Recombinant DNA and Genetic Engineering

Chapter 15

Lipoproteins
Form when certain blood proteins combine with cholesterol High-density lipoproteins (HDLs) Low-density lipoproteins (LDLs)

Familial Hypercholesterolemia
Gene encodes protein that serves as cells LDL receptor Two normal alleles for the gene keep blood level of LDLs low Two mutated alleles lead to abnormally high cholesterol levels & heart disease
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Example of Gene Therapy

Woman with familial hypercholesterolemia Part of her liver was removed Virus used to insert normal gene for LDL receptor into cultured liver cells Modified liver cells placed back in patient

Results of Gene Therapy

Modified cells alive in womans liver Blood levels of LDLs down 20 percent No evidence of atherosclerosis Cholesterol levels remain high Remains to be seen whether procedure will prolong her life
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Genetic Changes
Humans have been changing the genetics of other species for thousands of years
Artificial selection of plants and animals

Natural processes also at work

Mutation, crossing over
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Genetic Engineering
Genes are isolated, modified, and inserted into an organism Made possible by recombinant technology
Cut DNA up and recombine pieces Amplify modified pieces

Discovery of Restriction Enzymes

Hamilton Smith was studying how Haemophilus influenzae defend themselves from bacteriophage attack Discovered bacteria have an enzyme that chops up viral DNA
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Specificity of Cuts
Restriction enzymes cut DNA at a specific sequence Number of cuts made in DNA will depend on number of times the target sequence occurs
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Making Recombinant DNA

5 3 G C T T A A one DNA fragment A A T T C G another DNA fragment

G C

A A T T C T T A A G

3 5

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Making Recombinant DNA

nick 5 3 G A A T T C C T T A A G 3 5

nick DNA ligase action

G A A T T C C T T A A G

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Using Plasmids
Plasmid is small circle of bacterial DNA Foreign DNA can be inserted into plasmid
Forms recombinant plasmids Plasmid is a cloning vector Can be used to deliver DNA into another cell
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Using Plasmids

DNA fragments + enzymes

recombinant plasmids

host cells containing 13 recombinant plasmids

mRNA transcript

Making cDNA
mRNAcDNA hybrid

single-stranded cDNA

double-stranded cDNA
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Amplifying DNA
Fragments can be inserted into fast-growing microorganisms Polymerase chain reaction (PCR)

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Polymerase Chain Reaction

Sequence to be copied is heated Primers are added and bind to ends of single strands DNA polymerase uses free nucleotides to create complementary strands Doubles number of copies of DNA
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Polymerase Chain Reaction

DNA to be amplified

DNA is heated

Primers are added

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Polymerase Chain Reaction

Mixture cools Base pairing occurs

Complementary strand synthesized

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Primers
Short sequences that DNA polymerase recognizes as start tags To carry out PCR, must first determine nucleotide sequences just before and after the gene to be copied Complementary primers are then created

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The DNA Polymerase

Most DNA polymerase is denatured at high temperature Polymerase used in PCR is from bacteria that live in hot springs

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Temperature Cycles
DNA is heated to unwind strands Cooled to allow base-pairing with primers and complementary strand synthesis DNA is heated again to unwind strands Cycle is repeated over and over again
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DNA Fingerprints
Unique array of DNA fragments Inherited from parents in Mendelian fashion Even full siblings can be distinguished from one another by this technique

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Tandem Repeats
Short regions of DNA that differ substantially among people Many sites in genome where tandem repeats occur Each person carries a unique combination of repeat numbers
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RFLPs
Restriction fragment length polymorphisms DNA from areas with tandem repeats is cut with restriction enzymes Because of the variation in the amount of repeated DNA, the restriction fragments vary in size Variation is detected by gel electrophoresis
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Gel Electrophoresis
DNA is placed at one end of a gel A current is applied to the gel DNA molecules are negatively charged and move toward positive end of gel Smaller molecules move faster than larger ones

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Analyzing DNA Fingerprints

DNA is stained or made visible by use of a radioactive probe Pattern of bands is used to:
Identify or rule out criminal suspects Determine paternity

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Genome Sequencing
1995 - Sequence of bacterium Haemophilus influenzae determined Automated DNA sequencing now main method 3.2 billion nucleotides in human genome determined in this way

