Download Regulating Gene Expression

Today’s Plan: 2/16/11
 Bellwork: Talk about yesterday and
the test (30 mins)
 Casting gels for tomorrow (30 mins)
 DNA Tech Notes (the rest of class)
Today’s Plan: 2/18/2010
 Bellwork: Test discussion (15 mins)
 Transformation
 Notes, if time
Today’s Plan: 2/19/2010
 Bellwork: Flies and Test Corrections
(15 mins)
 AP Lab 6 Lab Bench Intro to the
Molecular Biology Lab (30 mins)
 Continue notes (the rest of class)
 Pack/Wrap-up (last few mins of class)
Today’s Plan: 2/22/10
 Bellwork: Cast Gels (30 mins)
 Count flies and look at bacteria (while
gel is dissolving
 Practice and run gels (45 mins)
 Continue with notes (the rest of the
period)
Today’s Plan: 2/23/10
 Bellwork: Read Gels/answer
questions/fly counts (30 mins)
 Set up for Lunch demos (30 mins)
 Continue notes (the rest of class)
Today’s Plan: 2/24/10
 Bellwork: Flies/Test Q&A (20 mins)
 DNA Tech test (the rest of class)
Today’s Plan: 9/15/09
 Bellwork: Intstructions (5 mins)
 Research for Discussion (30 mins)
 Bioethics discussion (the rest of class)
Today’s Plan: 9/16/09
 Bellwork: Last Presentation (10 mins)
 Senses Stations (50 mins)
 Continue with DNA Tech notes (the
rest of class)
Today’s Plan: 9/17/09
 Bellwork: Class Optional Taste Demo
(10 mins)
 Finish Senses Stations (50 mins)
 Finish DNA Tech notes (the rest of
class)
Today’s Plan: 9/18/09
 Bellwork: Test Q&A (15 mins)
 DNA Tech Test (as needed)
 If you finish your test early, work on
the senses questions.
Regulating Gene Expression
 Prokaryotes (Bacteria)
 Often live in erratic environments
 Need to turn on and off genes in response to the
environment
 Use Operons to regulate genes.
 These are DNA sections that are regulated by
repressors which can turn off the promoter site,
and keep transcription from happening.
 Operons contain several related genes, each
with its own start and stop codon
 The convenience of the operon is that there
only has to be 1 on/off switch for all of the
genes
Regulating Operons
 Operons contain a promoter region (consisting
of an attachment point for RNA polymerase and
an operator for the repressor to attach to when
not needed), and the genes for a specific job
 Repressors come from a regulatory gene at a
point away from the operon
 Repressible operons are always on unless a
repressor is bound (ex: trp operon-repressor is
inactive until bound to a tryptophan molecule)
 Inducible operons are off unless an inducer is
present to inhibit the repressor’s hold on the
DNA (ex: lac operon-repressor is active unless
bound to allolactose)
Figure 17-4
E. coli
Galactoside
permease
-Galactosidase
Glucose
Galactose
Lactose
Plasma membrane
Figure 17-8
lac operon
DNA
lacl
promoter
lacl
Promoter Operator
of lac operon
lacZ
lacY
lacA
Figure 17-7
Repressor present, lactose absent:
Repressor binds to DNA.
No transcription occurs.
The repressor blocks transcription
Repressor
synthesized
DNA
Normal
lacl gene
Repressor present, lactose present:
Lactose binds to repressor,
causing it to release from
DNA. Transcription occurs
Repressor
(lactose acts as inducer).
synthesized
Normal
lacl gene
lacZ
lacl+
RNA polymerase
bound to promoter
(blue DNA)
TRANSCRIPTION BEGINS
-Galactosidase Permease
mRNA
lacZ
lacl+
RNA polymerase
bound to promoter
(blue DNA)
No repressor present, lactose present
or absent:
Transcription occurs.
No functional
repressor synthesized
Mutant
lacl gene
lacY
lacY
Lactose-repressor
complex
TRANSCRIPTION BEGINS
mRNA -Galactosidase Permease
Lacl –
lacZ
RNA polymerase
bound to promoter
(blue DNA)
lacY
Figure 17-9
When tryptophan is present, transcription is blocked.
Repressor Tryptophan
No transcription
Operator
RNA polymerase
bound to promoter
When tryptophan is absent, transcription occurs.
TRANSCRIPTION
5 genes coding for enzymes involved
in tryptophan synthesis
RNA polymerase
bound to promoter
Figure 17-10
lac operon
trp operon
Catabolism
(breakdown of lactose)
Anabolism
(synthesis of tryptophan)
Repressor
Lactose
Repressor
Tryptophan
Tryptophan
binds to
repressor
Lactose binds
to repressor
Lactoserepressor
complex
releases from
operator
Operator
Tryptophanrepressor
complex binds
to operator
Operator
Transcription
of lac operon
TRANSCRIPTION
No more
transcription
of trp operon
Positive Gene Regulation
 Bacteria needs to sense whether or not glucose as
well as lactose are present in its environment.
 Bacteria prefer to use glucose for glycolysis, and
therefore only use lactose when there isn’t glucose
available
 Cyclic AMP (cAMP) accumulates when glucose is
scarce.
 cAMP binds to a regulatory protein, catabolite
activator protein (CAP) and the complex becomes an
activator that binds to the DNA just upstream of the
promoter.
 The activator makes it more likely that RNA
polymerase will attach to the operon and transcribe
Figure 17-14
Glucose inhibits the activity of the enzyme adenylyl cyclase, which catalyzes production of cAMP from ATP.
Glucose inhibits
this enzyme
ATP
Adenylyl cyclase
cAM
P
Two phosphate
groups
The amount of cAMP and the rate of transcription of the lac operon are inversely related to the concentration
of glucose.
CAP
HIGH
glucose
concentration
INACTIVE
adenylyl cyclase
LOW
cAMP
CAP does not
bind to DNA
Infrequent transcription
of lac operon
(Cell continues to use
glucose as energy source.)
cAMP
CAP
LOW
glucose
concentration
ACTIVE
adenylyl cyclase
HIGH
cAMP
CAP-cAMP complex
binds to DNA
Frequent transcription
of lac operon
(Cell uses lactose
if lactose is present.)
Prokaryote
Genomes
vs.
 Smaller Genome
 Fewer Genes
 Higher gene
density (more
genes in a smaller
segment of DNA
 Relatively few
noncoding regions
and protein genes
are continuous
Eukaryote




