Download Chapter 22 & 23

Chapter 18
Molecular Genetics
Goals for this Chapter:
1. Summarize the events and
experiments that led to the
discovery of the structure of
DNA
2. Explain how the interaction
between DNA and proteins
results in the accurate
replication of genetic
information
3. Design and construct models
to simulate the structure and
replication of DNA
Goals for this Chapter:
4. Explain how genetic
information is encoded in
DNA molecules
5. Describe the processes
through which genetic
information is expressed in
living cells
6. Design and perform a
simulation to illustrate the
steps of protein synthesis
7. Explain some of causes and
effects of DNA mutations
Goals for this Chapter:
8. Describe how random
changes in nucleotide
sequences provide a source
of genetic variability
9. Explain how nucleotide
sequences provide evidence
that different species are
related
10.Design and perform a
simulation to illustrate the
use of restriction enzymes
and ligases to create
recombinant DNA
Goals for this Chapter:
• Explain how the insertion of
new DNA sequences into cells
can transform organisms
• Describe some of the social,
environmental, and ethical
issues associated with
genetic technologies
18.1 – DNA Structure and
Replication
• In 1869, Friedrich Mieschner
coined the term “nucleic
acid” to describe the material
found in the nuclei of cells
• However, it took almost a
century for scientists to
understand the DNA was the
material that carried
hereditary information
Isolating the Material of Heredity
• In the early 1900s, Phoebus
Levene identified two
compounds in chromosomes
– proteins and DNA
• Scientists did not know what
part (the DNA or protein)
actually carried hereditary
information
• Two major experiments led
to the identification of DNA
as hereditary material:
Griffith’s Transforming Principle
Griffith’s Transforming Principle
• Griffith’s experiment
provided good evidence that
DNA was the material
responsible for passing on
traits
• However, scientists were not
prepared to accept this
explanation until more
evidence was gathered
Hershey & Chase
• Hershey and Chase
performed an experiment in
1952 that used radioactive
labeling of compounds to
trace hereditary material
• They used two radioactive
materials (sulfur-35, which
would be found in proteins
and phosphorus-32, which
would be found in DNA) to
label parts of a bacteriophage
Hershey and Chase
• In the case where
the phosphorus
marker on DNA
was used,
material was
found inside the
cell, while the
sulfur markers on
the proteins were
not
What is DNA?
• DNA is deoxyribonucleic acid
• It is a molecule used by cells
to carry genetic information
• The code in DNA is arranged
into genes
http://www.pbs.org
What is Found in DNA?
• DNA actually contains both
proteins and nucleic acids
• However, the proteins do not
contain the genetic code
• Our genetic code is contained
in the nucleic acids found
within the DNA structure
http://www.accelrys.com
The Structure of DNA
•
1.
2.
3.
4.
5.
6.
DNA consists of 6 chemicals:
Deoxyribose sugar
Phosphate
Adenine
Cytosine
Guanine
Thymine
•
The nitrogen bases are
always found in
complementary pairs
http://student.ccbcmd.edu
Chagraff’s Rule
• In the 1940s, Edwin Chagraff
determined that although
nucleotides were not found in
equal amounts, there are
roughly the same amounts of
complementary bases
• For instance, if a sample of
DNA has 15% thymine
bases…
Watson & Crick
• To understand how DNA
operates, its structure must
be understood
• James Watson & Francis
Crick determined the helical
structure of DNA at
Cambridge University in 1953
• Their analysis of X-ray
diffraction patterns of
crystallized DNA molecules
allowed them to determine
the structure of DNA
http://nitro.biosci.arizona.edu
http://genome.jgi-psf.org
Rosalind Franklin
• Rosalind Franklin
provided the X-ray
diffraction analysis of
crystallized DNA to
Watson & Crick
• Her work along with the
work of Chagraff
allowed Watson and
Crick to develop the
well-known doublehelix model of DNA that
we have today
A Closer Look at DNA
• As you can
see, DNA is
antiparallel,
which means
that the left
hand strand
runs the
opposite
direction of
the right
hand strand
mRNA vs. DNA
mRNA
Genes and the Genome
• Gene:
• Genome:
Placement of Genes
• Genes are not equally spaced
on chromosomes
• For instance, chromosome 4
is relatively long (200 million
bases), but has about 800
genes
• Chromosome 19 has only 55
million bases in comparison,
but has more than 1500
genes
The Replication of DNA
• The DNA molecule can make
copies of itself
• This is required to ensure
that two new cells that arise
from mitosis have the same
genetic code
• Replication occurs in a series
of steps
Initiation
• Replication starts at a
specific nucleotide sequence,
called the replication origin
• Our chromosomes have
multiple replication origins,
while the circular DNA of
bacteria only have a single
replication origin
“Unzipping the Helix”
• An enzyme known as DNA
helicase unwinds the DNA at
replication forks
• The action of helicase creates
a “replication bubble” where
the DNA has been unwound
• At each end of the “bubble”
are replication forks that
