sequence of the DNA.
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organisms have special systems of enzymes that can
repair certain kinds of alterations in the DNA.
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once the DNA sequence has been changed, DNA
replication copies the altered sequence just as it would
copy a normal sequence.
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provide the variation necessary for evolution to happen
in a given species.
Types of Mutations
Somatic mutations
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Occurs in cells not dedicated to sexual reproduction
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The mutant genes disappear when the cell in which it
occurred dies and can only be passed on through
asexual reproduction.
Germline mutations
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found in every cell descended from the zygote to which
that mutant gamete contributed.
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If an adult is successfully produced, every one of its
cells will contain the mutation.
Types of Mutations
Single-base Substitution/point mutation
 exchanges one base for another.
 If one purine [A or G] or pyrimidine [C or T] is
replaced by the other, the substitution is called a
transition.
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If a purine is replaced by a pyrimidine or vice-versa,
the substitution is called a transversion.
Original:
The fat cat ate the wee rat
Point Mutation: The fat hat ate the wee rat
Types of Mutations
Point mutations continued
 A change in a codon to one that encodes a
different amino acid and cause a small change in
the protein produced = missense mutation.
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Example sickle-cell disease
A → T at the 17th nt of the gene for the beta
chain of hemoglobin changes the codon GAG
(glutamic acid) to GTG (valine)
Therefore: 6th amino acid glutamic acid → valine
Missense mutation
Examples of Diseases caused by
point mutations
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Color blindness
Cystic fibrosis
Hemophilia
Phenylketonuria
Tay Sachs
Types of Mutations
Point mutations continued
 change a codon to one that encodes the same amino
acid and causes no change in the protein produced
= silent mutations.
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change an amino-acid-coding codon to a single
"stop" codon → an incomplete protein
= a nonsense mutation
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can have serious effects since the incomplete protein
probably won't function.
Nonsense Mutation
Types of Mutations
Insertion
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extra base pairs are inserted into a new place in the DNA.
Original: The fat cat ate the wee rat.
Insertion: The fat cat xlw ate the wee rat.
Deletion
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a section of DNA is lost, or deleted.
Original: The fat cat ate the wee rat.
Deletion: The fat
ate the wee rat.
Insertion mutation
Types of Mutations
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An example of a human disorder caused by insertion is
Huntington’s disease.
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In this disorder, the repeated trinucleotide is CAG,
which adds a string of glutamines (Gln) to the encoded
protein (called huntingtin).
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The abnormal protein increases the level of the p53
protein in brain cells causing their death by apoptosis.
Huntington’s
Deletion Mutation
Examples of Diseases caused by
deletions
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Cri du chat
De Grouchy syndrome
Shprintzen syndrome
Wolf-Hirschhorn syndrome
Duchenne muscular dystrophy
Types of Mutations
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Insertion and deletions involving one or two base pairs (or
multiples )
 can have devastating consequences to the gene because
translation of the gene is "frameshifted"
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DNA is read in sequences of three bases therefore the addition or removal of
one or more bases alters the sequence that follows as the bases all shifted.
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The entire meaning of the sequence has changed.
Frameshifts often create new STOP codons → nonsense
mutations
Original: The fat cat ate the wee rat.
Frame Shift: The fat caa tet hew eer at.
Frame shift mutation
Types of Mutations
Duplications
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Duplications are a doubling of a section of the
genome.
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During meiosis, crossing over between sister
chromatids that are out of alignment can produce one
chromatid with an duplicated gene and the other
having two genes with deletions.
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Example of disease :DM1 (Myotonic dystrophy)
Types of Mutations
Translocations
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Translocations are the transfer of a piece of one
chromosome to a nonhomologous chromosome.
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Translocations are often reciprocal; that is, the two
nonhomologues swap segments.
Types of Mutations
Translocations can alter the phenotype is several ways:
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the break may occur within a gene
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destroying its function
creating a hybrid gene.
translocated genes may come under the influence of
different promoters and enhancers so that their
expression is altered.
Types of Mutations
Inversion
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an entire section of DNA is reversed.
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A small inversion may involve only a few bases within a
gene, while longer inversions involve large regions of a
chromosome containing several genes.
Original: The fat cat ate the wee rat.
Insertion: The fat tar eew eht eta tac.
Inversion
Types of Mutations
Suppressor mutation
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partially or completely masks phenotypic expression of
a mutation but occurs at a different site from it
(i.e., causes suppression)
may be intragenic or intergenic.
It is used particularly to describe a secondary mutation
that suppresses a nonsense codon created by a primary
mutation.
Naming genes
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given an official name and symbol by a formal committee
The HUGO Gene Nomenclature Committee (HGNC) – US
and UK designates an official name and symbol (an abbreviation
of the name) for each known human gene.
Some official gene names include additional information in
parentheses, such as related genetic conditions, subtypes of a
condition, or inheritance pattern.
The Committee has named more than 13,000 of the estimated
20,000 to 25,000 genes in the human genome.
a unique name and symbol are assigned to each human gene,
which allows effective organization of genes in large databanks,
aiding the advancement of research.
How are genetic conditions named?
Disorder names are often derived from one or a combination of
sources:
 The basic genetic or biochemical defect that causes the condition
(alpha-1 antitrypsin deficiency)

One or more major signs or symptoms of the disorder (sickle cell
anemia)
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The parts of the body affected by the condition (retinoblastoma)
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The name of a physician or researcher, often the first person to
describe the disorder (Marfan syndrome - Dr. Antoine Marfan)
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A geographic area (familial Mediterranean fever)
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The name of a patient or family with the condition (Lou Gehrig
disease)
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Disorders named after a specific person or place are called eponyms.
References/ sources of images
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http://www.genetichealth.com/g101_changes_in_dna.shtml
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usmlemd.wordpress.com/2007/07/14/dna-replication/
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