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Lecture PowerPoint to accompany
Inquiry into Life
Twelfth Edition
Sylvia S. Mader
Chapter 24
GCU
DNA, RNA
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
1
24.1 DNA Structure and Replication
• Hershey-Chase Experiments (1952)
– Demonstrated that DNA is the genetic material that
directs all cells
– DNA stands for Deoxyribonucleic Acid
2
Hershey-Chase Experiments
http://highered.mcgraw-hill.com/olc/dl/120076/bio21.swf
3
24.1 DNA Structure and Replication
• Structure of DNA
– James Watson and Francis Crick determined
the structure of DNA in 1953
– DNA is a chain of nucleotides
– Each nucleotide is a complex of three subunits
• Phosphoric acid (phosphate)
• A pentose sugar (deoxyribose)
• A nitrogen-containing base (adenine, guanine,
cytosine, thymine)
4
24.1 DNA Structure and Replication
H
• Structure of DNA
– Four Possible Bases
• Adenine (A) - a purine
N
Sugar
• Cytosine (C) - a pyrimidine
– Complimentary Base Pairing
N
N
Adenine (A)
• Guanine (G) - a purine
• Thymine (T) - a pyrimidine
N H
N
O
N
N
Sugar
O
CH3
H N
N
O
Sugar
Thymine (T)
H
H N
N H
N
N
N H
H
Guanine (G)
O
N
Sugar
Cytosine (C)
• Adenine (A) always pairs with Thymine (T)
• Guanine (G) always pairs with Cytosine (C)
• http://highered.mcgrawhill.com/sites/dl/free/0072835125/126997/animation
12.html
5
Overview of DNA Structure
6
24.1 DNA Structure and Replication
• Replication of DNA
– Semi-conservative replication
• Each daughter DNA molecule consists of one new chain of
nucleotides and one from the parent DNA molecule
– The two daughter DNA molecules will be identical to
the parent molecule
A
C
T
A
G
T
G
A
T
C
A
C
T
A
G
T
G
A
T
C
(a) The parent molecule has two
(b) The first step in replication is
complementary strands of DNA.
separation of the two DNA
Each base is paired by hydrogen
strands.
bonding with its specific partner,
A with T and G with C.
A
C
T
A
G
T
G
A
T
C
A
C
T
A
G
T
G
A
T
C
(c) Each parental strand now
serves as a template that
determines the order of
nucleotides along a new,
complementary strand.
A
C
T
A
G
T
G
A
T
C
A
C
T
A
G
T
G
A
T
C
(d) The nucleotides are connected
to form the sugar-phosphate
backbones of the new strands.
Each “daughter” DNA
molecule consists of one parental
strand and one new strand.
7
24.1 DNA Structure and Replication
• Replication of DNA
– Before replication begins, the two strands of the parent molecule
are hydrogen-bonded together between the bases
– DNA helicase (enzyme) unwinds and “unzips” the doublestranded DNA
– New complementary DNA nucleotides fit into place along divided
strands by complementary base pairing. These are positioned
and joined by DNA polymerase (enzyme)
– DNA ligase (enzyme) repairs any breaks in the sugar-phosphate
backbone
– The Two double helix molecules identical to each other and to
the original DNA molecule
8
Overview of DNA Replication
http://highered.mc
grawhill.com/sites/dl/fr
ee/0072835125/1
26997/animation1
6.html
9
Ladder Configuration and DNA
Replication
10
24.2 Gene Expression
• Gene:
A segment of DNA that specifies the
amino acid sequence of a
polypeptide
• DNA does not directly control protein
synthesis, instead its information is
transcribed into RNA
• The “Central Dogma”:
11
24.2 Gene Expression
• RNA (ribonucleic acid)
12
24.2 Gene Expression
• Three Classes of RNA
– Messenger RNA (mRNA)
• Takes a message from DNA to the ribosomes
• strand
– Ribosomal RNA (rRNA)
• Makes up ribosomes (along with proteins)
• globular
– Transfer RNA (tRNA)
• Transfers amino acids to ribosomes
• Hairpin shape
13
24.2 Gene Expression
• Gene Expression Requires Two Steps:
• Transcription
– Is the synthesis of RNA under the direction of DNA
– Produces messenger RNA (mRNA)
• Translation
– Is the actual synthesis of a polypeptide, which occurs
under the direction of mRNA
– Occurs on ribosomes
http://highered.mcgrawhill.com/sites/dl/free/0072835125
/126997/animation1.html
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Ribonucleic Acid
• Why would the cell want to have an intermediate
between DNA and the proteins it encodes?