branch out to unpaired single
strands
Elongation
• Elongation:
• Elongation is carried out by
DNA polymerase enzymes
• They act based on place
placement of primers
“Primers”
• An enzyme known as primase
places RNA primers at the
sites where DNA replication
is to begin
“Polymerases”
• There are 2 significant DNA
polymerase enzymes
• polymerase III attaches base
pairs to the exposed DNA
strand in the 5’ to 3’ direction
(the 5’ and 3’ refer to the
carbons in the deoxyribose
sugar)
• One strand that is created is
continuous (known as the
leading strand), while other
strands (lagging strand) is
replicated in short segments
• These short segments are
known as Okazaki fragments,
and they will be sealed later
“Polymerases”
• The enzyme polymerase I
follows polymerase III and
removes the RNA primers,
replacing them with
nucleotides
• The result is two strands of
DNA that are identical to
their parent
“Sealing the Deal”
• At this point, the DNA still
has small “nicks” in it
• Another enzyme, known as
ligase, repairs those nicks
(assembles the Okazaki
fragments into a single long
DNA chain)
• The completion of the two
new DNA strands is known as
termination
Interactive Review
The Final Product
• As a result, we are left with
two strands of DNA
• DNA replication is
semiconservative – each new
strand has part of the older
parent strand
Gene Sequencing – Circa 1990s
• We can now map genes by
using restriction enzymes to
chop the DNA into small
segments
• Each of these enzymes cuts
at a specific DNA sequence
• This produces segments of
varying lengths, known as
RFLPs (Restriction Fragment
Length Polymorphisms)
• The RFLPs are then marked
with radioactive dyes
• Finally, the RFLPs are placed
on a thin layer of gel through
which a small electrical field
is applied
• Within the gel, the RFLPs are
pulled along by the electrical
field
• The smaller, lighter fragments
move the greatest distance
• This creates a distinct
banding pattern
• These bands can then
be used to map genes
• As well, this can be
used for “DNA
Fingerprinting” as each
person’s pattern of
bands is different
Modern Analysis
• Mapping genes using gel
electrophoresis takes an
incredibly long time
• Now, DNA is still cut into
fragments, but four different
colours of dyes are used
• A laser is run over the
fragments and a computer
records the reflected light
• Each of the colours
corresponds to a different
nitrogen base
• Therefore,
genes can be
now mapped by
computer at a
rate of over a
thousand base
pairs in a
minute (rather
than months of
work by hand)
http://bioweb.wku.edu
The Human Genome Project
• The first map of the human
genome was completed in
2000
• By 2003, a much more
complete and comprehensive
map was completed by an
international team of
scientists
18.2 – Protein Synthesis and Gene
Expression
• In the same year that Watson
and Crick published their
model of DNA, Frederick
Danger established that
proteins consist of long
chains of amino acids
• The sequence of the amino
acids determines the shape
and properties of the protein
• Ultimately, the interactions
between proteins drives how
cells operate
• Scientists began to wonder if
the sequence in DNA was
related to the sequence of
amino acids in a protein
• It was soon shown that the
genetic code in fact does
determine the sequence of
amino acids found in proteins
Gene Expression
• Genetic information flows
from DNA to RNA to protein
• This is known as the “central
dogma” of gene expression
DNA and Protein Synthesis
• Although DNA contains very few
different structural components, it
is responsible for coding for huge
amounts of information
(about 25, 000 genes in a human)
• The sequence of the base pairs is
the key to coding for different
proteins
• Because there are only 4 nitrogen
bases and 20 amino acids, 3 bases
together can code for different
proteins (two bases can only code
for 16, while three can code for 64
possible combinations)
Codons
• A codon is a 3-base pair
segment of DNA
• Each codon corresponds to a
particular amino acid, or it
also may correspond to an
initiator “go” or terminator
“stop” command
mRNA
• to produce proteins,
the DNA does not
leave the nucleus
• a carrier molecule
known as messenger
RNA (mRNA) is used
to carry the code to
the ribosomes which
produce protein
http://tigger.uic.edu
Transcription
•
•
•
•
The DNA strand “unzips”,
exposing the nucleotides
Nucleotides in the mRNA are
arranged using the
complementary nucleotides
on the DNA as a blueprint
The mRNA chain fuses and
is moved to the ribosome
The DNA strands rejoin
http://fig.cox.miami.edu
Translation and Protein Synthesis
• the single-stranded mRNA
attaches itself to the small
ribosome like a ribbon
• initiator codons in the mRNA
turn on protein synthesis
• transfer RNA (tRNA)
molecules in the cytoplasm
pick up amino acids and bring
them to the mRNA
Overview – Synthesis of Protein
•
•
•
•
•
•
DNA “unzips”
mRNA makes a complementary
copy of the DNA
mRNA is taken to the ribosomes
The ribosomes match the mRNA
with tRNA that carry amino acids
The amino acids form a chain,
which becomes a protein
the mRNA “stop” codon is read,
and synthesis stops
