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Ch. 11: Gene regulations
How is cloning possible?
Every cell has the same chromosomes
Then….. Why does a heart muscle cell look different
from a skin cell?
 Organisms respond to their environment by
altering gene expression
Central question: what regulates gene expression?
Differentiation
 Differentiation is controlled by turning specific
sets of genes on or off
DNA Packing
 eukaryotic chromosomes condense during
prophase of Mitosis
 helps regulate gene expression by preventing
transcription
– Nucleosomes
– Tight helical fiber =
– Supercoil = coiling of the tight helical fiber
Metaphase
chromosome
Tight helical fiber
(30-nm diameter)
DNA double helix
(2-nm diameter)
Linker
“Beads on
a string”
Nucleosome
(10-nm
diameter)
Histones
Supercoil
(300-nm diameter)
Animation: DNA Packing
700 nm
X-chromosome inactivation
– female mammals
– one of the two X chromosomes is highly
compacted and transcriptionally inactive (Barr
body)
– Occurs early in embryonic development, thus all
cellular descendants have the same inactivated
chromosome
– Tortoiseshell fur coloration is due to inactivation
of X chromosomes in heterozygous female cats
Early embryo
Two cell populations
in adult
Cell division
and random
X chromosome
inactivation
X chromosomes
Allele for
orange fur
Allele for
black fur
Active X
Inactive X
Orange
fur
Inactive X
Active X
Black fur
Eukaryotic gene expression
– Each gene has its own promoter and terminator
– Are controlled by interactions between numerous
regulatory proteins and control sequences
 Regulatory proteins
• Transcription factors - help RNA polymerase
bind to the promoter
• Activators –
• Silencers  Control sequences
– Promoter
– Enhancer
– Related genes located on different
chromosomes can be controlled by similar
enhancer sequences
Animation: Initiation of Transcription
Enhancers
Promoter
Gene
DNA
Activator
proteins
Transcription
factors
Other
proteins
RNA polymerase
Bending
of DNA
Transcription
Alternative RNA splicing
– Can involve removal of an exon with the introns
on either side
Animation: RNA Processing
Exons
1
DNA
RNA
transcript
1
1
2 3
5
4
3
2
RNA splicing
mRNA
4
3
2
5
or
5
1
2 4
5
Small RNAs control gene expression
 RNA interference (RNAi)
– small, complementary RNAs bind to mRNA
transcripts, blocking translation
 MicroRNA (miRNA)
– MicroRNA + protein complex binds to
complementary mRNA transcripts, blocking
translation
Animation: Blocking Translation
Animation: mRNA Degradation
Protein
miRNA
1
miRNAprotein
complex
2
Target mRNA
3
mRNA degraded
4
OR Translation blocked
 Control of gene expression also occurs with
– Breakdown of mRNA
– Initiation of translation
– Protein activation
– Protein breakdown
Ex. Insulin formation
Folding of
polypeptide and
formation of
S—S linkages
Initial polypeptide
(inactive)
Cleavage
Folded polypeptide
(inactive)
Active form
of insulin
Epigenetic Inheritance
This can be accomplished by acetylation or
methylation of histones
Regulation of Chromatin Structure
Chemical modification of histone tails can affect the
configuration of chromatin and thus gene expression
Histone
tails
DNA
double helix
(a) Histone tails protrude outward from a nucleosome
 Addition of methyl groups to certain bases
in DNA is associated with reduced transcription in
some species
Unacetylated histones
Acetylated histones
(b) Acetylation of histone tails promotes loose chromatin structure that
permits transcription
NUCLEUS
Chromosome
DNA unpacking
Other changes to DNA
Gene
Gene
Transcription
Exon
RNA transcript
Intron
Addition of cap and tail
Splicing
Tail
mRNA in nucleus
Cap
Flow through
nuclear envelope
mRNA in cytoplasm
CYTOPLASM
Breakdown of mRNA
Translation
Brokendown
mRNA
Polypeptide
Cleavage / modification /
activation
Active protein
Breakdown
of protein
Brokendown
protein
Why so much control over gene expression?
 It allows cells to respond appropriately to their
environment
 Signal transduction pathways convert messages
received at the cell surface to responses within the
cell via gene expression
 Three steps:
1. Reception –
2. Amplification/transduction –
3. Response - transcription factor is activated,
enters nucleus, transcribes specific genes
Signaling cell
Signaling
molecule
Plasma
Receptor membrane
protein
1
2
3
Target cell
Relay
proteins
Transcription
factor
(activated)
4
Nucleus
DNA
5
mRNA Transcription
New
protein
6
Translation
 Cloning: How? Nuclear transplantation
– Replacing the nucleus of an egg cell with a
nucleus from an adult somatic cell. Allow embryo
to form. Embryo can be used in:
– Reproductive cloning
– Therapeutic cloning
– Grow embryonic stem cells in culture
– Induce stem cells to differentiate and grow into
organs, tissues, etc.
Donor
cell
Nucleus from
donor cell
Reproductive
cloning
Implant blastocyst in
surrogate mother
Remove
nucleus
from egg
cell
Add somatic cell
from adult donor
Grow in culture
to produce an Therapeutic
early embryo cloning
(blastocyst)
Remove embryonic
stem cells from
blastocyst and
grow in culture
Clone of
donor is born
Induce stem
cells to form
specialized cells
To clone or not to clone….
 Benefits of reproductive cloning?
 Disadvantages of cloning?
