Download Document

Chapter 11: The Control of
Gene Expression
Life 1402: Principles of Biology
1. Cellular Differentiation during
embryonic development
46
23
23
a. a zygote is the first cell of an organism with a
full compliment of DNA
1. Cellular Differentiation during
embryonic development
b. the zygote undergoes mitosis giving rise to
genetically identical daughter cells
2. Cellular Differentiation during
embryonic development
c. with each mitotic division during
development, the daughter cells inherit
identical copies of DNA; therefore, every
diploid cell in an organism is genetically
identical unless mutations occur
2. Cellular Differentiation during
embryonic development
d. cellular differentiation is the process by
which genes are turned on and off, not
changed, during embryonic development
2. Cellular Differentiation during
embryonic development
e. cellular differentiation results in different
tissue types in a single organism
Turning Genes On and Off
http://www.youtube.com/watch?v=dSiSHR
wR49k
 http://www.youtube.com/watch?v=CkR53X
8vksY

3. The lac operon
a. Collection of genes that regulate protein
synthesis depending upon the cell’s needs
b. Described as found in E.coli
3. The lac operon
c. Operon is turned off in the absence of lactose
d. Operon is turned on in the presence of
lactose
4. Cloning
4. Cloning
a. A clone is a cell or organism that is genetically
identical; e.g., identical twins which result from
separation of an embryo before differentiation
occurs
b. Variable success has been realized in attempts to
clone organisms, even mammals
c. Both ethical and physical barriers exist when cloning
of humans is considered
4. Cloning
d. Basic process of cloning
1. nucleus is removed from an egg
2. nucleus is removed from an adult somatic cell and
injected into the egg which had its nucleus removed
3. the resulting cell is then grown in culture to produce a
blastocyst; i.e., an early embryo consisting of a ball of
app. 200 cells
4. the blastocyst can then be used to produce an entire
organism (reproductive cloning) or used to provide
embryonic stem cells which can be grown in culture
(therapeutic use)
5. Nuclear transplantation
a. the process of cloning described above
results from nuclear transplantation; i.e., a
nucleus from a differentiated adult cell is
transplanted into a non-differentiated egg
cell
b. nuclear transplantation experiments have
resulted in clones of plants and animals
5. Nuclear transplantation
c. the resulting clones are genetically identical to
the donor parent regardless of the adult cell type
used as a donor
d. differentiated adult cells must all have the same
DNA if clones result from nuclear transplantation
regardless of the donor cell type
5. Nuclear transplantation
e. if the genome were different among different
adult cell types, clones from these different
cells would not be the same
6. Application of reproductive cloning
a. Dolly is a sheep that was cloned from an adult cell
b. Since Dolly, reproductive cloning has been used to
establish clones for scientific, medical, and
agricultural uses
6. Application of reproductive cloning
c. Scientists have cloned pigs with a
gene "knocked out" that codes for a
protein making their hearts activate
our immune system --- What's the
application?
6. Application of reproductive cloning
d. Scientists have cloned farm animals with specific
sets of desirable traits
e. Scientists are cloning mammals that are
genetically engineered to synthesis valuable
drugs
Got Clones?
7. Stem cells
a. stem cells are cells that have not yet
differentiated
b. in the appropriate conditions, stem cells can
be stimulated to differentiate into specific
cell types; e.g., nerve cells, muscles cells,
etc. thus providing a source of tissue for
therapeutic use
Stem Cells
http://video.pbs.org/video/1506726820/
http://video.pbs.org/video/1511335379/
7. Stem cells
c. growth factors can turn on and turn off
particular genes and thus determine the fate
of a particular cell
d. embryonic stem cells are taken from early
embryos and have the potential to
differentiate into any cell type
7. Stem cells
e. adults have stem cells;
e.g., bone marrow cells
differentiate into
different blood cell types
and epidermal germ
cells differentiate into
different skin cell types
f. although adult stem cells
are typically more
difficult to work with,
they provide a potential
source of stem cells
without destroying
embryos
8. Cancer
a. cancer can be caused by mutations in genes that
control cell growth
b. genes that cause cancer are called oncogenes, these
are usually mutated genes that produce growth
factor to stimulate cell division
8. Cancer
c. genes that may become cancer causing are protooncogenes
d. tumor suppressor genes inhibit cell growth, mutations
of these genes can also cause cancer
8. Cancer
e. carcinogen – “cancer generator” factors in the
environment that can cause cancer
CHAPTER 12
DNA Technology and
the Human Genome
1.
Cloning of genes through
genetic engineering
a. genetic engineering = direct manipulation
of genes for practical purposes
 b. gene cloning = making identical copies of
genes (fig 12.1)

1.
Cloning of genes through
genetic engineering
c. recombinant DNA = joining of two
different sequences of DNA
 d. plasmid = small, circular DNA molecule
separate from the larger bacterial DNA

1.

