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The Genetic Code of
Genes and Genomes
DNA Structure: Double Helix
• DNA backbone forms righthanded helix
• Each DNA strand has polarity =
directionality
• The paired strands are oriented
in opposite directions =
antiparallel
Central
Dogma
Defects in an enzymatic pathway
Can produce in phenotypes
Black urine diease
(Alkaptonuria )
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Mutations
• Mutation refers to any heritable change in a gene
• The change may be: substitution of one base pair in
DNA for a different base pair; deletion or addition of
base pairs
• Any mutation that causes the insertion of an incorrect
amino acid in a protein can impair its function
Normal, wild type
Mutation inactivates enzyme
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Chapter 6
DNA Structure, Replication, and Manipulation
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DNA: Molecule of Heredity
• Inherited traits are
affected by genes that
are transmitted from
parents to offspring in
reproduction
• Genes are composed
of the chemical
deoxyribonucleic acid =
DNA
DNA: Molecule of Heredity
• DNA was discovered by Friedrich Miescher in 1869
• In 1920s microscopic studies with special stains
showed that DNA is present in chromosomes
• In 1944 Avery, McLeod and McCarty provided the first
evidence that DNA is the genetic material
Griffith's experiment 1928 demonstrating bacterial transformation
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Avery, McLeod and McCarty identified DNA as the chemical substance responsible for changing
rough, nonvirulent cells of S pneumoniae (R) into smooth encapsulated infectious cells (S)
Genome Size
• The genetic complement of a cell or virus constitutes its
genome
• In eukaryotes, this term is commonly used to refer to one
complete haploid set of chromosomes, such as that found
in a sperm or egg
• The C-value = the DNA content of the haploid genome
• The units of length of nucleic acids in which genome
sizes are expressed :
• kilobase (kb) 103 base pairs
• megabase (Mb) 106 base pairs
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Genome Size
• Viral genomes are typically in the range 100–1000 kb:
Bacteriophage MS2, one of the smallest viruses, has only four genes in
a single stranded RNA molecule of about 4000 nucleotides (4kb)
• Bacterial genomes are larger, typically in the range 1–10 Mb:
•
The chromosome of Escherichia coli is a circular DNA molecule of 4600
kb.
Eukaryotic genomes are typically in the range 100–1000 Mb:
The genome of a fruit fly, Drosophila melanogaster is 180 Mb
•
Among eukaryotes, genome size often differs tremendously, even among
closely related species
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The C-value Paradox
• Genome size among species of protozoa differ by
5800-fold, among arthropods by 250-fold, fish 350fold, algae 5000-fold, and angiosperms 1000-fold.
• The C-value paradox: Among eukaryotes, there is no
consistent relationship between the C-value and the
metabolic, developmental, or behavioral complexity of
the organism
• The reason for the discrepancy is that in higher
organisms, much of the DNA has functions other than
coding for the amino acid sequence of proteins
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DNA: Chemical Composition
• DNA is a linear polymer of four deoxyribonucleotides
• Nucleotides composed of 2'- deoxyribose (a five-carbon sugar),
phosphoric acid, and the four nitrogen-containing bases denoted A, T,
G and C
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DNA: Chemical Composition
• Two of the bases, A and G, have a double-ring structure; these are
called purines
• The other two bases, T and C, have a single-ring structure; these
are called pyrimidines
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DNA Structure
• The duplex molecule of DNA consists of two polynucleotide chains
twisted around one another to form a right-handed helix in which the
bases form hydrogen bonds
Adenine pairs with thymine; guanine with cytosine
A hydrogen bond is a weak bond
The stacking of the base pairs on top of one another also contribute to
holding the strands together
The paired bases are planar, parallel to one another, and perpendicular
to the long axis of the double helix.
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DNA Structure
• The backbone of each polynucleotide
strand consists of deoxyribose sugars
alternating with phosphate groups that
link 5 ' carbon of one sugar to the 3'
carbon of the next sugar in line
• The two polynucleotide strands of the
double helix run in opposite directions
• The paired strands are said to be
antiparallel
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DNA Replication
Watson-Crick model of DNA
replication:
• Hydrogen bonds between DNA
bases break to allow strand separation
• Each DNA strand is a template for the
synthesis of a new strand
• Template (parental) strand determines
the sequence of bases in the new strand
(daughter)= complementary base pairing
rules
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M. Meselson and F. Stahl
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Circular DNA Replication
•
Autoradiogram of the intact replicating circular chromosome of E. coli shows
that
– DNA synthesis is bidirectional
– Replication starts from a single site called origin of replication (OR)
•
The region in which parental strands are separating and new strands are
being synthesized is called a replication fork
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Replication of Linear DNA
• The linear DNA duplex in a eukaryotic chromosome also
replicates bidirectionally
• Replication is initiated at many sites along the DNA
• Multiple initiation is a means of reducing the total
replication time
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DNA Synthesis
• One strand of the newly made DNA is synthesized continuously
= leading strand
• The other, lagging strand is made in small precursor fragments
= Okazaki fragments
• The size of Okazaki fragments is 1000–2000 base pairs in
prokaryotic cells and 100–200 base pairs in eukaryotic cells.