Large Genome
Many more genes
Lower gene density
Many noncoding
regions (introns)
and protein genes
are not continuous
Eukaryotic Gene Regulation
 Recall that in complex organisms, the
complete genome is in all cells, but only the
genes necessary for the function of the
individual cell are turned on in that
individual cell
 In stead of regulating just transcription, as
bacteria do, eukaryotic cells can regulate
gene expression at any step from DNA to
protein
Figure 18-1
Nucleus
1. Chromatin remodeling
2. Transcription
Chromatin
(DNA-protein complex)
“Open” DNA
(Some DNA not closely
bound to proteins)
Primary transcript
(pre-mRNA)
3. RNA processing
Cap
Tail
Mature mRNA
Cytoplasm
4. mRNA stability
Degraded mRNA
(mRNA lifespan varies)
5. Translation
mRNA
Polypeptide
6. Post-translational
modification (folding,
transport, activation,
degradation of
protein)
Active protein
DNA Regulation

Chromatin is DNA that is packaged with proteins, called histones.
The basic unit of chromatin is called the nucleosome




DNA Methylation




Under normal conditions, the lysine tails of histones extend out from
the nucleosome and are attracted to other nucleosomes
Histone acetylation attaches acetyl groups to these tails, making them
no longer attracted to other histones, which loosens up the chromatin
to make transcription easier
It’s also been shown that methyl groups are also added to the histone
tails, which can promote condenstion of the chromatin
Methyl groups can be attached to cytosine, again causing condensation
of the chromatin
Some research shows that heavily methylated areas recruit
deacetylation enzymes, which dually promotes condensation
This appears to be an important regulatory step from embryo to
mature organism. In cases where a template strand is methylated, the
cell matches the methylation in the daughter strand after replication so
that the cell stays specialized
Epigenetic Inheritance

Inheritance of traits not directly involved with the DNA sequence, such
as alteration of methylation patterns
Figure 18-2
Nucleosomes in chromatin
Nucleosomes
DNA
Nucleosome structure
Linker
DNA
H1 protein attached
to linker DNA and
nucleosome
DNA
Nucleosome
Group of
8 histone
proteins
In some cases, nucleosomes may be grouped into
30-nanometer fibers.
30 nm
Figure 18-4
Condensed chromatin
Decondensed chromatin
Acetyl group
on histone
The Eukaryotic Gene
 Recall that even Eukaryotic genes
contain a promoter site, on which the
transcription initiation complex
assembles
 There are introns and exons within
the gene and control elements that
don’t code but bind proteins
Figure 18-7
Enhancer
Promoter
Start site
Enhancer
PromoterExon Intron Exon
proximal element
Intron Exon
Enhancer
Regulating Transcription

Transcription factors bind to the DNA and make it easier for
RNA polymerase to bind



These can be general transcription factors, if they are
necessary for all protein-coding genes, and if they result in a
low rate of transcription
Specific transcription factors are proteins that attach to only
certain genes and generally produced a high rate of
transcription in cells needing particular genes
Enhancers are generally found thousands of nucleotides
upstream from the gene and are called Distal control
elements



