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Chapter 16: Gene Technology
I. Introduction
A. New Techniques Developed to Manipulate DNA
B. Techniques Can Be Applied to Alter an Organism's Genes
II. Plasmids and the New Genetics
A. First Human Gene Inserted into Bacteria
1. Interferon
a. Increases
to viral infection
b. Rare, purification of small quantities is very expensive
2. Bacterial cells made to produce protein at high rate
a. Masses of cells
from original cells
b. Each cell a miniature interferon factory
c.
produced in the same manner
B. Beginning of Genetic Engineering
1. Ability to cut up DNA into
2. Recognize and cleave specific nucleotide
3. Segments inserted via
III. Restriction Enzymes
A. Bacteria Are Natural Source of Enzymes
1. Enzymes are
2. Recognize sequence, bind to DNA and cleave strand
3. allows
mapping and reconstruction of the
molecule
4. Two types of RE’s
a. simple cross cut
b. dyad symmetry
1)
bases
2)
cut
3) creates
a) allows DNA from
sources to be joined
b) nicks are linked by
forming a
c. creates
recombinant molecules
B. Host/Vector
1. produces a large amount of
in cells
a. vector carries the
2. Most common
a. plasmids – small
chromosomes
1) clones
pieces of DNA
a) replicated in
b) selectable marker – only cells with the plasmid
2) selection – only the desired
survives
3) screening – all
b. phages
1) larger and
virus based
a) require
b) linear – middle is removed and replaced with
C. Cohen and Boyer: First Artificial Bacterial Plasmid
1. Cut plasmid containing resistance transfer factor with EcoRI
a. Contained replication origin and tetracycline resistance gene
b. Complementary ends joined forming pSC101 plasmid
2. Same restriction enzymes used to cut frog genome
a. Frog DNA pieces added to open pSC101 circles
b. Added to bacteria, select for
resistance
c. Isolated cells with plasmids containing
genes
3.
: a molecule created in laboratory
D. DNA Library
1. a collection of
fragment repeats of all
of an organism
a. plasmids with different
b. phages with different
c. genomic library – repeats of the entire
in a vector
d. cDNA library genes only
1) reverse transcriptase – makes DNA from
2) called
3) often used for genes in specific tissues or
IV. Genetic Engineering
A. Four Stages of a Genetic Engineering Experiment
1. Stage 1:
a.
cleave DNA into fragments
b. differentiate fragments by different
c. fragments are separated by size using
2. Stage 2:
a. Fragments put into
3. Stage 3:
a. DNA fragments are introduced into cells, usually
b. each cell reproduces forming a
that contains the
4. Stage 4:
a. Identify clone line containing fragment of interest
b. Among most difficult and critical steps
B. Preliminary Screening of Clones
1. Eliminate bacteria not containing proper DNA fragment
2. Use genes conferring
3. Eliminate bacteria without vehicle
a. Culture clones on medium containing
b. Only bacteria
to antibiotic will grow on it
4. Eliminate bacteria with vehicle, but lacking fragment
a. Use vector with gene that enables cell to metabolize X-gal sugar
1) Metabolism of X-gal produces blue product
2) Cells with vector and functional gene will turn
C. Getting Enough DNA to Work With:
1. Produce
of DNA by adding
2. PCR used to
sequences or
sequences as primers to
cleaved DNA
3. Five steps in PCR process
a.
1) Primer of synthetic nucleotides mixed with DNA fragment
2) Temperature of mixture increased to 980 C
b.
1) Solution cooled to 60¯ C
2) Single strands of DNA reassociate into double strands
3) Fragment base-pairs with complementary primer nucleotide
4) Part of fragment still single stranded
c.
1) Heat stable DNA polymerase added along with supply of all four
2) Polymerase
rest of fragment as in DNA replication
3)
of original now exist
d.
1) Repeat heating and cooling in short cycles
2) Each cycle
amount of DNA
3) After twenty cycles one fragment can become more than one
4. PCR allows investigation of
samples of DNA
5. Has had enormous impact on all aspects of biology
D. Identifiying DNA – Southern Blot
1. Southern blot technique fig 19.8
a. Fragments spread apart by
b. Gel blotted with
, DNA transfers to sheet
c. Probe poured onto nitrocellulose sheet
d. Only fragments with proper gene
with probe
e. Probe may be radioactive chemical
E. Analysis of restriction fragment length polymorphisms
a. a point mutation, sequence repetition, and transposons with or withour
end recognition sites and will alter the
of the DNA
fragment
1) DNA from different individuals rarely have
the same
and distribution b. Cut DNA samples with particular restriction
1) Separate fragments according to
with
electrophoresis
.
