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11 Δεκ 2012 (πριν από 4 χρόνια και 8 μήνες)

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Principles of Biology I (BIO103)


Reading Assignment 2:
Genetic Engineering


Introduction



Of all the scientific discoveries of the 20
th

century, none has had a more profound
impact on our understanding of the physical universe, and our place in it, than
the
discovery of the genetic material and the
deciphering of the genetic code.

We now
understand that all living things pass information from one generation to the next with
DNA (deoxyribo nucleic acid), the molecule that contains the genetic code, and all

living
things, including viruses, use RNA (ribo nucleic acid) to manufacture the proteins of life.
As molecular biologists grow more fluent in genetic language, they have learned to edit
the genetic code, thus altering life itself.
What we can read, we ca
n now edit and write
anew. Our growing understanding is being applied in many ways, but genetic
engineering

which involves the alteration of a
genome
: to add, subtract, or to alter
genes in other ways

exemplifies the promise and the peril of genetic techno
logies.



The earliest efforts of genetic engineering focused on the large
-
scale production
of various proteins

essential in medicine, food production, and various kinds of basic
research. These proteins all employ single
-
celled bacteria that reproduce ver
y quickly,
providing many generations of bacteria for scientists to work with.


Restriction Enzymes



Hundreds of strains of bacteria and a few eukaryotic cells contain
restriction
enzymes

that cut DNA wherever a particular nucleotide sequence occurs.

Eco
RI cuts
between the G and the A of the sequence GAATTC. EcoRI and many other restriction
enzymes make staggered cuts. These cuts have single
-
stranded tails, or “sticky ends,” on
the cut fragments. Each tail can base
-
pair with the tail of any other DNA frag
ment that
was cut by the same enzyme, because their sticky ends match up.
DNA ligase

appropriated from replication and repair processes is used to “glue” restriction fragments
together. This result is a molecule of
recombinant DNA
.


Cloning Vectors



Bact
eria have one circular chromosome in their cells. Many also have
plasmids
,
much smaller circles of DNA with a few extra genes. Bacteria divide, making huge
populations of genetically identical cells. Before each division, replication enzymes copy
both the
chromosomal DNA and the plasmid DNA. Researched discovered that foreign
DNA inserted into a bacterial plasmid gets replicated and distributed into each daughter
cell along with the plasmid. Bacteria could thus be used to copy a small fragment of
DNA again
and again. A plasmid that accepts foreign DNA and slips into a
cell of a
host
bacteri
um
, yeast, or another kind of cell is called a
cloning vector
.

Cloning vectors
typically contain several unique restriction enzyme recognition sites that can be used for
c
loning. A cell that takes up a cloning vector with foreign DNA in it may establish a huge

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population of descendant cells, each with an identical copy of the vector and a “cloned”
copy of the foreign DNA.

DNA cloning is one tool in the molecular toolkit. It

helps
scientists to isolate and identify manageable sections of huge DNA molecules, and also to
obtain enough of each bit to be able to study it.


cDNA Cloning



F
iguring out where a gene is in chromosomal DNA is not by any means obvious.
Unlike a gene i
n chromosomal DNA
, an mRNA conveniently contains only a coding
sequence, minus introns, along with appropriate signal sequences for its translation. Also,
when a gene is active, its transcribed mRNA is present in good quantity. For these
reasons, researche
rs studying gene expression use mRNA as their starting material.



Genes used for protein expression in research and product development are
typically cloned, but mRNA cannot be cloned directly. Restriction enzymes will cut only
double
-
stranded DNA, so an

mRNA must be first converted into double
-
stranded DNA
before it can be spliced into a vector. A retroviral replication enzyme,
reverse
transcriptase
, is used to assemble a strand of cDNA, or
complementary

DNA
, on an
mRNA template.

A hybrid molecule, one s
trand of mRNA base
-
paired with one strand of
cDNA, is the outcome. DNA polymerase added to the mixture then strips the RNA away
as it copies the first strand of cDNA into a second DNA strand. The result is a double
-
stranded DNA copy of the original mRNA. B
ecause it is double
-
stranded, that copy may
be used for cloning.



A gene library is a mixed collection of host cells that have taken up cloned DNA.
A particular gene can be isolated from a library by using a probe, a short stretch of DNA
that can base
-
pa
ir with the gene and that is traceable with a radioactive or pigment label.
Probes help researchers identify one particular clone among millions of others. Base
-
pairing between strands of DNA or RNA from different sources is called
nucleic acid
hybridizati
on
.


The Polymerase Chain Reaction (PCR)



The polymerase chain reaction (PCR) is a way to rapidly copy particular pieces of
DNA. A DNA template is mixed with nucleotides, primers, and a heat
-
resistant DNA
polymerase. Each cycle of PCR proceeds through a
series of temperature changes that
doubles the number of DNA molecules. After 30 cycles, PCR amplification can result in
a billionfold increase in the number of DNA molecules.