Activators and repressors can bind to these elemets to
regulate the initiation of transcription by interacting with
mediator proteins
The DNA can also be bent so that these form a transcription
initiatinon complex
The actual number of activators is small, but it’s the
combination of control elements (proteins, activators, etc) that
is unique to each gene
Figure 18-10
THE ELEMENTS OF TRANSCRIPTIONAL CONTROL: A MODEL
Chromatin remodeling
complex (or HATs)
Regulatory
transcription
factor
1. Regulatory transcription
factors recruit chromatinremodeling complex, or
HATs. Chromatin
decondenses.
Exposed
DNA
Promoter-proximal
element
Promoter
Intron
Exon
Intron
Exon
2. When chromatin
decondenses, a region of
DNA is exposed, including
the promoter.
Transcribed portion of gene for
muscle-specific protein
3. Regulatory transcription
Regulatory
transcription
factors
Promoter-proximal element
Exon
Co-activators
Promoter
Intron
factors recruit proteins of
the basal transcription
complex to promoter.
Note looping DNA.
Exon
Basal transcription complex
4. RNA polymerase II
Intron
Exon
RNA polymerase II
Basal transcription complex
completes the basal
transcription complex;
transcription begins.
Do Eukaryotes have Operons?
 While there are co-expressed genes in
Eukaryotes, each gene has its own
promoters
 Some co-expressed genes are clustered,
while others are on different chromosomes
 Coordinate control of co-expressed genes
seems to be regulated by the genes having
the same combination of control elements
at the same time, usually in response to a
signal outside of the cell
Figure 18-14
Signaling
molecule
Cell-surface receptor
Inactive STAT protein
(two single polypeptide chains)
Cytoplasm
Activated STAT protein
(dimer of two polypeptide chains)
Enhancer
TRANSCRIPTION
Transcription activated
Nucleus
Post-transcriptional Gene
Regulation

RNA Processing-Alternative RNA splicing


mRNA Degradation


In Prokaryotes, mRNA is degraded within a few minutes, but
Eukaryotes’mRNA can last for days or weeks
Initiation of Translation



The same transcript may result in different mature RNA
depending on which segments are treated as introns
Proteins can block the 5’ end of an mRNA, preventing
attachment by the ribosome
In other cases, the poly-A tail is not synthesized long enough
until the organism is ready for the protein
Protein processing and Degradation