Cloning of genes through
genetic engineering
e. vector = virus or cell that transfers DNA to
another cell
2. Restriction
enzymes (fig 12.2)
a. enzyme = a substance, usually a
protein, that catalyzes (facilitates) a reaction
 b. restriction enzyme = an enzyme that
recognizes a specific sequence of DNA and
digests (cuts) the DNA at that recognition
site

2. Restriction enzymes
AATTC
G GAATTC
G GAATTC
AATTC

c. the resultant pieces of DNA after
digestion with a restriction fragment are
termed restriction fragments (if a linear
strand of DNA has two recognition sites,
digestion will produce three restriction
fragments)
2. Restriction enzymes
TGATCGTATCGATGCTAGCACATTATCGATCGATGCTAGCACA
TGAGCTAGCATCGATCGATAATGAGCTAGCATCGAATCGATCA
ATCGTTCGATGCTAGCACATTATCGAATCGATGCTAGCACA
TGAGCTAGCATCGATTCGATAATGAGCTAGCATCGAACGATCA

d. the cut ends of DNA are “sticky”; i.e.,
they will join together (anneal) with other
“sticky” ends cut with the same restriction
enzyme
2. Restriction enzymes
ATCGAATCGATGCTAGC
CTAGCATCGATTCGATAA
TGATCGAATCGATGCTAGCACATT
TGAGCTAGCATCGATTCGATCA
TCGATGCTAGCACATT
TGAGCTAGCATCGAAA

d. the cut ends of DNA are “sticky”; i.e.,
they will join together (anneal) with other
“sticky” ends cut with the same restriction
enzyme
2. Restriction enzymes

e. this is how the human insulin gene is
removed from human DNA and spliced into
the bacterial DNA
2. Restriction enzymes

f. restriction enzymes are also used in forensic
science to establish a DNA fingerprint = a set
of restriction fragments unique to an individual
2. Restriction enzymes (fig
12.2)
Bacterium
Bacterial
chromosome

Plasmid
g.
Overview of the technology behind
gene cloning using bacterial plasmids (fig
12.3)
1
Bacterium
Plasmid
isolated
2
3 Gene
Bacterial
chromosome
Plasmid
Cell containing gene
of interest
DNA
isolated
inserted
into plasmid
Gene of
interest
Recombinant DNA
(plasmid)
DNA
4
• 1. gene of interest; e.g., human insulin gene, is
removed from host DNA and inserted into
bacterial plasmid DNA thus making recombinant
DNA
Figure 12.3
1
Bacterium
Plasmid
isolated
2
DNA
isolated
3 Gene
Bacterial
chromosome
Plasmid
Cell containing gene
of interest
inserted
into plasmid
Gene of
interest
Recombinant DNA
(plasmid)
4
DNA
Plasmid put into
bacterial cell
Recombinant
bacterium
– 2. recombinant plasmid reinserted into
bacterium thus producing a recombinant
bacterium
Figure 12.3
1
Bacterium
Plasmid
isolated
2
DNA
isolated
3 Gene
Bacterial
chromosome
Cell containing gene
of interest
inserted
into plasmid
Plasmid
Gene of
interest
Recombinant DNA
(plasmid)
4
DNA
Plasmid put into
bacterial cell
Recombinant
bacterium
5 Cell multiplies with
gene of interest
Clones of cell
3. as recombinant bacterium reproduces, gene of interest is
cloned; i.e., the bacterium replicates the gene of interest along
with its own DNA prior to each mitotic division
1
Bacterium
Plasmid
isolated
2
DNA
isolated
3 Gene
Bacterial
chromosome
Cell containing gene
of interest
inserted
into plasmid
Plasmid
Gene of
interest
Recombinant DNA
(plasmid)
4
DNA
Plasmid put into
bacterial cell
Recombinant
bacterium
5 Cell multiplies with
gene of interest
Copies of gene
Gene for pest
resistance
inserted into
plants
Copies of protein
Clones of cell
Gene used to alter bacteria
for cleaning up toxic waste
Protein used to
make snow form
at higher
temperature
Protein used to dissolve blood
clots in heart attack therapy
4. bacteria now make human insulin for practical purposes
3.
Forensics and DNA technology:
a. Back ground
 1.
everyone has a unique sequence of
DNA (genome); although we are all human,
our nucleotide sequences vary considerably
3.
Forensics and DNA technology:
2. since each person’s genome differs, a given
restriction enzyme will digest each genome
differently and produce restriction fragments of
different sizes and number (the odds of two people
having the same DNA fingerprint are slim and next
to none, one in a billion!)
3.
Forensics and DNA technology:
b. Crime scene analysis
1. a suspect’s DNA can be compared to
crime scene DNA to determine if the
suspect was present (DNA fingerprinting at
work)
3.
Forensics and DNA technology:
2. some DNA sample; e.g., blood, semen, or
hair, that is found at some crime scene is
compared with a DNA sample from a
suspect by digestion with the same
restriction enzymes
Restriction
enzymes
CK A
B A+B M
A
B 10 kb
A 8 kb
2 kb
B 7 kb
3 kb
A 5 kb
+ 3 kb
B 2 kb
3. digested DNA (restriction fragments) are “run” on an
electrophoresis gel (fig 12.12) which separates the
fragments based on size and charge; i.e., the smaller
fragments travel further in the gel and opposite charges
attract
3.
Forensics and DNA technology:
Defendant’s
blood
Blood from
defendant’s
clothes
Victim’s
blood
4. DNA fingerprints of the suspect and crime
scene sample are compared, and, if
found identical, a match is made
BioClue
Mr. Bodie is dead!

