CHAPTER 16

Studying and Manipulating Genomes

IMPACTS, ISSUES: GOLDEN RICE, OR FRANKENFOOD? 124 million children around the world

have vitamin A deficiencies

Golden rice –Rice plants engineered with genes from

daffodils allowing it to produce beta-carotine in its seeds (rice)

–Beta carotine is the precursor to Vitamin A

Rice is the main food for 3 billion people

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

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

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

MAKING RECOMBINANT DNA

5’

3’

G

C T T A A

A A T T C

G

G A A T T C

C T T A A G3’

5’

one DNA fragment another DNA fragment

3’

5’

MAKING RECOMBINANT DNA

nick

5’

3’

3’

5’

G A A T T C

C T T A A G

nick

GA A T T C

C T T A AG

DNA ligase action

Fig. 16-2, p.244

Stepped ArtGC T T A A

A A T T CG

3’

5’ 3’

5’

C T T A A

A A T T CG

cut fragments

G

DNA ligase action

nick

nickGC T T A A

A A T T CG

3’

5’ 3’

5’

another DNA fragment

A A T T C

3’

5’ 3’

5’G5’

one DNA fragment

3’

3’

5’ G

C T T A A

3’

5’

enzyme recognition site

GC T T A A

AAT T CG 3’

5’

USING PLASMIDS

Plasmid is small circle of bacterial DNA

Foreign DNA can be inserted into plasmid

Forms recombinant plasmidsPlasmid is a cloning vectorCan deliver DNA into another

cell

Fig. 16-3b, p.244

Plasmids

Restriction enzyme cuts molecule of chromosomal DNA or cDNA

recombinant plasmids containing foreign DNA

host cells containing recombinant plasmids

Same enzyme cuts same sequence in plasmid DNA

Foreign DNA, plasmid DNA, and modificationenzymes are mixed

DNA or cDNAfragments with sticky ends

plasmid DNA with sticky ends

Stepped Art

Fig. 16-4, p.245

a A restriction enzymecuts a specific base sequence everywhere it occurs in DNA.

c The same enzyme cuts the same sequnece in plasmid DNA.

d The plasmid DNA also has sticky ends

e The DNA fragments and the plasmid DNA are mixed with DNA ligase.

f The result? A collection of recombinant plasmids that incorporate foreign DNA fragments.

g Host cells that can divide rapidly take up the recombinant plasmids.

b The DNA fragmentshave sticky ends.

Fig. 16-4, p.245

Using Plasmids

MAKING CDNA

Fig. 16-5, p.245

GENE LIBRARIES

Bacteria that contain different cloned

DNA fragments

Genomic library

cDNA library

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

Will base-pair with gene of

interest

a Bacterial colonies, each derived from a single cell, grow on a culture plate. Each colony is about 1 millimeter across.

b A nitrocellulose or nylon filter is placed on the plate. Somecells of each colony adhere to it. The filter mirrors how the colonies are distributed on the culture plate.

c The filter is lifted off and put into a solution. Cells stuck to it rupture; the cellular DNA sticks to the filter.

d The DNA is denatured to single strands at each site. A radioactively labeled probe is added to the filter. The probe binds to DNA with a complementary base sequence.

e The probe’s location is identified by exposing the filter to x-ray film. The image that forms on the film reveals the colony that has the gene of interest.

Fig. 16-6, p.246

Use of a Probe

FAMILIAL HYPERCHOLESTEROLEMIA

Gene encodes protein that serves as cell’s 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

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

AMPLIFYING DNA

Fragments can be inserted into fast-growing microorganisms

Polymerase chain reaction (PCR)

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

POLYMERASE CHAIN REACTION

Double-stranded DNA to copy

DNA heated to 90°– 94°C

Primers added to base-pair with ends

Mixture cooled; base-pairing of primers and ends of DNA strands

DNA polymerasesassemble new DNA strands

Fig. 16-6, p. 256

Stepped Art

POLYMERASE CHAIN REACTION

Stepped Art

Mixture heated again; makes all DNA fragments unwind

Mixture cooled; base-pairing between primers and ends of single DNA strands

DNA polymerase action again doubles number of identical DNA fragments

Fig. 16-6, p. 256

RECORDING THE SEQUENCE

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

T C C A T G G A

T C C A T G G

T C C A T G

T C C A T

T C C A

T C C

T C

T

electrophoresisgel

one of the many fragments of DNA migratingthrough the gel

one of the DNA fragmentspassing through a laser beam after moving through the gel

T C C A T G G A C C A

•DNA is placed on

gel•Fragments move off

gel in size order;

pass through laser

beam•Color each

fragment fluoresces

is recorded on

printout

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

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

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

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

Fig. 16-9b, p.249

GEL ELECTROPHO

RESIS

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

Identify bodies

Determine paternity

BOZEMAN VIDEO—DNA FINGERPRINTING

http://www.youtube.com/watch?v=DbR9xMXuK7c

GENOME SEQUENCING

1995 - Sequence of bacterium Haemophilus influenzae determined

Automated DNA sequencing now main method

Draft sequence of entire human genome determined in this way

GENOMICS

Structural genomics: actual mapping and sequencing of genomes of individuals

Comparative genomics: concerned with possible evolutionary relationships of groups of organisms

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

ENGINEERED PROTEINS

Bacteria can be used to grow medically

valuable proteins

Insulin, interferon, blood-

clotting factors

Vaccines

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

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

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

THE TI PLASMID

Researchers replace tumor-causing genes with beneficial genes

Plasmid transfers these genes to cultured plant cells

foreign genein plasmid

plant cell

Fig. 16-13, p.253

a A bacterial cell contains a Ti plasmid (purple) that has a foreign gene (blue).

b The bacterium infects a plant and transfers the Ti plasmid into it.

c The plant cell divides.

d Transgenic plants.

e Young plants with a fluorescent

gene product.

The Ti plasmid

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

TRANSGENIC MICE

Fig. 16-15, p.254

CLONING DOLLY

1997 - A sheep cloned from an adult cell

Nucleus from mammary gland cell was inserted into enucleated egg

Embryo implanted into surrogate mother

Sheep is genetic replica of animal from which mammary cell was taken

DESIGNER CATTLE

Genetically identical cattle embryos can be grown in culture

Embryos can be genetically modified

create resistance to mad cow disease

engineer cattle to produce human serum albumin for medical use

XENOTRANSPLANTATION

Researchers knockout the Ggta1genes in transgenic piglets

Ggta1 gene produces proteins that human antibodies recognize

Pig’s organs are less prone to rejection by a human

Fig. 16-14c, p.254

Genetically Modified Animals

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

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

ETHICAL ISSUES

Who decides what should be “corrected”

through genetic engineering?

Should animals be modified to provide organs

for human transplants?

Should humans be cloned?