Many protiens require post-translation modifications, such as
reversible phorphorylation
Some proteins are tagged with ubiquitin, which alerts the
proteasomes to their presence and degrades them
Figure 18-12
Tropomyosin gene
Intron
Exon
Intron
Exon
Processed mRNAs
Skeletal muscle
Smooth muscle
Intron
Exon
Exon
Some exons are specific to tropomyosin
in skeletal or smooth muscle; some
exons are common to both muscle types
Noncoding RNAs
 These are other molecules, like tRNA
and rRNA
 Many more RNAs are discovered
frequently and have a variety of
functions within the cell
 Apparently, not all DNA is supposed
to code for proteins, and in fact,
doesn’t
MicroRNAs
 These are small pieces of RNA that are complimentary
to mRNA
 Called miRNA
 Formed from a large primary transcript that bends
into one or more hairpin turns
 An enzyme, called a “dicer” cuts these away, forming
double-stranded mRNA
 One strand degrades, while the other forms a
complex with a protein
 These complexes can bond with and interfere with
mRNA (if they’re complimentary at some part), and
can degrade it (if they’re complimentary along the
length)
 Another type of RNA, small interfering RNA (siRNA)
can also interfere with mRNA’s function. These are
formed from larger, double-stranded precursor RNA
molecules
 Collectively, this is called RNAi (RNA interference)
Figure 18-13
miRNAs TARGET CERTAIN mRNAS FOR DESTRUCTION
RNA
hairpin
DNA
1. Transcription of
a microRNA gene.
RNA
polymerase
2. Initial transcript is
Precursor
miRNA
processed into a precursor
micro RNA (miRNA).
Cytoplasm
3. Enzyme in cytoplasm
Enzyme
cuts out hairpin loop,
forming a mature miRNA.
Mature
miRNA
Single-stranded
miRNA
RISC
protein
complex
4. miRNA becomes
single-stranded and binds
to RISC protein complex.
5. miRNA, held by RISC,
binds to complementary
sequence on target mRNA.
Target
mRNA
6. Enzyme inside RISC
cuts mRNA.
Gene Expression and Embryonic
Development
 The genome (including the
cytoplasmic genome) contains a
program for cell differentiation in the
embryo
 Cytoplasmic determinants (RNA and
DNA in the cytoplasm-matrolineal)
get divided unevenly, which may
contribute to cellular differentiation
 The embryo’s own cells may also
induce changes in the other
embryonic cells
Sequential Regulation of Gene
Expression during Differentiation
 Once a cell begins the process of
differentiation, it is irreversible-even
if the cell is moved to another part of
the embryo
 Each cell type produces its own
tissue-specific proteins from
transcribed mRNA in genes that are
turned on
Figure 21-7
VISUALIZING mRNAs BY IN SITU HYBRIDIZATION
1. Start with a singleDNA
probe
Label
stranded DNA or RNA
probe, complementary
in sequence to target
mRNA.
2. Add label to probe
Embryo
(a radioactive atom
or an enzyme that
catalyzes a colorproducing reaction).
3. Preserve the
DNA
probe
specimen (in this
case, a Drosophila
embryo).
4. Treat preserved
DNA
probe
Target mRNA
cells or tissues to
make them permeable
to probe. Add many
copies of probe.
5. Probe binds to
target mRNA. Labeled
probe that does not
bind to target mRNA
is excess, and is
washed away.
Target mRNA
6. In this case, target
Anterior
mRNAs are concentrated in the anterior
end of the embryo.
The label shows up as
Posterior black in this image.
Setting up the body plan
 Pattern formation is the organization of the body and
is regulated by cytoplasmic determinants and
inductive signals from neighboring cells
 Early on, positional information, such as where the
“head” and “tail” are to be, is established
 In Drosophila, a series of homeotic (hox) genes
control the segmentation of the body and position of
body parts
 Body Axis is determined by maternal effect geneswhen there is a mutant in the mother, there are
mutations in the offspring, regardless of the
offspring’s genotype
 Bicoids are two-tailed mutants come from mutations
in these maternal effect genes
 These genes produce Morphogens, that concentrate
in certain segments and determine what that
segment will become
Figure 21-6
A normal fruit-fly embryo
Head
Thoracic
segments segments
Abdominal
segments
A bicoid mutant
Abdominal
segments
Abdominal
segments
Figure 21-13
The location of Hox genes on the fly chromosome correlates
with their pattern of expression in fly embryos.
Hox genes
Fly
embryo
Head
Thorax
Abdomen
The location of Hox genes on the mouse chromosome
correlates with their pattern of expression in mouse embryos.
Hox genes
Mouse
embryo
Figure 21-12
Homeotic mutant
Normal fruit fly
Homeotic mutant
Antennae
Haltere
Wings in place of halteres
Legs in place of antennae
Wrapping up Eukaryotic Genomes
 Eukaryotic Genomes consist of many noncoding and repetitive sequences that
scientists now suspect actually serve
important purposes within the cells
 Transposable Elements-”Jumping Genes”
 Transposons are genes that move via a DNA
intermediate
 Retrotransposons are what most transposable
elements are and they move via an RNA
intermediate
Figure 20-5
HOW LINE TRANSPOSABLE ELEMENTS SPREAD
Gene for reverse transcriptase
Gene for integrase
1. A long interspersed
nuclear element
(LINE) exists in DNA.
DNA
Cytoplasm
Original location of LINE (1–5 kb)
2. RNA polymerase
LINE
protein
transcribes LINE,
producing LINE mRNA.
LINE mRNA
3. LINE mRNA exits
RNA polymerase
Ribosome
LINE mRNA and LINE proteins
nucleus and is
translated.
Reverse transcriptase
4. LINE mRNA and
Integrase
proteins enter nucleus.
cDNA
5. Reverse
mRNA
transcriptase makes
LINE cDNA from mRNA,
then makes cDNA
double stranded.
Reverse
transcriptase
Integrase
6. Integrase cuts
chromosomal DNA
and inserts LINE cDNA.
7. New copy of LINE is
Original copy
New copy
integrated into genome.
Figure 20-8
GENE DUPLICATION BY UNEQUAL CROSSOVER
1
2
3
4
5
1. Start with two homologous
6
chromosomes containing the
same genes (numbered 1–6).
Homologous
chromosomes
1
2
3
4
1
2
3
4
1
3
2
5
5
6
2. The genes misalign during
6
meiosis I. Crossing over and
recombination occur.
4
5
6
Gene deletion
1
2
4
5
6
Gene duplication
1
2
3
3
4
5
6
3. Gene 3 has been deleted
from one chromosome and
duplicated in the other
chromosome.
The Molecular Biology of Cancer
 As we learned before, cancers are unregulated cells.
 Scientists think that that oncogenes-are the cancercausing genes as well as random mutation from DNA
damage
 Proto-oncogenes are the normal genes that code for
proteins that regulate the cell cycle
 Tumor supressor genes inhibit cell division
 There are generally 3 ways that protooncogenes
become oncogenes
 DNA movement w/in a genome
 Point mutations
 Amplification of a proto-oncogene
The development of Cancer
 Cancer requires multiple mutations and at
least 1 oncogene
 Cancers can begin as benign polyps,
tumors, etc, but the longer that these exist,
the longer there is for the necessary
mutations to accumulate
 Viruses also play a role in the development
of some cancers
 Retroviruses have oncogenes that can be
donated to the host cell
 The viral DNA may also be inserted in such a
way that it disrupts a tumor-supressing gene.
What about Genetic Predisposition?
 It makes sense, that if oncogenes are partially
responsible for cancer, that certain cancers should run
in families
 Examples of cancers with a strongly-heritable
component are colorectal cancer and breast cancer
 In Breast cancer, mutations in the BRCA1 or BRCA2
genes appear to be responsible for many breast
cancers
 These genes play a role in the cell’s DNA damage
repair proteins
 It makes sense, then, that avoiding mutagens would
lower the risk of cancer, even if one has the
mutations in his/her genome
Viruses
 At their simplest, these are a piece of genetic material
with a protein coat (called the capsid)
 These are considered non-living b/c they have no
metabolism, homeostasis, growth, and require a host
cell to carry out their functions
 Are extraordinarily small, since they are active inside
of cells.
 They can contain traditional, double-stranded DNA,
single-stranded DNA, or even RNA
 Recall that they’re specific to their hosts-the capsid
must fit into a receptor on the host cell in order to
infect the cell, and there’s a lot of variety in the
capsid
 A few, like influenza, have a viral envelope, derived
from the membranes of their host cell
Figure 35-7
Nonenveloped virus
Genome (in this
case, DNA)
Capsid (protein)
Enveloped virus
Viral protein
Genome (in this
case, RNA)
Host protein
Capsid (protein)
Envelope
(phospholipid
bilayer)
Figure 35-17
Figure 35-6
Bacteriophage T4
Tobacco mosaic virus
Adenovirus
Influenza virus
Figure 35-21
Healthy leaf
Leaf infected with virus
Figure 35-1-1
Brain and CNS
encephalitis
rabies
polio
herpes zoster
yellow fever
Ebola
dengue
West Nile
Lymphatic and
immune systems
Epstein-Barr
HIV
paramyxovirus
(e.g., measles)
Trachea and lungs
parainfluenza
RSV
influenza
adenovirus
Heart
coxsackie
Figure 35-1-2
Digestive track
and liver
hepatitis A, B, C, D, E
rotavirus
Blood vessels and
blood cells
erythrovirus
Ebola
hantavirus
Reproductive organs
herpes 2
papillomavirus
Skin
rubella
variola
papillomavirus
herpes 1
molluscum
contagiosum
Skeletal muscles
coxsackie
Peripheral nerves
rabies
Figure 35-15-Table 35-2
Viral Cycles