He was found murdered in the library of
the Bodie mansion.
Murder Weapon?
#1

#2
Near his body you find a candlestick and
a lead pipe, both test positive for blood
using phenolphthalein. You collect a
sample from the candlestick and label it
sample #1 and a sample from the pipe
labeled #2

There were signs of a struggle and a
suspicious trail of blood drops leading
away from the scene. A sample of the
blood drops, and Mr. Bodie’s blood,
have already undergone analysis and
you are awaiting the results.
#4
#5
Suspects
#6
#3
Prof. Grape
Mrs Pheasant

Col Ketchup
Miss Violet
Four people beside you were in the
mansion at the time of the murder,
Misses Pheasant, Professor Grape, Miss
Violet and Colonel Ketchup
DNA

•
Analyze
samples
collected with
Highlightthe
allDNA
of the
GCG sequences
using
the restriction enzymes CT and AT
the probe.
with the probe GCG.
GCAGCATCGATCGTAGCATGCTAGCTAGCTAGCTGACTGACTGCGCATCGATG
GCAGCATCGATCGTAGCATGCTAGCTAGCTAGCTGACTGACTGCGCATCGATGC
CATGCTAGCTAGCTAGCTAGTCGACTGAGGCGCGCTATAGCGATACGCATAGC
ATGCTAGCTAGCTAGCTAGTCGACTGAGGCGCGCTATAGCGATACGCATAGCA
AGATGCAGCGGATCGAGCTAGCATCGAGCGGCGCGGATAGCGATCGAGC
GATGCAGCGGATCGAGCTAGCATCGAGCGGCGCGGATAGCGATCGAGC
DNA

Now, find all of the sequences where the
restriction enzyme AT will cut the
fragment.
GCAGCAT|CGAT|CGTAGCAT|GCTAGCTAGCTAGCTGACTGACTGCGCAT|CGAT|G
GCAGCATCGATCGTAGCATGCTAGCTAGCTAGCTGACTGACTGCGCATCGATG
CATGCTAGCTAGCTAGCTAGTCGACTGAGGCGCGCTATAGCGATACGCATAGC
CAT|GCTAGCTAGCTAGCTAGTCGACTGAGGCGCGCTAT|AGCGAT|ACGCAT|AGC
AGATGCAGCGGATCGAGCTAGCATCGAGCGGCGCGGATAGCGATCGAGC
AGAT|GCAGCGGAT|CGAGCTAGCAT|CGAGCGGCGCGGAT|AGCGAT|CGAGC
DNA
This leaves the following fragments.
 The ones that are not marked with the
probe will not appear in the gel.

