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STUDY GUIDE: DNA. PROTEIN SYNTHESIS AND BIOTECHNOLOGY
I hate being a DNA
molecule! There’s
so much to
remember!
I. DNA Structure :
The DNA molecule is made of two strands of repeating units called nucleotides. A nucleotide is
composed of three parts: a sugar group, a phosphate group and one of four nitrogen containing bases.
Nucleotides are bonded together in both vertical and horizontal dimensions to give the famous double helix
shape. See Fig. 10-6 & 10-8
II. DNA replication - the process by which two identical strands of DNA are made from one original.
A. The double helix is unzipped by the enzyme DNA helicase
B. Weak attractions called hydrogen bonds are broken between the bases (A &T I G & C)
C. Each exposed strand acts as a template to refigure the missing half.
D. Free floating nucleotides in the cytoplasm find their complement base and reestablish hydrogen
bonds. See Fig. 10-10
Example:
DNA: A T T C C G G C T
to
DNA: T A A G G C C G A
(Template strand)
(new strand)
Protein Synthesis.
I. Transcription - the process of transferring the DNA message to mRNA (messenger RNA).
mRNA is necessary because DNA cannot leave the nucleus , but yet the message it has needs to get to the
ribosomes, which are in the cytoplasm.
A. Hydrogen bonds between portions of the DNA strand are broken, as before during replication,
exposing the sense strand. The sense strand is that side of the ''DNA ladder" that acutally codes
for the protein.
B. The enzyme RNA polymerase takes freely floating RNA nucleotides and forms temporary
bonds with their complementary DNA nucleotide. The base pairing rules are similar to DNA
replication but with one important difference . The base Thymine is not found in RNA. It is
replaced with Uracil, a base that will also bond with Adenine.
Example: DNA : A T C C G A G C T A
to
mRNA: uA G G C U C G A U
C. The single stranded mRNA leaves the nucleus for the cytoplasm where its instructions will be
translated.
II. Translation - the process of constructing the protein that mRNA codes for.
Can you see any recognizable message here?
ateukerseethecatatetheratbakudieoneue
A. Codon - a series of three nucleotides that code for a particular amino acid. Codons are the
"words" of the genetic language. See Table 10-1 on page 207. What conclusions can you make?
B. Amino acid - the building blocks of proteins. All proteins are made from a combination of 20
different amino acids. Each of the 20 amino acids has one or more codons.
C. t (transfer)RNA - another type of RNA that decodes the mRNA. Called tRNA, because they
transfer the appropriate amino acid from the cytoplasm to the ribosome I mRNA complex where
proteins are made.
D. anti codon - a series of three bases that are complementary to mRNA codons. A tRNA
anticodon I mRNA codon match ensures the proper amimo acid gets put in the correct sequence of
the protein.
E. peptide bond - strong bonds that glue one amino acid to the next. A protein is a long chain of
amino acids glued together with peptide bonds. The sequence of amino acids was determined by
the sequence of bases in DNA.
Study Figure 10-16 on pages 208 and 209 for a good visual summary of the process of protein synthesis.
DNA message
transcribed to
mRNA by
complementary
base painng.
C-G and A-U
I
mRNA leaves
nucleus and is
translated by
tRNA. mRNA
codon bonds with ·
tRNA anticodon
I
occurs in the
cytoplasm at
the ribosome
occurs m
the nucleus
,r
DNA
tRNA brings
amino acids
to the ribosome
amino acids
are joined together
by peptide bond formation
v
mRNA
tRNA
Protein
Trait
Gene Mutations
A. Mutation - an error or change in the DNA message. Mutations may involve entire pieces of a chromosome or
may be a "spelling" error in a particular gene.
Point Mutations - result from the substitution, deletion or additon of a nucleotide in a particular gene.
Normal
Substitution
<no effect)
Substitution
(with effect) 1111!!'!!!!!!!!!!!!!!!!!
Met
Insertion
Deletion
Examples of Gene I Point Mutations That Lead to Human Health Disorders
Sickle cell anemia - This is a blood disorder in which a base substitution occurs in the gene for hemoglobin.
