Analysing and ENGINEERING DNA, genes, cells and

organisms (Ch. 19ish)

 The genetic code (AAA, GCG) is near universal
Complimentary base-pairing (C with G, A with T) is universal
All organisms copy (replicate) DNA the same way
All organisms produce proteins pretty much the same way
(transcription/translation)

We can make use of this BASIC knowledge =

genetic engineering or biotechnology

Transgenic salmon with a mouse promotor

controlling a rat growth hormone gene

Tobacco plant with Fluorescent pig with

firefly gene jellyfish gene
 Transgenics how do we move genes between
different organisms (Fig 19.3 in Freeman)

Human?

Escherichia
coli

Human?

Bacteria have plasmids small circular DNA molecules that replicate

separately from the bacterial chromosome

DNA from a different species (e.g. human) can be inserted into bacterial
plasmid and re-inserted into recombinant bacterium

Let bacteria replicate to form clones (populations) of cells with many

copies of the inserted section of human gene = cDNA library
WHY? Use bacteria to make genes or proteins normally
made by, and useful to, humans (or any other organism)
How do you insert foreign DNA into a bacterial plasmid?
cutting and pasting genes

Restriction enzymes
(endonucleases from bacteria) cut
DNA in a very specific manner

Single stranded sticky ends

(TTAA) allow for complimentary
base-pairing

Foreign DNA is cut with same

Foreign DNA
Human? restriction enzyme producing
complimentary sticky end (AATT)

Complimentary base pairing

occurs inserting foreign DNA

DNA ligase is used to seal DNA

strands
 Storing cloned genes in cDNA libraries

Each clone contains copies of different

fragments of the foreign genome

Different clones of - these are stored in libraries in 384-well

recombinant bacteria plates
carrying different
recombinant plasmids How do we find the clone with the section
with different genes of DNA or gene we are interested in?
Goin fishin with a nucleic acid probe that utilises
complimentary base pairing

Probe and complimentary

DNA hybridise

Bathe membrane in
Transfer few cells
solution containing
from each clone
radiolabeled probe

Known DNA sequence 5 CTCATCACCGGC 3

Synthetic probe sequence 3 GAGTAGTGGCCG 5

 You can make lots of DNA using bacteria or you can
mimic DNA replication: polymerase chain reaction (PCR)

PCR machines are thermocyclers

Heating denatures DNA separating

double strands 1

Cooling allows primers to anneal (bind)

to complimentary DNA 2

Extension phase DNA polymerase

adds nucelotides to 3 end 3

30 cycles = a billion DNA copies!

Gel electrophoresis is used to separate out
mixes of nucleic acids or proteins (Fig. 20.9)

Gel = polymer (e.g. agarose) that acts

like a molecular sieve

Separates nucleic acids or proteins on

the basis of size or electrical charge

All negatively charged nucleic acids

move towards anode (+) but at different
speeds

Longer, larger DNA fragments move

more slowly

Can use this to compare DNA of

different people = DNA fingerprinting
 Create a DNA Fingerprint
It's the case of the licked lollipop, and you have to solve it. Fortunately,
you have the latest forensic technology on your side: DNA profiling. This
interactive feature, part of the NOVA: "The Killer's Trail" Web site, guides
you through the process of creating DNA profiles of several criminal
suspects and tissue evidence left at the crime scene. Then you'll
compare the profiles you've created and, hopefully, find the criminal --
just like forensic investigators do.

[Link]
 Is genetic engineering good or bad?

Are genetically-modified organisms (GMOs)

good or bad?

While scientific progress on molecular biology has great potential

. it should not be used as justification to turn the environment into
a giant genetic experiment by commercial interests

The release of GMOs is 'genetic pollution' and is a major threat

because GMOs cannot be recalled once released into the
environment

The biodiversity and environmental integrity of the world's food

supply is too important to our survival to be put at risk

[Link]
 DNAs structure was discovered in 1953
- so how long ago we did start genetically-modifying organisms?

Domestic hen

Red junglefowl
Zea maize

Teosinte

25 g egg
4-7 eggs 60 g egg
Maize was developed > 9000 years ago >300 eggs

Weve been modifying plants and animals for a long, long time!!
Golden Rice the poster organism for genetic engineering
Vitamin A deficiency causes blindness and death in
hundreds of thousands of children under 5 each year

Golden Rice has 2 genes added

(transgenics)
1 from daffodil
1 from soil bacterium

Makes the rice express beta-carotene, a

precursor of vitamin A (provitamin), in the
edible part of the grain

Does it work?

Philippines 2013:
anti-GMO activists
pull up golden rice
plants
 Glow-in-the-dark pets: good idea or bad idea?