– The DNA can then stay pristine and protected,
away from the caustic chemistry of the
cytoplasm.
– Gene information can be amplified by having
many copies of an RNA made from one copy of
DNA.
– Regulation of gene expression can be effected
by having specific controls at each element of
the pathway between DNA and proteins.
– The more elements there are in the pathway,
the more opportunities there are to control it in
different circumstances.
15
24.2 Gene Expression
• Transcription
– During transcription, a segment of the DNA serves as a template
for the production of an RNA molecule
– Messenger RNA (mRNA)
• RNA polymerase (enzyme) binds to a promoter (“start”
sequence)
• DNA helix is opened so complementary base pairing can
occur
• RNA polymerase joins new RNA nucleotides in a sequence
complementary to that on the DNA, in a 5’ to 3’ direction
16
Transcription of DNA to form mRNA
17
Messenger RNA
• mRNA - of the 64 possible 3-base combinations:
– 61 code for the twenty different amino acids
– 3 code for "stop"; i.e. chain termination
• Specific nucleotide sequences call for “start” of
transcription (usually AUG = methionine) = PROMOTOR
sequence
• “stop” of mRNA synthesis = TERMINATION sequence
(UAA, UGA, UAG)
• Finished mRNA strands are ~500-10,000 nucleotides
long
18
• During transcription
– The gene determines the sequence of bases along
the length of an mRNA molecule
Gene 2
DNA
molecule
Gene 1
Gene 3
DNA strand 3
(template)
A C C A A A C C G A G T
5
TRANSCRIPTION
mRNA
U G G U U U G G C U C A
5
3
Codon
TRANSLATION
Protein
Figure 17.4
Trp
Amino acid
Phe
Gly
Ser
19
24.2 Processing of mRNA
• After Transcription
• Primary “Pre-”mRNA must be modified into
mature mRNA
– Introns are intragene segments (often, junk)
– Exons are the portion of a gene that is expressed
• Intron sequences are removed, and a poly-A tail is
added
– Ribozyme splices exon segments together
– http://highered.mcgrawhill.com/sites/dl/free/0072835125/126997/animation20.html
20
mRNA Processing
pre-RNA must be modified before translation
21
The Functional and Evolutionary Importance of Introns
• The presence of introns
– Allows for alternative RNA splicing
– Animations of RNA processing:
http://highered.mcgrawhill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/00
72437316/120077/bio25.swf::Processing%20of%20Gene%20Infor
mation%20-%20Prokaryotes%20versus%20Eukaryotes
– http://highered.mcgrawhill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/00
72437316/120077/bio30.swf::How%20Spliceosomes%20Process%
20RNA
22
24.2 Gene Expression
• Translation
– The Genetic Code
• Triplet code: each 3-nucleotide unit of a mRNA molecule is
called a codon
• There are 64 different mRNA codons
– 61 code for particular amino acids
» Redundant code; some amino acids have numerous code
words
» Provides some protection against mutations
– 3 are stop codons signal polypeptide termination
23
Messenger RNA Codons
24
Overview of Gene Expression
Protein synthesis
http://highered.mcgrawhill.com/olcweb/cgi/plugi
npop.cgi?it=swf::535::53
5::/sites/dl/free/0072437
316/120077/micro06.sw
f::Protein%20Synthesis
25
24.2 Gene Expression
• Transfer RNA
– tRNA transports amino acids to the
ribosomes (creates polypeptide chain)