All cycles begin with the virus binding to the host cell


Some are taken in by endocytosis
Others inject their genome

Also called virulent phages, because these infect, degrade the
host’s DNA, reproduce, and kill the host cell right away
(rhinovirus, influenza, T4 bacteriophage)
Lytic Viruses
Lysogenic Viruses


These are called temperate phages because these inject their
genome (prophage), and integrate it within the host’s DNA, so
they can “hide” inside of the host until they’re triggered (ex:
HSV, HPV, lambda phage)
Retroviruses

These are special lysogenic viruses whose prophage is made of
RNA, so they must inject reverse transcriptase (rt) as well as
the prophage in order to integrate with the host’s
chromosomes (ex: HIV)
Figure 35-8a
LYTIC REPLICATION RESULTS IN A NEW GENERATION OF VIRUS PARTICLES AND THE DEATH OF THE HOST CELL.
Host-cell
genome
Virus particle
DNA mRNA Protein
1. Viral genome enters
2. Viral genome
host cell.
is transcribed; viral
proteins are produced.
DNA
Protein
3. Viral genome is replicated.
6. Free particles
in tissue or
environment
are transmitted
to new host.
5. Particles exit
4. Particles assemble
to exterior.
inside host.
Figure 35-8b
LYSOGENIC REPLICATION RESULTS IN VIRUS GENES BEING TRANSMITTED TO DAUGHTER CELLS OF THE HOST.
1. Viral genome
2. Viral genome
3. Host-cell DNA
4. Cell divides. Virus is transmitted
enters host cell.
integrates into hostcell genome.
polymerase copies
chromosome.
to daughter cells.
Figure 35-14
cDNA
RNA template
First, reverse transcriptase
synthesizes cDNA from RNA
Double-stranded DNA
cDNA
template
Then, reverse transcriptase
synthesizes double-stranded
DNA from cDNA
Preventing Viruses
 Some cells have evolved defenses
against these viruses in the form of
restriction enzymes that can destroy
the viral DNA
 Vaccination helps animals avoid viral
infection
 Being infected allows the immune
system to learn to detect and fight
existing strains of viruses
Figure 35-9
HOW VACCINATION WORKS
The antigens are usually
protein components of a
virus capsid or envelope
The cells that produce specific
antibodies remain active for a
long time—years or decades
Virus
1. Viral antigens
2. Antigens bind
3. These cells stimulate
4. Later, if the host organism
5. Viruses that are
(in red) are introduced into the
body.
to receptors on
certain immune
system cells.
other immune system cells
to produce antibodies
(in green) to the virus.
is exposed to actual virus
particles, the antibody-producing
cells are activated. The virus
particles become coated with
antibodies.
coated with antibodies
are destroyed by
immune system cells.
Emerging Viruses
 New Viruses occur because of 3 main
causes:
 Mutation of existing viruses-especially RNA
viruses, which mutate faster
 Viruses coming from a small, isolated human
population
 Viruses jumping from one species to anotherespecially in closely-related species
 Epidemic=emergence of a new strain of an
existing virus
 Pandemic=global epidemic
Plants and viruses
 Yes, plants get viruses too
 Transmission occurs in 1 of 2 ways:
 Horizontal transmission-plant is infected by an
external source of virus, especially if the
epidermis of the plant is damaged (herbivore
damage is especially bad b/c herbivores can act
as horizontal transmitters)
 Vertical transmission-plant inherits the virus
from the parent. The virus can spread through
the plasmodesmata
Viroids and Prions
 Viroids=circular pieces of RNA that infect
plants
 These reproduce inside of the plant’s cells and
cause errors in the regulation of growth
 Infected plants typically exhibit stunted growth
 Prions=infectious proteins (ex: BSE=mad
cow disease)
 These develop slowly (up to 10 year incubation
period)
 These are indestructible
 Scientists believe that these are abnormallyfolded proteins, that, when they enter a cell that
has the normal proteins, corrupt these
DNA Technology
 Involves a number of techniques for
identifying, copying, cutting, and
modifying DNA
 These are all part of the field of
biotechnology
 Genetic engineering-directly
manipulating the DNA of an
organism, is also part of
biotechnology
DNA Cloning
 Involves copying DNA-useful for studying specific
genes, since you can keep a library of cloned genes,
rather than search an entire genome for them
 Most cloning is done with bacterial plasmids-circular
pieces of DNA in a bacteria that contain only a few
genes and are separate from the bacteria’s main
chromosome (these are called cloning vectors)
 In recombinant DNA, a plasmid is removed from the
bacteria and spliced with a new piece of DNA.
 This can be re-inserted into the bacteria, which will
both express the gene and copy it every time the cell
divides
 The gene we inserted is called the donor gene
 The process of putting the gene back into the
bacteria is called transformation
Figure 19-2
GENES CAN BE CLONED BY INSERTING THEM INTO PLASMIDS
Recognition
site
5
3
3
5
Recognition
site
5
3
Restriction
endonuclease
(EcoR1)
3
5
Plasmid
Plasmid
Recombinant
plasmid
Sticky end
1. Plasmid DNA
2. Attach the same
3. A restriction endonuclease
4. Sticky ends on
5. Use DNA ligase to
contains a recognition
site for a restriction
endonuclease.
recognition site to the
gene that will be
inserted into the
plasmid.
makes staggered cuts at each
of the recognition sites,
creating “sticky ends.”
plasmid and on gene to
