Chapter 13

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Tobin and Dusheck: Asking About Life, 3E





__________ Chapter 13

Copyright 2005

Brooks/Cole

Thomson Learning

Genetic
Engineering and
Recombinant DNA

Chapter 13

Tobin and Dusheck: Asking About Life, 3E





__________ Chapter 13

Copyright 2005

Brooks/Cole

Thomson Learning

Key Questions


What does it mean to
“genetically engineer” an
organism?


What steps must biologists take
to insert a gene into the genome
of another organism?


How are transgenic plants and
animals useful to biologists and
others?

Tobin and Dusheck: Asking About Life, 3E





__________ Chapter 13

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PCR


Selectively copying a short segment
of DNA


Tobin and Dusheck: Asking About Life, 3E





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PCR
Depends on
Primers

Tobin and Dusheck: Asking About Life, 3E





__________ Chapter 13

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Biotechnology


Use of living organisms for
practical purposes


Traditional examples: domestic
animal breeding, plant breeding,
brewing, cheese making


Today increased precision and
speed, but only those traits that
are biochemically understood,
not complex traits

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Natural and Induced Variation


Biotechnology selects useful traits
from a range of variation


Original penicillin mold produced
antibiotic at low levels


Select most productive strains


Mutagenize, with X
-
rays, chemicals


Select more productive strains


Eventually increase production
100X

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Alter Biochemical Pathways


Tomatoes ripen by inducing
ethylene gas as a hormone


Damage inducing gene,
tomatoes make flavor
components but do not soften or
turn red


Treat artificially with ethylene
before selling


flavorful and red
but can be shipped

Tobin and Dusheck: Asking About Life, 3E





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Recombinant DNA


DNA molecule with segments not
found together in nature


Cut and paste DNA fragments


Or replace single nucleotides


Use viruses or plasmids to move
into new cells


Make copies in bacteria or other
cells

Tobin and Dusheck: Asking About Life, 3E





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Recombinant DNA Technology

Tobin and Dusheck: Asking About Life, 3E





__________ Chapter 13

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Restriction Enzymes


Cut DNA only at specific sequences


Sequence is a restriction site


Restriction fragments are DNA
pieces that all have same end
sequences


Restriction map is the sites on a
DNA molecule cut by a particular
restriction enzyme

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Restriction
Digestion

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Sticky Ends


Staggered cuts
leave sticky ends
that can be joined
by ligase


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Cloning Human Insulin


Gene for human insulin joined to
the promoter and operator of lac
operon by ligase


Transform E. coli with new
plasmid


Grow E. coli in presence of
lactose to induce promoter,
make insulin protein

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Using Recombinant Vectors


Build plasmid with


Origin of replication


Genes for antibiotic resistance


Any gene of interest


Use vector (plasmid or virus) to
carry gene into a cell


Cell uses new gene to make
protein

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Eukaryotic Genes in Bacteria


Eukaryotic genes have introns, but
bacteria cannot process mRNA


Use processed mRNA for cloning


RNA copied into DNA with reverse
transcriptase enzyme


Resulting DNA is a copy of mRNA,
or cDNA, without introns


Join to vector; put into a cell

Tobin and Dusheck: Asking About Life, 3E





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Multiplying Recombinant DNA


Recombinant plasmids are
copied as host cells replicate


Or, PCR used to make millions of
copies of a specific sequence,
without constructing
recombinant DNA molecule


PCR requires knowledge of the
sequence

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Modified Proteins


Some eukaryotic proteins are
modified after translation,
bacteria cannot do modifications


Insert new genes into eukaryotic
cells:


Moth, baculovirus system


Yeast


Mammalian cells in culture

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Recombinant DNA Products


Human insulin,
growth hormone


Tissue plasminogen
activator


Erythropietin


Vaccines


Cytokines

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Thinking About the Possibilities


Would you mind eating a
genetically engineered tomato?
Why or why not?


What kinds of genes are added
to transgenic crop plants?