GCAGCAT
CGAT
CGTAGCAT
GCTAGCTAGCTAGCTGACTGACTGCGCAT
CGAT
GCAT
GCTAGCTAGCTAGCTAGTCGACTGAGGCGCGCTAT
AGCGAT
ACGCAT
AGCAGAT
GCAGCGGAT
CGAGCTAGCAT
CGAGCGGCGCGGAT
AGCGAT
CGAGCd
DNA

Count the bases in each of the marked
fragments
GCTAGCTAGCTAGCTGACTGACTGCGCAT - 29
GCTAGCTAGCTAGCTAGTCGACTGAGGCGCGCTAT-35
GCTAGCTAGCTAGCTAGTCGACTGAGGCGCGCTAT
AGCGAT-6
AGCGAT
GCAGCGGAT-9
GCAGCGGAT
CGAGCGGCGCGGAT-14
CGAGCGGCGCGGAT
AGCGAT-6
AGCGAT
DNA
If fragment has an odd number of bases, round
up to the next even number.
GCTAGCTAGCTAGCTGACTGACTGCGCAT - 30
29
GCTAGCTAGCTAGCTAGTCGACTGAGGCGCGCTAT-36
GCTAGCTAGCTAGCTAGTCGACTGAGGCGCGCTAT-35
AGCGAT-6
GCAGCGGAT-10
GCAGCGGAT-9
CGAGCGGCGCGGAT-14
AGCGAT-6
DNA

Now, find all of the sequences where the
restriction enzyme CT will cut the
fragment.
GCAGCATCGATCGTAGCATGCTAGCTAGCTAGCTGACTGACTGCGCATCGATG
CATGCTAGCTAGCTAGCTAGTCGACTGAGGCGCGCTATAGCGATACGCATAGC
AGATGCAGCGGATCGAGCTAGCATCGAGCGGCGCGGATAGCGATCGAGC
GCAGCATCGATCGTAGCATGCT
AGCT
AGCT
AGCT
GACT
GACT
GCGCATCGATGCATGCT
AGCT
AGCT
AGCT
AGTCGACT
GAGGCGCGCT
ATAGCGATACGCATAGCAGATGCAGCGGATCGAGCT
AGCATCGAGCGGCGCGGATAGCGATCGAGC
GCGCATCGATGCATGCT - 17
GAGGCGCGCT - 10
ATAGCGATACGCATAGCAGATGCAGCGGATCGAGCT -36
AGCATCGAGCGGCGCGGATAGCGATCGAGC -30
GCGCATCGATGCATGCT - 18
17
GAGGCGCGCT -10
ATAGCGATACGCATAGCAGATGCAGCGGATCGAGCT -36
AGCATCGAGCGGCGCGGATAGCGATCGAGC - 30
DNA
If fragment has an odd number of bases, round
up to the next even number.
GCGCATCGATGCATGCT - 18
GAGGCGCGCT -10
ATAGCGATACGCATAGCAGATGCAGCGGATCGAGCT -36
AGCATCGAGCGGCGCGGATAGCGATCGAGC - 30
4. PCR
Polymerase Chain Reaction is a series of
chemical reactions that copy small DNA
samples so that there is enough material to
be analyzed
4. PCR
a. Denaturation – heating the DNA to 94 C for
1 minute separates the 2 strands
4. PCR
b. Annealing – DNA is cooled to 54 C and
primers are added that select the strand to
be replicated
4. PCR
c. Extension – DNA is heated to 72 C the
polymerase replicates the strand
4. PCR
d. This process is
repeated and the
sample grows
exponentially, doubling
every cycle
Human Genome Project
Sequencing the Genetic code of a human
 Completed in 2003
 Humans ~ 31,000 genes

Rapid Sequencing
Rapid Sequencing
“Shotgun sequencing”
 Sequencing many genes at the same time
 Computers look for overlapping strands
and put them in order

CTGTAGCTGATGTCGTAGCTGA
GTAGCTGATGCTGATCGATGCTGA
ATACGTAGGCATGCTGT
Rapid Sequencing
“Shotgun sequencing”
 Sequencing many genes at the same time
 Computers look for overlapping strands
and put them in order

CTGTAGCTGATGTCGTAGCTGA
GTAGCTGATGCTGATCGATGCTGA
ATACGTAGGCATGCTGT
Rapid Sequencing
“Shotgun sequencing”
 Sequencing many genes at the same time
 Computers look for overlapping strands
and put them in order

ATACGTAGGCATGCTGTAGCTGATGTCGTAGCTGATGCTGATCGATGCTGA