be inserted bind by
complementary base
pairing.
catalyze a phosphodiester
bond at points marked by
green arrows, “sealing” the
inserted gene.
Restriction Enzymes
 These are enzymes that cut DNA at specific
recognition sequences (usually palindromic)
 Useful for many biotechnology applications
because we know their recognition
sequences
 Each resulting restriction fragment (DNA
cut with a restriction enzyme), has stickyends so that it is easy to splice
Storing Cloned Genes
 Genomic Library=cell clones containing the
recombinant plasmid
 Sometimes, phages are used as genomic libraries b/c
they can carry bigger inserts
 Scientists have also found mRNA extracts useful in
producing libraries b/c of the poly-A tail
 The tail is a useful primer for reverse transcriptase,
and can be used to make cDNA (complimentary DNA)
 The cDNA can then be inserted into the cloning
vector
 Bacterial Artificial Chromosomes (BAC) can also act as
libraries
 This is simply a large plasmid which contains the
inserts and genes necessary for replication
Figure 19-3-1
CREATING A cDNA LIBRARY THAT CONTAINS THE HUMAN GROWTH HORMONE GENE
Singlestranded
cDNA
mRNA
Doublestranded
cDNA
mRNA
Reverse
transcriptase
1. Isolate mRNAs from cells in pituitary
2. Use reverse transcriptase to
3. Make the cDNA double-
gland.
synthesize a cDNA from each
mRNA.
stranded.
Screening a Library for a Gene
 This involves creating a nucleic acid
probe that has a complimentary
sequence to the DNA we’re looking
for
 We can then see where this probe
hybridizes to find the gene
Figure 19-4
USING A DNA PROBE TO FIND A TARGET SEQUENCE IN
A COLLECTION OF MANY DNA SEQUENCES
Labeled probe
1. Single-stranded
DNA probe has a
label that can be
visualized.
2. Expose probe
to collection of
single-stranded
DNA sequences.
3. Probe binds to
complementary
sequences in target
DNA—and only to
that DNA. Target
DNA is now labeled
and can be isolated.
Expressing Eukaryotic cloned DNA
 Eukaryotic expression in bacteria is sometimes
difficult b/c the promoters and control sequences are
often different
 Scientists use an expression vector, a vector that has
a very active promoter region upstream from the
donor gene
 Scientists also occasionally need to use cDNA donor
genes b/c of the presence of introns in the eukaryotic
genes, making them unwieldy
 Yeasts can be used as cloning vectors to completely
bypass this problem
 Yeast Artificial Chromosomes (YACs) combine the
necessary origin for DNA replication, centromeres,
and telomeres, with the donor genes
 Sometimes, you need to use a eukaryotic vector b/c
only it is capable of the post-translational protein
modifications necessary for the protein to function
PCR
 Polymerase Chain Reaction allows the
scientist to amplify a sample of DNA
 Produces results within hours, rather
than days
 Involves thermal cycling to denature
(unzip) the DNA molecule with heat,
then cooling to promote annealing
(hydrogen bonding), and uses a heatstable DNA polymerase molecule
Figure 19-6
PCR primers must be located on either side of the target
sequence, on opposite strands.
5
3
Primer
3
5
Primer
Region of DNA to
be amplified by PCR
When target DNA is single stranded, primers bind and allow
DNA polymerase to work.
5
3
3
5
3
Primer
Primer
5
3
5
Figure 19-7
THE POLYMERASE CHAIN REACTION IS A WAY TO
PRODUCE MANY IDENTICAL COPIES OF A SPECIFIC GENE
3
dNTPs 5
3
5
1. Start with a solution
containing template
DNA, synthesized
primers, and an
abundant supply of
3 Primers
the four dNTPs.
5
2. Denaturation
Heating leads to
denaturation of the
double-stranded DNA.
5
3
5
5
5
5
3
5
3
5
3
5
5
3
3
3
3. Primer annealing
At cooler temperatures,
the primers bind to the
template DNA by
complementary base
pairing.
4. Extension
During incubation,
Taq polymerase uses
dNTPs to synthesize
complementary DNA
strand, starting at the
primer.
5. Repeat cycle of three
steps (2–4) again,
doubling the copies of
DNA.
6. Repeat cycle again,
up to 20–30 times, to
produce millions of
copies of template DNA.
DNA Sequences
 Gel Electrophoresis
 Uses charge and size to pull fragments of DNA
across a Gel
 Useful for generating characteristic banding
patterns, but also for looking at differences in
sequences, as the DNA fragments are cut with
restriction enzymes
 Southern Blotting
 Is a combination of gel electrophoresis and DNA
hybridization
 Probe is radioactive
Figure 20-7b
A gel showing minisatellite seqences from unrelated
and related individuals
X M
B U
U
U
Lane sources:
X: An unrelated individual
M: A mother
B: A boy the mother claims is her own
U: Undisputed children of the mother
Figure 19-8l
SOUTHERN BLOTTING: ISOLATING AND FINDING A TARGET DNA IN A LARGE COLLECTION OF
DNA FRAGMENTS
Location of restriction
endonuclease cuts
Samples from
four individuals
Sample 1
1
2
3
4
Doublestranded
DNA
Power
supply
Double-stranded
DNA
1. Restriction endonucleases cut
2. A sample consists of
3. During electrophoresis,
DNA sample into fragments of
various lengths. Each type of
restriction endonuclease cuts a
specific sequence of DNA.
all the DNA fragments of
various lengths. The
sample is loaded into a
gel for electrophoresis.
a voltage gel separates
DNA fragments by size.
Small fragments run faster.
Figure 19-8r
SOUTHERN BLOTTING: ISOLATING AND FINDING A TARGET DNA IN A LARGE COLLECTION OF
DNA FRAGMENTS
1
2
3
4
Singlestranded
DNA
Stack of