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Gene Libraries


Thousands or millions of restriction
fragments from a single genome


Each fragment combined with a
vector


Recombinant molecule introduced
into bacteria


Library contains at least 1 copy of
every sequence in the genome

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cDNA Library


Constructed from mRNA


Contains only expressed genes
from a specific cell type or tissue

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Finding a Gene in a Library


Use a probe, a short single
strand of DNA complementary to
part of the gene wanted


Or, if the protein product is
known, use an antibody that
recognizes the protein


Find 1 colony in 500,000 with
the gene of interest

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Using a
DNA Library

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Southern Blotting


Used to look at a small number of
fragments in a complex mixture


Separate restriction fragments by
electrophoresis


Immobilize fragments on filter
paper


Hybridize filter with radioactive
probe


Expose to X
-
ray film

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Southern
Blot
Method

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Types of Blots


Southern: visualize DNA
fragments


Northern: visualize RNA
molecules to determine gene
expression in a cell type


Western: visualize proteins using
antibodies as probes

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DNA Microarrays


Used to study expression of thousands of
genes at 1 time


Thousands
of DNA
fragments
arranged
on a filter
or chip


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Using Microarrays


Isolate mRNA from cells, copy
into cDNA with fluorescent tag


Hybridize cDNA with microarray


Expressed genes light up spots
on the chip


Can be quantitated


bright
spots, high expression levels in
cell

Tobin and Dusheck: Asking About Life, 3E





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Uses of Microarrays


Compare patterns of gene
expression in cancer and normal
cells


which genes are turned
on in cancer cells?


Cluster analysis


find groups of
genes that are regulated
together


Analysis is called bioinformatics

Tobin and Dusheck: Asking About Life, 3E





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Transgenic Organisms


Organisms that carry recombinant
DNA in their genomes


New DNA inserted into fertilized
egg


during development all cells
in the body will have new gene


Inefficient process


1% success


Requires normal expression and
must not disrupt other genes

Tobin and Dusheck: Asking About Life, 3E





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First Transgenic Animals


Mice were given
human growth
hormone, grew
twice as big


Later


pigs,
goats, sheep
and monkeys

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Process of
Making
Transgenic
Animals

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Knockout Animals


Experimental
animals with
inactivated gene


Used to study
genetic
diseases


Determine
function of a
gene


Many surprises


Long haired mice


Or no effect

Tobin and Dusheck: Asking About Life, 3E





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Genetically Engineered Plants


Easier to modify plants than
animals


Ti plasmid vector carries genes
into plants


Gold pellets coated with DNA
shot into cells with a gene gun


Cells grown in culture can
become viable plants

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Engineered
Plants


Bt corn contains
bacterial toxin


kills insects


Fungus resistant
soy beans


Round
-
up Ready
plants resistant
to herbicide
Round
-
up

Tobin and Dusheck: Asking About Life, 3E





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Bioengineered Products


High
-
oil corn


Soy beans with increased lysine
and methionine (low levels in
grain)


Sunflowers with more oil


Tobacco plants make human
antibodies


Tomatoes make serum albumin for
burn victims

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Risks of Genetically Modified
(GM) Plants


Gene exchange with wild plants
(herbicide resistance)


Dispersing viral genes that cause
plant diseases


Dispersing genes for antibiotic
resistance

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Example of Escaping Gene


Rapeseed (source of canola oil)
resistant to herbicide fed to bee
larvae


Bacteria and yeast from gut of
bee carried genes for herbicide
resistance


Engineered genes can move
between organisms

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Economic Issues


GM plants are patented


Farmers must buy new seed
each year


Cannot save seed from 1 crop to
plant the next year


Farmers in developing countries
become economically dependent

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__________ Chapter 13

Copyright 2005

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Key Concepts


Genetic engineering started with
selective breeding, used natural
and induced variation


Can create genes that do not
exist in nature


Can be used in both prokaryotes
and eukaryotes to make proteins


Can make new plants and
animals