DNA Sequencing



Once a targeted DNA molecule has been isolated

in sufficient quantity, the order
of its nucleotide bases may be determined with
DNA sequencing

another technique in
the molecular toolkit. As DNA polymerase is copying a template DNA, progressively
longer fragments stop growing when one of four kinds of
modified, fluorescent
nucleotides becomes attached.
Gel electrophoresis

separates the resulting labeled DNA

3

fragments into bands according to length. The order of the colored bands as they migrate
through the gel reflects which fluorescent base was added t
o the end of each fragment,
and so indicates the template DNA nucleotide sequence.



Tandem repeats

are multiple copies of a short DNA sequence that follow one
another along a chromosome. The number and distribution of tandem repeats, unique in
each perso
n, can be revealed by gel electrophoresis; they form a
DNA fingerprint
.



Application of Genetic Engineering



Bacteria have very simple genomes that contain genetic information. In the
simplest genetic engineering protocols, bacterial cells are expos
ed to strands of DNA
with a desirable gene on them. The DNA can be from any other organism

a human
being, a plant, or even a virus, because we sometimes can get a virus to incorporate a new
protein. It’s difficult to target the correct part of a bacterial
genome, so when you insert
new DNA material, it may be incorporated into the genome, or it may not. Sometimes it
is incorporated into a place where it can’t be expressed, and it goes unnoticed.



But once in a while, if you expose the bacterium to a speci
fic gene, that gene may
be incorporated in such a way that it can be expressed, and the bacterium makes some of
the protein
for which that gene codes. Given enough bacteria, a few cells may be
successful. Then it just becomes a brute
-
force method where you

expose lots and lots of
cells to the desired gene, and then extract the few cells that have successfully
incorporated that particular gene.



Once you have a single bacterium that has the correct gene and expresses it, then
that bacterium can be moved, an
d isolated, and it starts multiplying on its own, making
more and more copies of itself. Where you had one bacterium before, you’re going to
have countless numbers, if you give them the appropriate environment in which to grow
and develop.



Dramatic earl
y successes of this kind included the development of
E. coli

strains
that were engineered to contain specific genes for components of human insulin and
synthetic growth hormone. Prior to this technology, diabetics had to take insulin from
another animal, o
ften pig insulin, which is not identical to human insulin. But now you
can engineer insulin that is identical to human insulin, so it’s much more effective for
diabetics. Pharmaceutical companies culture these bacteria in huge vats to produce the
desired p
roteins in commercial quantities. You have lots and lots of bacteria, which are
literally chemical factories producing the specific desired proteins.

All you need to begin
is one successful bacterium, one successful microbe that duplicates itself over and
over
again. On a much smaller scale, scientists obtain significant quantities of rare proteins by
a similar technique.



Bacteria can manufacture huge quantities of whatever protein you want, as long
as you can get them to start doing this technique. Then

the proteins can be purified, and
then you can make crystals of them by purifying them, extracting and crystallizing them

4

out. You can do x
-
ray structure studies, for example, on hemoglobin or on other proteins.
This is an important way of discovering pro
tein structures

by starting with microbes.
This whole technique has proven especially valuable in studies of defective proteins

that
are designed intentionally to incorporate one or more amino acid mistakes. If you
understand the behavior of a mutant prote
in, then you can gain insight on the function of
enzymes when they’re working correctly. You also can get a feeling for what causes
genetic diseases, which are nothing more than genes that are producing defective
proteins.



For example, in mice, damaging

one protein causes the animals to become
gluttonous

(voracious eating habit). They overeat and begin to get fatter and fatter

just
by altering a single gene.
Eventually, the mice gain so much weight they can hardly move
around in their cages. We now know
that particular target proteins acts as a hunger
suppressant of some kind, and similar genes occur in humans; this may be a key to
certain overeating diseases.



This research would not be possible without genetic engineering; you can
engineer bacteria to

incorporate the defective mouse protein. Because they reproduce so
fast, the bacteria quickly produce lots of the target protein, so it can extracted and
analyzed and understood in a way that you never could if using mammals, which
reproduce much, much mo
re slowly.



You can insert appropriate genes into a cell’s genome in plants as well.

Because
plants have a similar characteristic, you can grow an entire plant from a single cell. Under
the proper growth environments, the entire plant can be cloned. If y
ou modify a single
cell in a plant, and put it in the proper environment, you can then produce a whole new
plant that has the modification built into every cell. In one historic series of experiments,
plants were infected with engineered bacteria that cont
ained a gene for proteins that were
toxic to insects; an insect repellent. The bacteria infected plants, and some of those
bacteria transformed some of their genetic material into the plant through a tumorous
growth.

By extracting some of the genetically m
odified cells from the tumor, it was
possible to grow new plants that had the insect resistant capability originally present in
the engineered bacterium.