blotting paper
Labeled
probe DNA
Filter
Gel
Sponge in
alkaline solution
4. The DNA
5. Blotting. An alkaline
6. Hybridization with labeled
7. Visualize
fragments are
treated to make
them single
stranded.
solution wicks up through
the gel into blotting paper.
DNA fragments from the gel
are carried to the
filter, where they are
permanently bound.
probe. The filter is put into a
solution containing labeled
probe DNA. The probe binds
to DNA fragments
containing complementary
sequences.
fragments bound by
probe. Fluorescence
or autoradiography
(see BioSkills 7) is
used to find label.
DNA Sequencing
 This is when the sequence of bases
on the molecule is determined
 Mostly, this is automated now.
 Dideoxy Chain Method of Sequencing
is one of these methods, using
fluorescent dyes and can sequence a
segment up to about 800 bps
Figure 19-9
DIDEOXY SEQUENCING
Smaller fragments
5 end
Template DNA
3
5
5
Normal
dNTP
(extends 3
DNA strand)
Larger fragments
3 end
5
ddCTP’s
ddNTP
(terminates
synthesis) 3
ddATP’s
No OH
ddTTP’s
5
3
Labeled primer
Non-template DNA
Non-template DNA 5
3
Template DNA 3
5
ddGTP’s
1. Incubate a large number of normal dNTP’s with a small
number of ddNTP’s (in this case starting with ddGTP’s),
template DNA, a primer for the target sequence, and DNA
polymerase.
2. Collect DNA strands that are produced. Each
3. Repeat process three more
4. Line up different-length strands by size using gel
strand will end with a ddGTP (corresponding to
a C on the template strand).
times using ddCTPs, ddATPs, and
ddTTPs, which will terminate
synthesis where G’s, T’s, and A’s
occur on the template strand,
respectively.
electrophoresis to determine DNA sequence.
DNA
sequence
Figure 19-10
FLUORESCENT MARKERS IMPROVE SEQUENCING
EFFICIENCY.
Long
fragments
Template DNA
DNA polymerase
1. Do one sequencing reaction
2. Fragments of newly
instead of four. Reaction mix
contains ddATP, ddTTP, ddGTP,
ddCTP with distinct fluorescent
markers. (With radioactive labels,
four reactions are needed—one
labeled ddNTP at a time.)
synthesized DNA that result
have distinctive labels.
Short
fragments
Capillary
Output
tube
3. Separate fragments via
electrophoresis in massproduced, gel-filled capillary
tubes. Automated
sequencing machine reads
output.
Sequencing Whole Genomes
 HGP was set up to create chromosome
maps of the human genome
 This was done with a 3-step approach
 The first step was to create a linkage map (like
we did with Sordaria
 Next, a physical map was constructed, using
linkage mapping data
 Finally, the genes were sequenced (dideoxy
sequencing)
 Shotgun sequencing
 Uses cut-ups of human DNA, inserted into
bacteria for cloning, then analysis of the small
sequences and reconstruction
Figure 20-2
SHOTGUN SEQUENCING A GENOME
160 kb fragments
1. Cut DNA into fragments of 160 kb, using
sonication.
Genomic DNA
2. Insert fragments into bacterial artificial
BAC library
BAC
Main bacterial
chromosome
chromosomes; grow in E. coli cells to obtain
large numbers of each fragment.
3. Purify each 160-kb fragment, then cut
1-kb fragments
each into a set of 1-kb fragments, using
sonication, so that 1-kb fragments overlap.
4. Insert 1-kb fragments into plasmids; grow
“Shotgun
clones”
in E. coli cells. Obtain many copies of each
fragment.
5. Sequence each fragment. Find regions
Shotgun
sequences
where different fragments overlap.
6. Assemble all the 1-kb fragments from
each original 160-kb fragment by matching
overlapping ends.
Draft sequence
7. Assemble sequences from different BACs
(160-kb fragments) by matching overlapping
ends.
Analyzing Gene Expression
 Northern Blotting
 Same basic procedure as Southern Blotting, but we’re
looking for mRNA in cells at different stages of
development to see if the protein we’re studying is
needed at these steps
 Reverse-transcriptase PCR
 Will accomplish the same thing as Northern Blotting,
but uses rt to make cDNA from the mRNA, which is
then put through PCR and run on a gel
 The gene we’re observing only occurs in samples that
contained the mRNA with that gene
 DNA Microarray Assays
 Hybridization of cDNA with a pre-fixed slide of mRNA
 This helps scientists to see which genes may be
turned on at the same time and thus working
together
Figure 20-11
Microarray slide
Exon
286
Each spot on
the slide contains
many singlestranded copies
of a different
exon
Exon
287
Exon
288
Figure 20-12
PROTOCOL FOR A MICROARRAY EXPERIMENT
Normal
temperature
High
temperature
1. Use reverse transcriptase
to prepare single-stranded
cDNA from mRNA of control
cells and treatment cells.
cDNA
mRNA
Microarray computer output:
2. React with labeled nucleotides
cDNA
probes
to add fluorescent green label to
control cDNA and fluorescent
red label to treatment cDNA.
3. Probe a microarray with the
labeled cDNAs. Probe cDNA will
bind and label spots containing
complementary sequences.
Microarray
4. Shine laser light to induce
fluorescence. Analyze the pattern
of hybridization between the two
cDNAs and the DNA on the
microarray.
Green =
genes
transcribed
in
control cells
Yellow =
genes
transcribed
equally in
both cells
Dark =
low
gene
expression
Red =
genes
transcribed
in
treatment cells
Determining Gene Function
 Usually, scientists disable a gene
which has been identified by DNA
tech, then observe the consequences
in the cell
 This is called in vitro mutagenesis
Cloning Organisms