2)Use
to identify fragments
3) Obtain
in gel
c. Called
1) Used in
2) Used as
3) using several probes,
identity is established
or ruled out
V. Bio technology: A Scientific Revolution
A. Pharmaceuticals
1. Most obvious commercial application of gene technology
2. Bacteria can produce gene products in
a. Several forms of
b. Manufacture valuable nonhuman enzymes
3. Produce medically important proteins
a.
: regulate blood pressure, kidney function
b. Tissue plasminogen activator:
4. Must separate desired protein from bacterial proteins
a. Time-consuming and expensive
b. Produce RNA transcripts of genes
c. Make proteins directly in cell-free culture
B. Piggyback Vaccines
1. Subunit vaccines for herpes virus and hepatitis viruses
a. Protein-polysaccharide coat genes isolated
b. Spliced to
c. Live vaccinia added to cell culture with fragments
d. Recombinant virus carries coat genes of other
e. Infected animal produces antibodies to outer surface of virus
2. Make
against virus without
to it
3. DNA vaccine
a. infected cells are
when they stick a foreign
protein on the outer surface
b.
attack
C. Agriculture
1. Plants cells don’t possess as many
as bacteria
2. Currently use
a. Infects broad leaf plants but not cereal plants
b. Attach other genes to this plasmid
3. Development of Flavr Savr tomatoes
a. Contain
antifreeze gene
b. Produce ethylene glycol from ethylene
c. Lack of ethylene delays ripening of
D. Herbicide Resistance
1. Broadleaf plants engineered to be resistant to
2. Glyophosate is the active ingredient in
herbicide
3. Extra copies of EPSP synthetase gene via Ti plasmid
4. Plants overproduce enzyme
5. Overcome
6. Advantages
a. Crops would not need to be
b. Wide variety of weeds killed and desired crop
c. Glyphosate readily
E. Insect Resistance
1. Insects presently controlled via
2. Engineer plants for resistance to
a.
insecticidal protein genes
b. Ingested by tomato hornworm, converted to poison
c. Harmless to animals with different stomach enzymes
d. Genes introduced into plants via
e. Plants safe from attack by insects that eat them
3. Examples:
a. Genetically altered potato kills
b. Cotton resistant to
c. Corn resists
4. Isolation insect-killing enzyme from a fungus
a.
disrupts insect gut membranes
b. Fungal gene inserted into a variety of crops
c. Kills variety of insects including
5. Introduce insecticidal protein into root bacteria
a. B. thuringiensis does not normally inhabit roots
b. Protect roots from various pests, including Pseudomonas
F. Nitrogen Fixation
1. Insert proper niF genes into
2. Plants use ammonia to make
and other nitrogen molecules
3. potential to lower crop cost of
G. Farm Animals
1. Somatotropin growth hormone (BST) synthetically produced
2. Added to diary cow's diet to increase yield
3. Potential to increase
4. Human tests to increase size of hormonal dwarfs
5. Public resistance to BST in milk
a. Generalized fears of gene technology
b. BST is a protein, digested in stomach
6. Development of transgenic animals
H. Other Applications
1. Create strains of bacteria to eat
2. Grow synthetic
3. Forensic use
4. Identification of individuals
I. Promise of Genetic Engineering
1. % of soybeans are
to be herbicide resistant
a. less tillage and
b. cultivate crops more cheaply and
2. Golden Rice – problems overcome
a. too little iron – transferred a
gene from beans
b. inhibition of iron absorption by intestines – transfer a
gene from a fungus
c. too little S for efficient iron absorption – transfer a sulfur rich
from wild rice
d. add genes for the
missing enzymes to make
from a
J. Ethics and Regulation
1. Concerns regarding tampering with genetic material
a. selectable marker still
b. could become an
c. no current
crop contains protein that acts as an
2. Harm to the Environment
a. pest induced loss of wildlife is more
damaging than
side effects
b. weeds are important
sources for insects
c. resistance – use of insecticides creates a pressure favoring
that make the pest
1) could become resistant to
2) farmers must plant
% non Bt crops next to Bt crops to
prevent
3. Gene Flow
a. round 1 – no available relative to receive the gene
b. round 2 – the risk increases