This mutation causes the red blood cells to become sickle shaped. The shape of these cells do not allow proper
circulation through small blood vessels. In addition, these cells do not carry oxygen as well as normal red blood
cells. Individuals with this disease, on average do not live beyond their mid twenties. This disease is most
common in African Americans. Treatment: (1) Possible gene therapy at embryonic stage of development (2)
Medications that restore the cells to their normal shape. (3) blood transfusions
Hemophilia - A blood disorder in which the individual is missing the blood clotting protein, Factor VIII.
Simple cuts or scrapes could be fatal because the bleeding can not be stopped. Treatment: (1) Supply
individual with an external source of Factor Vlll serum which is injected into the body (2) Skin grafts that
contain cells which can produce enough Factor VIII for the body's needs. (3) Gene therapy at early stages of
development.
Diabetes - A metabolic disorder in which tie individual lacks sufficient amounts of insulin, a hormone that
controls the amount of sugar in the blood. Treatment In the past, insulin was extracted from the pancreas of
pigs and other animals;an expensive and inefficient way of getting insulin. In 1982.the gene for human insulin
was isolated and put into bacteria cells which produce it by the gallon in large fermentation vats.
Tay - Sachs - A disorder of the central nervous system in which the individual lacks a chemical that breaks
down fatty acids in the brain and surrounding nerve cells. The result is that the brain cells and surrounding
nervous tissues "clog up" with fat and become useless. lndividual s with Tay-Sachs rarely live beyond -their
teens. This disease is most common in Jewish people. Treatment: Currently there is no cure. Research is
being done to develop a medication that will take the place of the missing chemical. In the future, scientists hope
the find the gene mutation responsible for this and correct it at a cellular level.
PKU IPhenylketonuria) - A metabolic disorder in which the individual can not break down the amino acid,
(g)
phenylalanine. Over time, the concentration of phenylalanine increases to such a point that it impairs the normal
development of -the brain. if unchecked , PKU can lead 1:0 severe mental retardation in young children.
Treatment: (1) A diet low in phenylalanine. (2) Produce an enzyme that breaks down phenylalanine in the lab
which can be injected into the individual.
Cystic Fibrosis - One of the most common genetic diseases known. One in 2,000 babies born are affected by
CF while 1in 20 Caucasians carry this gene. People who suffer from CF can not expel salt from their lung
cells. As a result a thick mucus develops in the lungs, making breathing difficult. This abnormal mucus
clogs the lungs and leads to fatal infections. The mucus also obstructs the pancreas , preventing enzymes from
reaching the intestines to digest food. This disease is most common in caucasian people. Treatment: (1)
Medications such as Pulmozyme (developed in 1993) and high doses of ibuprofen have been helpful. (2) Gene
therapy is currently being tested on a number of patients with posi tive results.
Huntingtons Disease - A degenerative central nervous system disorder showin g itself during middle age.
Treatment: Unfortunately , there is no treatment and no cure. However, a test has been developed that can tell
an individual whether they will have the disease or not. Although knowing this will not help the person directly,
at least it can help them determine if they should have children or not.
* see other examples of point mutations on Table 12-1 of your text (page 246).
Chromosome Mu t ations:
Changes in chromosome structure are another type of chro­
mosomal mutation. Some but not all changes in chromo­
some structure can be detected microcop1cally (Fig. 12.11).
Various agents in the environment. such as radiation, certain
organic chemicals, or even viruses, can cause chromosomes
to break. Ordinarily, when breaks occur in chromosomes,
the two broken ends reunite to give the same sequence of
gens. Sometimes, however, the broken ends of one or more
chromosomes do not rejoin in the same pattern as before,
and the result is various types of chromosomal mutation.
Changes in chromosome structure include deletions,
translocations, duplications, and inversions of chromosome
segments, as illustrated in Figure 12.11.
A deletion occurs when an end of a chromosome
breaks off or when two simultaneous breaks lead to the Joss
of an internal segment. Even when only one member of a
pair of chromosomes is affected, a deletion often causes ab­
normalities.
A translocation is the movement of a chromosome seg­
ment from one chromosome to another, nonhomologous
chromosome. In 5% of cases,a translocation that occurred in
a previous generation between chromosomes 21 and 14 is
the cause of Down syndrome. In other words, because a
portion of chromosome 21 is now attached to a portion of
chromosome 14, the individual has three copies of the alleles
that bring about Down syndrome when they are present
in triplet copy. In these cases, Down syndrome is not related
to the age of the mother, but instead tends to run in the
family of either the father or the mother.