This is a way of engineering plants

by actually exposing them to bacteria that
may cause an individ
ual plant to get sick, but that plant produces a whole new strain of
plant cells that contain that modified genome. Continuing experiments of this kind now
focus on increasing crop yields for foods and fibers, extending the shelf life of fruits and
vegetab
les, and improving resistance to drought and diseases.



Today, in your grocery store, you can probably buy

some of these chemically
engineered products. The Calgene Corporation has produced a new line of tomatoes.
Tomatoes normally begin to rot when they
’re pulled off the vine, because as soon as the
tomato is pulled off the vine, a mechanism is triggered that starts forming a protein
causing the tomato to rot from the inside. A tomato, after all, wants to rot; it wants to
distribute its seeds as soon as
possible. The Calgene tomato turns off that gene so the

5

tomato does not start rotting and stays fresh longer. You can haul these tomatoes longer
distances; they stay fresher on the grocery shelves.



Not long ago the World Health Organization made an esti
mate that 124 million
undernourished children around the world have vitamin A deficiency. These children
may become permanently blind and develop other disorders. To help these children,
geneticists transferred genes from daffodils into rice plants. The pl
ants began making
beta
-
carotene, a yellow pigment converted in the body to vitamin A.
B
eta
-
carotene is
stored in the plant seeds

the grains of “golden rice.”




Other crops that are more resis
tant to drought or poor soils, or act as natural
herbicides, ha
ve been designed. We have frost
-
resistant strawberries. We have plants that
manufacture plastics by inserting a gene that forms one of the basic polymers that will
make plastics. It’s amazing what you can do with genetic engineering, and we are only
just b
eginning to learn this process.



There are, of course, new ethical concerns that are raised from these kinds of
genetic experiments. Technology to insert healthful genes into bacteria might just as
easily be used to create highly toxic microorganisms to
be used as biological weapons.
As genetic technologies become very routine, how are we going to protect ourselves?
More troubling to some people are the unintended consequences of introducing what they
see as essentially a new species, or new varieties of
life, into stable ecosystems. If you
introduce a plant that’s resistant to insects, is that going to change
the natural balance of
an ecosystem? Complex systems, like ecosystems, may be changed in dramatic and
unexpected ways just by introducing small chan
ges.



Of course, on the other hand, nature is constantly introducing new varieties
through natural processes, through mutations and so forth.
You have to ask yourself, is
there a real difference between what nature does and what humans engineer? It’s a r
eal
philosophical puzzler.



The genetic engineering of animals is extremely difficult because we haven’t yet
learned to duplicate animals from the genetic material of a single cell. Bacteria and plants
can often be modified by brute force and then regrow
n from a single cell that’s been
modified. You can’t clone animals easily from just any single cell.



Some of the difficulties associated with genetic engineering were captured in
Michael Crichton’s book,
Jurassic Park
, which dealt with bringing back the
dinosaurs.
The novel’s conceit involved finding ancient amber that preserved insects such as
bloodsucking mosquitoes.

That allowed scientists to go into the mosquito’s gut, pull out
some of the blood cells it had been sucking, and to actually recover a com
plete set of
DNA from a dinosaur.



It’s a fascinating idea, but it’s not so easy. The most basic problem in bringing
back any ancient life form is obtaining the complete genome: How do you get a complete
set of chromosomes? For DNA to work properly, every

base pair has to be in line on the

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complete sequence, and you have to have the complete set of chromosomes. The reality is
that DNA tends to degrade over time; you can’t preserve a genome for millions of years.
Effects of heat and radiation disturb it. As

soon as an animal dies, the DNA starts
degrading, and that’s an irreversible process. This is also true for DNA in your body. It
suffers thousands of hits every day, in every cell; but the living body has many different
repair mechanisms. It continuously
reverses this damage in every cell of your body. As
soon as the organism dies, the repair mechanisms stop, but the damage goes on.



More realistically, you might be able to preserve libraries of genetic material for
organisms now threatened with extinctio
n

whales, condors, tigers, many endangered
rainforest species. You can preserve them in the form of a few cells, each with a complete
copy of the unique genome. Then maybe someday we’d be able to figure out what to do
with those genomes to bring those crea
tures back, if they do go extinct.



By far the most urgent applications of genetic engineering are the cure of human
diseases, inherited genetic diseases. More than 3,000 inherited human genetic diseases
are now recognized: cystic fibrosis, muscular dyst
rophy, sickle
-
cell anemia, juvenile
arthritis, hemophilia. There are hereditary forms of cancer and numerous other genetic
ailments that are now understood at the molecular level. Each of these diseases arises
from a different gene defect. Each one has to
be investigated independently, individually.
In some instances, it’s a single mistake in a gene’s nucleotide sequence that leads to a
defective protein.



Someday, as the variants of each gene are discovered, DNA analysis may be able
to provide everyone w
ith a quick, in
-
depth genetic medical screening

perhaps even
before birth.