Plants can be cloned using single-cell cultures




Differentiated cells from the root can be grown in culture and
become entire organisms, genetically identical to the parent
When mature cells are capable of dedifferentiating and
redifferentiating, they’re called totipotent
Recall that through propogation, plants are cloned as well!
Animals can be cloned via nuclear transfer



Originally, an unfertilized egg was used, which worked, except
that the ability of the new nucleus to control the resultant
clone decreased with donor nucleus age
Dolly was different because she was made from an alreadydifferentiated mammary cell. Dedifferentiation was
accomplished by culturing the cell in a nutrient poor medium
Dolly died at age 6, when she was euthanized after suffering
from a lung disease that usually effects much older sheep,
leading scientists to speculate that clones weren’t as vigorous
as the original organism.
Figure 21-3
CLONING A SHEEP
Mammary-cell Egg-cell
donor sheep donor sheep
1. Start with two
female sheep. Each
will donate one cell.
2. Culture mammarygland cells. Remove
nucleus from egg cell.
Mammary cells
Egg cell
3. Fuse the
mammary-gland cell
to enucleated egg cell.
Fused cell
Early embryo
Surrogate
mother
4. Egg cell now
contains nucleus
from mammarygland cell.
5. Grow in culture.
Embryo begins
development.
6. Implant early
embryo in uterus
of third sheep.
7. Embryo develops
Cloned
sheep
“Dolly”
normally, resulting
in lamb that is
genetically identical
to mammary-cell
donor.
This result supports the hypothesis that mature
cells contain all the genes in the genome.
Problems with Organismal Cloning
 Cloning is inefficient-only a small
percent of cloned embryos develop
normally, and there are often defects
(like pneumonia, obesity, liver failure,
and premature death)
 Scientists are working to improve the
efficiency of cloning by studying
systematic changes to the chromatin
as the nucleus matures
Stem Cells
 These are unspecialized cells
 Ultimately, this is what scientists
would like to achieve through cloning
for the treatment of disease
 The most common place to find these
is in embryos (these are pluripotentcan develop into a wide variety of cell
types), although, there are some less
flexible stem cells in adults
Applications of Biotechnology
 Medical Applications
 Diagnosis of disease
 Gene Therapy
 Pharmaceuticals
 Forensic Evidence
 Environmental Clean-up
 Ag Apps
 “old school”=selective breeding
Ethics Issues with Biotechnology
 Safety questions about GMOs
 Problems with the technologies
leading to “super bugs” and
maldeveloped mutants
 Creating organisms with medical
issues since clones aren’t as vigorous
 Obtaining Stem Cells
 Where to “draw the line” with
research?