A duplication is the presence of a chromosomal seg­
ment more than once in the same chromosome. Duplica­
tion is known to occur as a re:.ult of an inversion in which
asegment of a chromosome is turned around 180°
a. Deletion
b. Translocallon
c.Dupl cation
d. Inversion
Figure 12.11 Types of chromos;,mal mutations.
a. Deletionis the loss of a chromosome piece.b.Translocation is the
exchange of chromosome pieces between nonhomologous pairs.
c. Duplication occurs when the same piece is repeated within the
chromosome .d.Inversion occurs when a piece of chromosome breaks
loose and then rejoins in the reversed direction.
d
Human Syndromes
Changes in chromosome structure occur in humans and
lead to various syndromes, many of which are just now be­
ing discovered. Sometimes changes in chromosome struc­
ture can be detected in humans by doing a karyotype. They
may also be discovered by studying the inheritance pattern
of a disorder in a particular family.
deletion
e
9
h )
i
9 .,
a.
+
Deletion Syndromes
Williams syndrome occurs when chromosome 7 loses a tiny
end piece (Fig. 12.13). Children who have this syndrome
look like pixies, with turned-up noses, wide mouths, a
small chin, and large ears. Although their academic skills
are poor, they exhibit excellent verbal and musical abilities.
The gene that governs the production of the protein elastin
is missing, and this affects the health of the cardiovascular
system and causes their skin to age prematurely.Such indi­
viduals are very friendly but need an ordered life, perhaps
because of the loss of a gene for a protein that is normally
active in the brain.
Cri du chat (cat's cry) syndrome is seen when chromo­
some 5 is missing an end piece. The affected individual has
a small head, is mentally retarded, and has facial abnormal­
ities. Abnormal development of the glottis and larynx re­
sults in the most characteristic symptom-the infant's cry
resembles that of a cat.
lost
b.
Figure 12.13 Deletion.
a. When chromosome 7 loses an end piece, the result is Williams
syndrome. b.These children, although unrelated, have the same
appearance, health, and behaviora l problems.
Translocation Syndromes
A person who has both of the chromosomes involved in a
translocation has the normal amount of genetic material and
is healthy, unless the chromosome exchange breaks an allele
into two pieces. The person who inherits only one of the
translocated chromosomes will no doubt have only one
copy of certain alleles and three copies of certain other al­
leles. A genetic counselor begins to suspect a translocation
has occurred when spontaneous abortions are common­
place and family members suffer from various syndromes.
A special microscopic technique allows a technician to de­
termine that a translocation has occurred.
Figure 12.14 shows a father and son who have a
translocation between chromosomes 2 and 20. Although
they have the normal amount of genetic material, they have
the distinctive face, abnormalities of the eyes and internal
organs, and severe itching characteristic of Alagille syn­
drome. People with this syndrome ordinarily have a dele­
tion on chromosome 20; therefore, it can be deduced that the
translocation disrupted an allele on chromosome 20 in the
father. The symptoms of Alagille syndrome range from mild
to severe, so some people may not be aware they have the
syndrome. This father did not realize it until he had a child
with the syndrome.
translocation
a.
b.
Figure 12.14 Translocation.
a. When chromosomes 2 and 20 exchange segments, (b) Alagille
syndrome, with distinctive facial features, sometimes results because
the translocation disrupu an a llele on chromosome 20.
B. Mutagens - Anything that can change the base sequences of DNA.
.
1. Chemicals and drugs
a. thalidomide - causes severe birth defects
2.Radiation
a. ultraviolet from the sun (ozone protects us to a large degree, but for how long? )
b. X-rays
c. gamma rays (emitted from nuclear weapons)
3. Heat - can break hydrogen bonds between bases
* Summary notes about mutations:
1. Mutations are rare, usually hannful and can be lethal.
2. Mutations can be "silent". That is, you and your spouse may be carrying mutations that
show up only in your offspring.
3. Mutations can accumulate throughout your life depending on the lifestyle you choose, so live
wisely!
Biotechnology (Genetic Engineering) - customizing genes in one or more species to suit human
desires.