– Single stranded nucleic acid that
correlates a specific nucleotide sequence
with a specific amino acid
– Amino acid binds to one end, the
opposite end has an anticodon
– the order of mRNA codons determines
the order in which tRNA brings in amino
acids
Protein synthesis
http://highered.mcgrawhill.com/olcweb/cgi/pluginp
op.cgi?it=swf::535::535::/si
tes/dl/free/0072437316/12
0077/micro06.swf::Protein
%20Synthesis
26
Transfer RNA: Amino Acid Carrier
27
rRNA
– Ribosomal RNA is the most abundant type of RNA in
cells
– Ribosomes: comprised of subunits 2/3 RNA, 1/3
protein
• Two populations of
ribosomes are evident
in cells, Free and
bound
• Free ribosomes in
the cytosol initiate the
synthesis of all
proteins
28
• The ribosome has three binding sites for
tRNA
– The P site
– The A site
– The E site
P site (Peptidyl-tRNA
binding site)
A site (AminoacyltRNA binding site)
E site
(Exit site)
Large
subunit
E
mRNA
binding site
Figure 17.16b
P
A
Small
subunit
(b) Schematic model showing binding sites. A ribosome has an mRNA
binding site and three tRNA binding sites, known as the A, P, and E sites.
This schematic ribosome will appear in later diagrams.
29
• Concept 17.4: Translation is the RNAdirected synthesis of a polypeptide: a closer
look
Quicktime movie:
http://carbon.cudenver.edu/~bstith/transla.MOV
Narrated animation: http://highered.mcgrawhill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/
120077/micro06.swf::Protein%20Synthesis
Interactive practice:
http://learn.genetics.utah.edu/content/begin/dna/transcribe/
30
24.2 Gene Expression
• Ribosome and Ribosomal RNA
– Ribosome has a binding site for mRNA and
for 2 tRNAs
– Facilitates complementary base pairing
– Ribosome moves along mRNA and new
tRNAs come in and line up in order
– This brings amino acids in line in a specific
order to form a polypeptide
– Several ribosomes may move along the
same mRNA
• Multiple copies of a polypeptide may be made
• The entire complex is called a polyribosome
31
Polyribosomes
• A number of ribosomes can translate a single
mRNA molecule simultaneously
– Forming a polyribosome
Completed
polypeptide
Growing
polypeptides
Incoming
ribosomal
subunits
Start of
mRNA
(5 end)
End of
mRNA
(3 end)
(a) An mRNA molecule is generally translated simultaneously
by several ribosomes in clusters called polyribosomes.
Ribosomes
mRNA
0.1 µm
Figure 17.20a, b
(b) This micrograph shows a large polyribosome in a prokaryotic
cell (TEM).
32
Translation (Building a polypeptide)
requires Three Steps:
– Initiation (requires energy)
– Elongation (requires energy)
– Termination
Amino end
Growing polypeptide
Next amino acid
to be added to
polypeptide chain
tRNA
3
mRNA
5
Codons
(c) Schematic model with mRNA and tRNA. A tRNA fits into a binding site when its
anticodon base-pairs with an mRNA codon. The P site holds the tRNA attached to
the growing polypeptide. The A site holds the tRNA carrying the next amino acid to
be added to the polypeptide chain. Discharged tRNA leaves via the E site.
33
Summary of Gene Expression
34
24.2 Gene Expression
• Genes and Gene Mutations
– A gene mutation is a change in the sequence of
bases within a gene.
– Gene mutations can lead to malfunctioning proteins in
cells.