Human stem cell research
 Ethical concerns with reproductive cloning
 Ethical concerns with therapeutic cloning?
 Benefits:
 Human embryos – have the greatest potential
to give rise to all cell types
– Adult stem cells (bone marrow) or cord blood
cells
– can give rise to many but not all
types of cells
Ch 12: DNA Technology
1. DNA profiling
2. Genetically modified organisms/recombinant DNA
technology
3. Gene therapy
4. Genomics
1. DNA profiling = analysis of DNA fragments
to determine whether they come from a
particular individual
 3 steps:
1..
2.Amplify (copy) markers for analysis –
3.Compare sizes of amplified fragments by
gel electrophoresis
1. Select genetic marker to analyze
 Short tandem repeats (STRs) are genetic
markers used in DNA profiling
– STRs =
– STR analysis compares the lengths of STR
sequences at specific regions of the genome
– Current standard for DNA profiling is to analyze
13 different STR sites
STR site 1
STR site 2
Crime scene DNA
Number of short tandem Number of short tandem
repeats match
repeats do not match
Suspect’s DNA
2. Amplify the DNA sample
 Polymerase chain reaction (PCR) = method
of amplifying a specific segment of a DNA
molecule
 Relies upon a pair of primers =
 Repeated cycle of steps for PCR:
1. Sample is heated to separate DNA strands
2. Sample is cooled and primer binds to specific
target sequence
3. Target sequence is copied with DNA
polymerase
Cycle 1
yields 2 molecules
Genomic
DNA
3
1
3
5
3
Target
sequence
5
5
5
3
Cycle 2
yields 4 molecules
5
5
2 Cool to allow
3
Heat to
primers to form
separate
DNA strands hydrogen bonds
with ends of
target sequences
5
3
5
3
Primer
3
5
DNA
polymerase adds
nucleotides
to the 3 end
of each primer
5
3
New DNA
Cycle 3
yields 8 molecules
3. Gel electrophoresis
 separates DNA molecules based on size
– DNA samples placed at one end of a porous gel
– Current is applied and DNA molecules move
from the negative electrode toward the positive
electrode
– DNA fragments appear as bands, visualized
through staining or radioactivity or fluorescence
Video: Biotechnology Lab
Mixture of DNA
fragments of
different sizes
Power
source
Longer
(slower)
molecules
Gel
Shorter
(faster)
molecules
Completed gel
Crime scene
1 DNA isolated
2 DNA of selected
markers amplified
3 Amplified DNA
compared
Suspect 1
Suspect 2
Mixture of DNA
fragments
A “band” is a
collection of DNA
fragments of one
particular length
Longer
fragments
move slower
Shorter
fragments
move faster
DNA attracted to +
pole due to PO4– groups
Power
source
Applications of DNA profiling
 Forensics - to show guilt or innocence
 Establishing paternity
 Identification of human remains
 Species identification
– Evidence for sale of products from endangered
species
2. Recombinant DNA technology/ Genetically
Modified organisms
– Recombinant DNA is formed by joining DNA
sequences from two different sources:
1. .
2. .
– Bacterial Plasmids (small, circular
DNA molecules independent of the
bacterial chromosome) are often used
as vectors
Recombinant cells and organisms can
mass-produce gene products
– Common prokaryotic host: E. coli bacterium
– Has many advantages in gene transfer, cell
growth, and quantity of protein production
– Common eukaryotic hosts:
– Yeast: S. cerevisiae
– “Pharm” animals
– Will secrete gene product in milk
 Advantages of recombinant DNA products
 Genetically modified (GM)
 Transgenic organisms contain at least one
gene from another species
Agrobacterium tumefaciens
Plant cell
DNA containing
gene for desired trait
1
Ti
plasmid
Insertion of gene
into plasmid
3
2
Recombinant
Ti plasmid
Introduction
into plant
cells
Regeneration
of plant
DNA carrying new gene
Restriction site
Plant with new trait
Pros?
 GM plants
 GM animals
Cons?
3. Gene therapy
 One possible procedure:
– insert functional gene into a virus
– virus delivers the gene to an affected cell
– Viral DNA & gene insert into the patient’s
chromosome
– Return the cells to the patient for growth and
division
Cloned gene
(normal allele)
1
Insert normal gene
into virus
Viral nucleic acid
Retrovirus
2
Infect bone marrow
cell with virus
3
Viral DNA inserts
into chromosome
Bone marrow
cell from patient
Bone
marrow
4
Inject cells
into patient
4. Genomics
 Genomics =
 Applications:
– Evolutionary relationships: Genomic studies
showed a 96% similarity in DNA sequences
between chimpanzees and humans
– Medical advancement: Functions of human
disease-causing genes have been determined
by comparisons to similar genes in yeast
Human Genome Project
 Goals:
– To determine the nucleotide sequence all
DNA in the human genome
– To identify the location and sequence of
every human gene
 Results of the Human Genome Project
– 21,000 genes in 3.2 billion nucleotide pairs
– Only 1.5% of the DNA codes for proteins
– The remaining 88.5% of the DNA contains
– Control regions (promoters, enhancers)
– Unique noncoding DNA
– Repetitive DNA
Exons (regions of genes coding for protein
or giving rise to rRNA or tRNA) (1.5%)
Repetitive
DNA that
includes
transposable
elements
and related
sequences
(44%)
Introns and
regulatory
sequences
(24%)
Unique
noncoding
DNA (15%)
Repetitive
DNA
unrelated to
transposable
elements
(15%)