Protein Synthesis
Animation
The Genome and Proteome
• Genomics is the study of
entire genomes and how the
genes interact
• However, study of the
proteome (the proteins
produced by the genome) is
often more important
because they are the
functional parts of the
genome
Mutations and Genetic
Recombination
• Genomes are not constant
• Mutations occur from time to
time
• Mutations occurring in body
cells are called somatic cell
mutations
• However, only mutations
occurring in reproductive
cells (germ line mutations)
will be passed on to offspring
Errors and Mutations
• If there are 3 billion base
pairs in the DNA of each of
your cells, even 1 mistake in
1000 could cause up to
3,000,000 mutations during
each replication
• However, mistakes in
duplicating DNA are very
infrequent
• This is because
“proofreading” enzymes look
for mismatched base pairs
and make repairs
Types of Mutations
• Point Mutation
• Silent Mutation
Types of Mutation
• Mis-sense Mutation
• Nonsense Mutation
Types of Mutations
• Frameshift Mutation
• Chromosomal Mutation
Causes of Mutations
• Some mutations occur
naturally (spontaneous
mutations)
• These mutations may be
caused by incorrect base
pairing by DNA polymerase
during replication
• Some mutations, however,
are caused by substances or
events known as mutagens
Physical Mutagens
• Physical mutagens are events
that change DNA sequences
Chemical Mutagens
• Chemical mutagens are
chemicals that cause changes
in the DNA sequence
• Many of these are
carcinogenic (cancercausing)
Mutations and Variation
• Genetic variation is a result
of mutations
• This is because changes in
the DNA are the only source
of variation at a heritable
level
• This variation can eventually
become an adaptation if
there is a change in the
environment that favors that
new variation
Mitochondrial DNA
• Mitochondrial DNA (mtDNA)
is a short genome found in
the mitochondria
• This may be a holdover from
a time where mitochondria
may have been free-living
organisms
• mtDNA is always identical
between mother and child,
and can therefore be used to
trace maternal lineage
Gene Recombinations
• In a laboratory, restriction
and ligase enzymes can be
used to put genes into small
organisms such as bacteria to
study individual genes
Restriction Enzymes (Endonucleases)
• Restriction enzymes,
such as Eco R1, cut
up DNA at specific
sites
• These sites have
“sticky ends” which
tend to bond with
“sticky ends” that
are created by other
restriction enzymes
DNA Fingerprinting
• DNA fingerprinting is carried
out using RFLPs (Restriction
Fragment Length
Polymorphisms)
• These RFLPs, which are
“chunks” of DNA are
produced when restriction
enzymes (which are found in
bacteria) cut up DNA into
small segments
• The length of the RFLPs differ
from person to person
DNA Fingerprinting
• The DNA fragments are
transferred to a gel that has a
current run through it
• The current pulls the DNA
fragments through the gel
• The smallest fragments move
the furthest, so a set of
bands is produced that is
unique to each individual
Gene Sequencing
Gel Electrophoresis Animation
Ligase Enzymes
• The ligase enzymes are
then used to reassemble
the DNA segment, often
in a vector plasmid
• These plasmids are then
introduced to bacteria,
which take in the new
DNA and incorporate it
into their own
Uses of Recombinant DNA
• Bacteria can now be used to
produce many human
products
• Insulin, erythropoietin,
clotting factors, antibodies,
GH, and proteins that fight
cancers are all being
produced by using bacteria
• The advantage to using
bacteria is that they
reproduce quickly (ensuring
significant levels of the
products), and they are
cheap to grow
Oncogenes
• most cancer cells show
nitrogen base substitution
• cancer-causing genes
(oncogenes) seem to turn on
cell division
• the oncogenes seem to be
present in normal DNA
strands, but they are not
active
Regulator Genes
• one theory to explain this is
that the oncogene must be
transposed to the proper site
on the chromosome to
become active
• most genes on the
chromosomes are structural
genes, they produce required
proteins
• these structural genes are
controlled by regulator
genes that produce proteins
that turn other genes “on” or
“off”
http://www.brooklyn.cuny.edu
• the most common oncogene, ras,
is found in 50% of colon cancer
cases and 30% of lung cancer
cases
• Ras makes a protein that acts as a
“on” switch for cellular division
• however, the oncogene produces
a protein that prevents this gene
from turning “off”
• this may occur if the regulator and
structural genes, which are
normally adjacent to each other,
are separated
18.4 – Genetics and Society
• Biotechnology allows us to
create new products and
technologies from natural
biological systems
• However, biotechnology has
also raised a large number of
ethical, social, and legal
issues
Gathering and Managing Genetic
Information
• Computers now allow us to
analyze and store large
amounts of genetic
information
• There are computerized gene
banks and DNA libraries that
provide researchers access to
large amounts of genetic
information
DNA Microarray
• Microarrays work in a 4-step
sequence:
1.