35
24.2 Gene Expression
• Genes and Gene Mutations
– Causes of Mutations
• Errors in replication
– Rare
– DNA polymerase “proofreads” new strands and errors
are cleaved out
• Mutagens
– Environmental influences
– Radiation, UV light, chemicals
– Rate is still fairly low because DNA repair enzymes
monitor and repair DNA
36
Transposons
Transposons
• “jumping genes”
• Can move to new locations and disrupt gene
sequences
37
Types of Gene Mutations
Point Mutations
– The substitution of one nucleotide for another
• Missense mutations
– a point mutation in which a single nucleotide is changed, resulting in a
codon that codes for a different amino acid
– Missense mutations are responsible for about 75% of the mutations in
the p53 gene. Mutations of this gene are responsible for about 30-50%
of cancers in humans
• Silent mutations
• Nonsense mutations
– mutations that change an amino acid to a stop codon
38
Types of Gene Mutations
• Frameshift Mutations
– One or more nucleotides
are inserted or deleted
– Results in a polypeptide
that codes for the wrong
sequence of amino acids
– Codons must be read in
the correct reading frame
for the specified
polypeptide to be
produced
39
DNA Technology
• In laboratory experiments
– Genes can be transcribed and translated after
being transplanted from one species to another
• Called “Recombinant
DNA” technology
• Can be produced via
“Genetic Engineering”
(laboratory manipulation)
40
Overview: Understanding and Manipulating Genomes
• One of the greatest achievements of
modern science has been the sequencing
of the human genome, which was largely
completed by 2003
• DNA sequencing accomplishments
– Have all depended on advances in DNA
technology, starting with the invention of
methods for making recombinant DNA
– DNA sequencing animation
41
DNA Cloning
• Concept 20.1: DNA cloning permits
production of multiple copies of a specific
gene or other DNA segment
• To work directly with specific genes
– Scientists have developed methods for
preparing well-defined, gene-sized pieces of
DNA in multiple identical copies, a process
called gene cloning
42
24.3 DNA Technology
• The Cloning of a Gene
– Cloning:
Production of many identical copies of
an organism through some asexual means.
– Gene Cloning:
The production of many identical
copies of a single gene
– Two Ways to Clone a Gene:
– Recombinant DNA
– Polymerase Chain Reaction
43
Using Restriction Enzymes to Make Recombinant DNA
• Bacterial restriction
enzymes
– Cut DNA molecules at
a limited number of
specific DNA
sequences, called
restriction sites
44
Restriction Enzymes and Sticky Ends
Step through animation of cut/splice using EcoRI
Narrated animation
45
Cloning of a Human Gene / Recombinant DNA
– Restriction enzymes breaks
open a plasmid vector at
specific sequence of bases
“sticky ends”
– Foreign DNA that is to be
inserted is also cleaved with
same restriction enzyme so
ends match
– Foreign DNA is inserted into
plasmid DNA and “sticky ends”
pair up
– DNA ligase seals them together
– Narrated animation of “Cloning
a Gene”
46
24.3 DNA Technology
• Polymerase Chain Reaction
– Amplifies a targeted DNA sequence
– Requires DNA polymerase, a set of primers, and a
supply of nucleotides
• Primers are single stranded DNA sequences that start
replication process
–
–
–
–
Amount of DNA doubles with each replication cycle
Process is now automated
Narrated animation
Step by step animation
47
Polymerase Chain Reaction (PCR)
48
24.3 DNA Technology
• DNA Fingerprinting
– Permits identification of individuals and their relatives
– Based on differences between sequences in
nucleotides between individuals
– RFLPs : restriction fragment length polymorphisms
– Narrated animation
– Detection of the number of repeating segments (called
repeats) are present at specific locations in DNA
• Different numbers in different people
• PCR amplifies only particular portions of the DNA
• Procedure is performed at several locations to identify repeats
49
DNA Fingerprints
DNA fragments (after digest with
restriction enzymes) can be
separated through
gel ELECTROPHORESIS
See How:
Step-by-step electrophoresis
Another walk-through explanation
50
Forensic Evidence
• DNA “fingerprints”
obtained by analysis of
tissue or body fluids
found at crime scenes
Defendant’s
blood (D)
Blood from
defendant’s
clothes
4
D
Jeans
g
8
shirt
Victim’s
blood (V)
g
V
– Can provide definitive
evidence that a suspect is
guilty or, more specifically,
not guilty
– Is a specific pattern of
bands of RFLP markers
on a gel
Figure 20.17
51
DNA fingerprinting
Can also be used in
establishing paternity
Figure: Electrophoresis of PCRamplified DNA fragments. (1)
Father. (2) Child. (3) Mother. The
child has inherited some, but not all
of the fingerprint of each of its
parents, giving it a new, unique
fingerprint.