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How fast is the fork going?
E. coli can replicate in about 20 minutes under optimal conditions.
E. coli genome contains 4.5*106 basepairs
4.5*106 basepairs/2 replication forks/1200 s =
1875 basepairs/replication fork/s
By comparison….
1 deck of cards/26 pairs
1875 pairs * 1 deck of cards/26 pairs = 72 decks of cards
To move as fast as a replication fork you would have to be able to
sort 72 decks of shuffled cards….
pairing every club with a spade and every heart with a diamond…
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each second.
The error rate of replication is ~ 1
error/ 1010 bases
How much is 1010?
Typical single space typewritten page 3000 characters/sheet
1 sheet/3000 characters * 1010 characters = 3,333,333 sheets
1 ream/500 sheets * 3,333,333 sheets = 6666 reams
1 box/10 reams * 6666 reams = 666 boxes
666 boxes of single-spaced typed sheets would fill the front of
this room up to the ceiling with only a single spelling error. 29
How many mistakes are made each time the cell replicates?
E. coli genome 4.5*106 basepairs
(1 cell/1 genome)*(1 genome/4.5*106 basepairs) * (1*1010 basepairs/1 error)=
~2222 cells/1 error
H. sapiens genome 3.1*109 basepairs
(1 cell /2 genomes)*( 1 genome/ 3.1*109 basepairs) * (1*1010 basepairs/1 error)=
~1.6 cells/ 1 error
What would happen in E. coli if mismatch repair did not occur?
What would happen in humans if mismatch repair did not occur?
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Nucleic Acid Hybridization
• DNA denaturation: Two DNA strands can be separated by
heat without breaking phosphodiester bonds
• DNA renaturation = hybridization: Two single strands that
are complementary or nearly complementary in sequence
can come together to form a different double helix
• Single strands of DNA can also hybridize complementary
sequences of RNA
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Restriction Enzymes
• Restriction enzymes cleave duplex DNA at particular
nucleotide sequences
• The nucleotide sequence recognized for cleavage by a
restriction enzyme is called the restriction site of the
enzyme
• In virtually all cases, the restriction site of a restriction
enzyme reads the same on both strands A DNA
sequence with this type of symmetry is called a
palindrome
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Southern Blot Analysis
• DNA fragments on a gel can often be visualized by staining with
ethidium bromide, a dye which binds DNA
• Particular DNA fragments can be isolated by cutting out the small
region of the gel that contains the fragment and removing the DNA
from the gel.
• Specific DNA fragments are identified by hybridization with a probe =
a radioactive fragment of DNA or RNA
• Southern blot analysis is used to detect very small amounts of DNA
or to identify a particular DNA band by DNA-DNA or DNA-RNA
hybridization
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Southern Blot Analysis
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Polymerase Chain Reaction
• Polymerase Chain Reaction (PCR) makes possible the
amplification of a particular DNA fragment
• Oligonucleotide primers that are complementary to the
ends of the target sequence are used in repeated round
of denaturation, annealing, and DNA replication
• The number of copies of the target sequence doubles in
each round of replication, eventually overwhelming any
other sequences that may be present
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Polymerase Chain Reaction
• Special DNA polymerase is used in PCR = Taq
polymerase isolated from bacterial thermophiles
which can withstand high temperature used in
procedure
• PCR accomplishes the rapid production of large
amounts of target DNA which can then be identified
and analyzed
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Polymerase
chain
reaction
(PCR)
Heat
1)
Needs only
the smallest
amount of
DNA
2) Short DNA
primers (that
you can
synthesize)
Cool
Allows you
to amplify
(generate a
ton of) any
gene or
sequence
that you
need
Polymerize
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DNA Sequence Analysis
•
DNA sequence analysis
determines the order of bases
in DNA
•
The dideoxy sequencing
method employs DNA
synthesis in the presence of
small amounts of fluorescently
labeled nucleotides that
contain the sugar
dideoxyribose instead of
deoxyribose
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DNA Sequencing:
Dideoxy Method
•
Modified sugars cause chain termination
because it lacks the 3’-OH group, which is
essential for attachment of the next
nucleotide in a growing DNA strand
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The products of DNA synthesis are then
separated by electrophoresis. In principle,
the sequence can be read directly from the
gel
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DNA Sequencing: Dideoxy Method
•
•
•
•
Each band on the gel is one base longer than the previous band
Each didyoxynucleotide is labeled by different fluorescent dye
G, black; A, green; T, red; C, purple
As each band comes off the bottom of the gel, the fluorescent dye
that it contains is excited by laser light, and the color of the
fluorescence is read automatically by a photocell and recorded in
a computer
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