2.
3.
4.
• The microarrays allow
scientists to study the action
of thousands of genes at
once
• Scientists can use these to
compare the expression of
genes in different
environments
Public Benefits of Genetic Research
• Most of the important
benefits of genetic research
are the development of new
treatments for genetic
disorders
• It is also now possible to
study how genes affect the
activity of medications
• As well, the information
gathered from the Human
Genome Project is publicly
available for anyone who
wants to perform research
Ownership of Genetic Information
• Many companies have
patented genes and
genetically modified
organisms
• This presents some
controversy, as some people
do not believe that one
should be able to patent a
living thing
Biotechnology Products
• Many products can be
produced through genetic
modification of organisms
• Transgenic organisms are
organisms that have a gene
from a different species
spliced into their genome
• Ex:
Medicinal Bacteria
• In 1982, insulin produced by
transgenic bacteria was
approved for medical use
• Bacteria are ideal for the
production of hormones
because they are easy to put
genes into, and they are
cheap to grow
• Bacteria can also be used for
bioremediation (cleanup of
environmental toxins by
living things)
Transgenic Plants
• Recombinant crops now
account for more than half of
the corn and canola produced
in North America
• Transgenic plants can also be
grown in new places
• Sometimes, the new
transgenic plants can
combine nutritional value of
more than one plant in one
(such as golden rice, which is
sent to developing countries)
Cloning
Assessing Risks
•
1.
2.
3.
4.
When considering proposals for
approving transgenic products in
Canada, the following criteria are
used:
Potential social, environmental,
and economic costs and benefits
The process by which the product
is made, including the source of
the genetic material
The biological characteristics of
the transgenic product
The potential health effects of
the product
Objections to the use of GMOs
1. Environmental threats (such
as herbicide-resistant
“superweeds”
2. Health effects (not enough
research is done on longterm effects of consuming
transgenic products)
3. Social and economic issues
(is the cost of research
better spent somewhere
else?)
Diagnosis and Treatment of
Genetic Disorders
•
Prenatal screening can be
carried out using either:
1. Amniocentesis
2. Chorionic Villus Sampling
Treating Human Genetic Disorders
• We now have a complete
map of the human genome (it
was completed in 2000)
• Therefore, it is now possible
to locate damaged genes
based on their DNA sequence
• But how can this be done?
Viral Transduction & Transformation
• Viruses are simply strands of
DNA or RNA within a protein
shell
• They work by injecting their
genetic material into a cell’s
genome
• When the cell reads its own
DNA, it also then reads the
virus DNA
• As a result, more virus
particles are formed
The Virus “Life Cycle”
• However, we can use this to our
advantage
• If a therapeutic gene is spliced
into viral DNA, then the virus
will insert the therapeutic gene
into the cell’s DNA as well
• As a result, the new cell will
have a functional gene that has
replaced the damaged gene
• In theory, if germ-line cells
were targeted by these viruses,
then modifications could be
passed on to the next
generation
Biological Warfare
• biological warfare has been
used since 600 BC
• generally, a bioweapon is
considered to be a diseasecausing living organism, or
the toxins produced by a
living organism
• one of the favored biological
agents is anthrax, a bacteria
(bacillus antractis) that
forms spores which protect it
from environmental factors
http://www.safebiology.com
Anthrax
• if inhaled into the lungs,
anthrax bacteria can be fatal
• however, anthrax cannot be
passed from one human to
another
http://www3.niaid.nih.gov
http://www.postgradmed.com
“Good” & “Bad” Agents:
• Some organisms, such as HIV, do
not make good biological weapons
(they need to enter the body in
ways that make it difficult to
deliver)
• Others produce symptoms and
death so quickly that they are not
easily spread (the agent “burns”
itself out)
• The “best” organisms to use for
biological weapons would be
those that can be delivered easily
(usually this means an airborne
agent), and have a high mortality
rate
• Smallpox is such an example,
because it is easily transmitted
and kills many of the people it is
infected with
• As well, by using genetic
engineering, viruses such as
smallpox can become even more
deadly by preventing successful
immunization
• It is also possible that the genes
could be manipulated so that a
virus that is normally not easily
transmitted could become
airborne (such as Ebola)
http://www.lewrockwell.com
http://webs.wichita.edu
Global Guide to Bioweapons