Fluorescent felines glow for

science: it is a rite of passage
for any sufficiently advanced
genetically-modified organism; at
some point scientists will splice in
a gene that makes you glow

Gene = green fluorescent protein (GFP)

Model for HIV/AIDs research

Cats have feline immunodeficiency Glofish
disease a close relative of HIV
 Are GMOs good or bad?
Widespread starvation globally
800M people have nutritional deficiencies
Popn growth will require 40% increase in food production

Advantages

Higher crop yields, foods with better nutrition value

Less pesticide use; plants engineered to be healthier, more resistant
Less deforestation; converting land to agriculture
New products, e.g. naturally decaffeinated coffee beans
Development of drought- or cold-resistant crops

Disadvantages

Concerns about human health

Harmful effects on non-target species
Transgene escape to wild plants
Spread of novel, genetically-superior pests and weeds
Higher cost of GMO seed, etc might not help developing countries
Social concerns: organic food, 100 mile diets
Cellular engineering (playing with cells): good or bad?

In vitro fertilisation (IVF)

an egg is fertilized by sperm outside
the body (in vitro cf. in vivo)
IVF is a major treatment for infertility
first successful birth of a "test tube
baby in 1978; now > 5 million IVF
births

What DNA does the embryo inherit

during IVF?

Embryos get both nuclear and

mitochondrial DNA (mtDNA) from
the mothers egg cell

Mutations in mtDNA cause birth

defects every 1:6500 cases
Blindness, muscular disorders,
learning disabilities, diabetes
A solution (or another problem)? IVF with mitochondrial
transfer
Designer babies playing God in the womb

Are we ready for three-parent babies?

Social issues, legal

issues???

Procedure approved June 2013 in UK

 Cloning - producing an identical copy of an organism
All multicellular organisms start life as single cells
(embryos)

So why cant we simply take a single cell and grow up a

whole new (identical) organism?

Single, mature fully differentiated plant cells (e.g. from root)

can de-differentiate and then give rise to all other
specialised cell types (e.g. stem, leaf cells) = pluripotent (or
totipotent) cells

But in animals, only embryonic and adult stem cells

retain the ability to differentiate into a wide variety of
different cell types, i.e. pluripotent animal cells are rare
 So cloning of a whole carrot plant from a single
carrot root cell is relatively easy (talk to Dr. Zamir Punja)

Shoot

Root

Single differentiated cells from mature plants are pluripotent i.e.

capable of generating all the tissues of a whole new plant
 But cloning of animals proved far more difficult
first done in 1997 (Fig 21.2 in Freeman)

Dolly

Mammals can be cloned by transplanting the nucleus from a differentiated

animal cell into an egg (which has had its nucleus removed)
Less than 20 years after cloning of Dolly safe, genetic
preservation and cloning of your pets is a reality
 5-10 years ago Playing God with embryonic stem
cells was highly controversial: good or bad?

Embryonic stem (ES) cells

reproduce indefinitely and can
differentiate into any of the 200
human cell types
= pluripotent

ES cells are isolated from the inner cell mass of 4-5

day old blastocysts consisting of 50150 cells
 Only a few years ago finding adult stem cells was
huge news!

Adult stem cells were thought to be rare and they can differentiate into
fewer, specific cell types; useful for tissue regenerative therapies

[Canadian!] Blood stem cell discovery may

revolutionise transplants July 2011

[Canadian!] Pioneering heart attack stem cell

treatment trial treats 1st patient CBC, Sept 2013

Self-organised vascular networks from human

pluripotent stem cells transplants PNAS Sept.
2013
 Induced pluripotent stem cells (iPS): reprogramming
cells in vivo

Nature
4 Sept 2013
ES

iPS
in vitro

iPS
in vivo

Take mature cells, de-differentiate them to stem-cell state, then re-program

them to develop into the cells you want and do this is a live animal!
 Stem cells: Close encounters with full potential
(Nature Sept 18 2013)

Induced pluripotent stem cells

Conferring stem-cell potential on mature cells is not easy. A decisive

impediment to this process has now been identified, and its elimination
allows almost all mature cells to efficiently adopt a stem-cell identity
 Food for thought
Are stem-cell hamburgers a good idea?

Muscle cells harvested from a living cow

Cells are fed and nurtured so they multiply to create muscle tissue
Cells grow into strands; 20,000 small strands of meat = 140g burger
The first burger costs $333,000!

It is biologically exactly the same meat tissue that comes from a cow

No genetic modification is involved in this process

CRISPR/Cas9 system: a powerful gene-editing technology, the
biggest game changer to hit biology since PCR
Part of prokaryotic immune system that cuts up foreign genetic material for
viruses, etc

[Link]

cheap, quick and easy, allowing researchers to quickly change the DNA
of nearly any organism including human embryos (Nature 520, 593595; 2015)
is the field's breakneck pace leaving little time for addressing the ethical
and safety issues from such experiments?
- a newspaper!
 Cool story: A flatworm never forgets even when it
loses its head

If you cut an earthworm in half

do you get TWO earthworms?

Flatworms can regenerate a new head

and brain from a tail section

And they regenerate memories

associated with their old brain
No! You get TWO halves of a
DEAD earthworm e.g. associated food with light

J Exp Biol 2013 216:3799-3810