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Nucleotides for Sequencing

Standard nucleotides (A,T,C, G) Modified versions of these nucleotides
Labeled so they fluoresce Structurally different so that they stop DNA synthesis when they are added to a strand

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Reaction Mixture
Copies of DNA to be sequenced Primer DNA polymerase Standard nucleotides Modified nucleotides
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Reactions Proceed
Nucleotides are assembled to create complementary strands When a modified nucleotide is included, synthesis stops Result is millions of tagged copies of varying length

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Recording the Sequence

DNA is placed on gel
T TC

TCCATGGACC TCCATGGAC TCCATGGA TCCATGG TCCATG TCCAT TCCA TCC electrophoresis gel

Fragments move off gel in size order; pass through laser beam Color each fragment fluoresces is recorded on printout

one of the many fragments of DNA migrating through the gel

one of the DNA fragments passing through a laser beam after moving through the gel

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T C C A T G G A C C A

Gene Libraries
Bacteria that contain different cloned DNA fragments
Genomic library cDNA library

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Using a Probe to Find a Gene

You want to find which bacteria in a library contain a specific gene Need a probe for that gene
A radioisotope-labeled piece of DNA It will base-pair with the gene of interest

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Use of a Probe

Colonies on plate

Cells adhere to filter

Cells are lysed; DNA sticks to filter

Probe is added

Location where probe binds forms dark spot on film, indicates colony with 34 gene

Engineered Proteins
Bacteria can be used to grow medically valuable proteins
Insulin, interferon, blood-clotting factors Vaccines

Human gene is inserted into bacteria, which are then grown in huge vats
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Cleaning Up the Environment

Microorganisms normally break down organic wastes and cycle materials Some can be engineered to break down pollutants or to take up larger amounts of harmful materials
Break down oil, sponge up heavy metals
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Basic Research
Recombinant DNA technology allows researchers to:
Investigate basic genetic processes Reconstruct lifes evolutionary history Devise counterattacks against rapidly mutating pathogens

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The Ti plasmid
Researchers replace tumorcausing genes with beneficial genes Plasmid transfers these genes to cultured plant cells
plant cell

foreign gene in plasmid

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Engineered Plants
Cotton plants that display resistance to herbicide Aspen plants that produce less lignin and more cellulose Tobacco plants that produce human proteins Mustard plant cells that produce biodegradable plastic
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First Engineered Mammals

Experimenters used mice with hormone deficiency that leads to dwarfism Fertilized mouse eggs were injected with gene for rat growth hormone Gene was integrated into mouse DNA Engineered mice were 1-1/2 times larger than unmodified littermates
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More Mouse Modifications

Experiments showed that human growth hormone genes can be expressed in mice Human genes are inserted into mice to study molecular basis of genetic disorders, such as Alzheimers disease Variety of methods used to introduce genes
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Cloning Dolly
1997 - A sheep cloned from an adult cell
Nucleus from mammary gland cell was inserted into enucleated egg from another sheep Embryo implanted into surrogate mother Sheep is genetic replica of animal from which mammary cell was taken
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Designer Cattle
Genetically identical cattle embryos can be grown in culture Embryos can be genetically modified
Experimenters are attempting to create resistance to mad cow disease Others are attempting to engineer cattle to produce human serum albumin for medical use
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The Human Genome Initiative

Goal - Map the entire human genome
Initially thought by many to be a waste of resources Process accelerated when Craig Ventner used bits of cDNAs as hooks to find genes Sequencing was completed ahead of schedule in early 2001
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Using Human Genes

Even with gene in hand it is difficult to manipulate it to advantage Viruses usually used to insert genes into cultured human cells but procedure has problems Very difficult to get modified genes to work where they should
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Eugenic Engineering
Selecting desirable human traits Who decides what is desirable? 40 percent of Americans say gene therapy to make a child smarter or better looking would be OK

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Where Do We Go Now?
Can we bring about beneficial changes without harming ourselves or the environment? Gene therapy is not harmless
A young man died after gene therapy that used an adenovirus

Gene therapy can save lives

Infants with disabled immune systems are now healthy
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Can Genetically Engineered Bacteria Escape?

Genetically engineered bacteria are designed so that they cannot survive outside lab Genes are included that will be turned on in outside environment, triggering death
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Effects of Engineered Organisms

Opposition to any modified organisms What if engineered genes escape into other species?

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