http://en.wikipedia.org/wiki/Polymerase_chain_reaction#Paternity_testing
52
24.3 DNA Technology
• Biotechnology
– Biotechnology uses natural biological systems to
create a product or to achieve a goal desired by
humans.
– “Model Organisms” favored for genetics research
– Transgenic organisms have a foreign gene inserted
into their DNA
53
– Have been engineered to be pharmaceutical
“factories”
Figure 20.18
“Pharm” Animals
54
Pharmaceutical Products
55
24.3 DNA Technology
• Transgenic Bacteria
– Medical Uses: Production of Insulin, Human Growth
Hormone, Hepatitis B Vaccine
– Agricultural Uses: Bacteria that protects plants from
freezing, bacteria that protect plant roots from insects
– Environmental: Bacteria that degrade oil (clean up
after oil spills), bacteria that remove sulfur from coal
56
Environmental Cleanup
• Genetic engineering can be used to modify
the metabolism of microorganisms
– So that they can be used to extract minerals
from the environment or degrade various types
of potentially toxic waste materials
57
24.3 DNA Technology
• Transgenic Plants
– Plants have been engineered to secrete a toxin that
kills insects (ex: Bt corn)
– Plants have been engineered to be resistant to
herbicides (ex: Roundup Ready)
58
Agricultural Applications
• DNA technology
– Is being used to improve agricultural
productivity and food quality
59
Genetic Engineering in Plants
• Agricultural scientists
– Have already endowed a
number of crop plants with
genes for desirable traits
Bt corn (right)
60
24.3 DNA Technology
• Transgenic
Animals
– Fish, cows, pigs,
rabbits and sheep
have been
engineered to
produce human
growth hormone in
order to increase
size of the animals
61
Animal Husbandry and “Pharm”
Animals
• Transgenic animals
– Contain genes from other organisms
– Sometimes called “chimeras”
– Fig 1. transgenic mouse lines expressing GFP known as “green
mice.”
62
Animal Husbandry and “Pharm” Animals
• “Knockout” mice
http://www.nca-nl.org/English/Newsletters/Nb13/nl13txt.html
Since cancer is a multistage process, it is obvious that transgenic or knock out animals, which have
already undergone one step in the cancer process, may be a sensitive alternative for the
standard bioassay.
A number of mice models have been developed: either possessing an inactivated tumor suppressor
gene (p53), an activated oncogene (Tg.AC), over-expression of a (human) oncogene (rasH2)
or being deficient in nucleotide excision repair (Xpa, de Vries et al., 1995).
These mice models have several advantages:
•
the number of animals needed for one study is 120 instead of 400-500
•
the duration of the study is 6-9 instead of 24 months leading to less distress of the animals
•
the transgenic mouse model is considered more discriminating hence improving the accuracy
and reliability of human carcinogen identification.
63
Safety and Ethical Questions Raised by DNA Technology
• The potential benefits of genetic engineering
– Must be carefully weighed against the potential
hazards of creating products or developing
procedures that are harmful to humans or the
environment
• Today, much public concern about possible
hazards
– Centers on genetically modified (GMOs)
organisms used as food
– Gene “escape”
64
Transgenic Animals
65