A.
Recombinant DNA - the process of combining DNA from one
individual with another. How its done: See Fi2 13-6 on page
259.
1. isolate the desired gene and cut it out with restriction enzyme ; a
genetic scissors that cuts DNA at a specific site, producing sticky ends.
cut open a section of bacterial DNA called a plasmid, with the same
restriction enzyme mentioned above.
2.
splice the desired gene into the plasmid, connecting the sticky ends together
with an enzyme called ligase.
3.
4.
Reintroduce the modified plasmid into a bacterial cell
Let bacteria reproduce; only now every time the bacteria reproduces, it will
make more copies of the desired gene within it. More importantly, the bacteria is
manufacturing the desired protein.
5.
6.
Harvest the protein produced by the bacteria
NOTE: SOMETIMES THE PROTEIN IS HARVESTED FROM THE BACTERIA.
OTHER TIMES, THE ALTERED GENE IS PUT DIRECTLY INTO THE
ORGANISM. REFER TOTHE NOTES ON GENE THERAPY AT THE END OF
THIS STUDY GUIDE AND READINGS DONE IN CLASS
B.
Commercial applications of recombinant DNA.
1. Insulin - in production since 1982. Treatment for diabetes
2. Interferon
- a natural defense against viral infections and certain types of
cancer. Formerly available in small amounts; can now be produced in
large amounts.
3. Factor
8 and 9 - clotting factors that are needed by hemophiliacs
4. Human
growth hormone - treatment for dwarfism
5. Flavor-Saver
Tomato -designed to stay fresh longer
C.
Other applications of genetic engineering
l.
Transgenic animals (chimeras) - splicing the DNA of one animal species with
another.
pigs and cows having human genes in them; act as living human protein
factories. May even produce human organs in the near future.
a.
2. Improved
agricultural yields, producing more food using less resources, and
resistance to disease.
3. Vaccines
4.
for diseases such as AIDS
Gene therapy - fixing the defective gene that causes a specific disease and
then inoculating the individual with doses of the healthy gene.
a. Cystic Fibrosis patients inhale a genetically engineered virus to replace
defective cells in their
b.
A cell killer gene known as p53 is introduced into cancer cells to stop
them from dividing
* John Sulston's work on the C. elegans worm led to the discovery of p53 in
people!
*Read pages 258 - 270 in your text as well as the following pages in this handout to get an
idea of the products that are here now, those that are being developed, and the science I
society issues surrounding this topic.
Young Victor Frankenstein stays after school
After flicking on the light Professor Zurkowitz is caught
of his efforts to cross-breed flying fish and piranha
off guard by the overnight success
®
270
p a r t II
16-l
16.2 Biotechnology Products
Today, bacteria, plants, and anima ls are genetically
engineered- that is, genetically altered to make biotechnol­
ogy products (Fig. 16.3). Organisms that have had a foreign
gene inserted into them are called transgenic organisms
[L. trans, across, through, and Gk., -genie, producing].
Transgenic Bacteria
Recombinant DNA technology is used to produce transgenic
bacteria, which are grown in huge vats called bioreactors.
The gene product is collected from the medium. Biotechnol­
ogy products now on the market that are produced by bacte­
ria include insulin, human growth hormone, t-PA (tissue
plasminogen activator), and hepatitis B vaccine. Transgenic
bacteria have many other uses as well. Some have been pro­
duced to promote the health of plants. For example, bacteria
that normally live on plants and encourage the formation of
ice crystals have been changed from frost-plus to frost-minus
bacteria. Also, a bacterium that normally colonizes the roots
of corn plants has now been endowed with genes (from
another bacterium) that code for an insect toxin. The toxin
protects the roots from insects.
Bacteria can be selected for their ability to degrade a
particular substance, and this ability can then be enhanced
by genetic engineering. For instance, naturally occurring
bacteria that eat oil can be genetically engineered to do an
even better job of cleaning up beaches af ter oil spills.
Figure 16.3 Biotechnology products.
Products such as clotting factor VIII, which is administered to
hemophiliacs, can be made by transgeni c bacteria, plants, or animals.
After being processed and packaged , the product is sold
commercially.
Genetic Basis of Life
Industry has found that bacteria can be used as biofilters to
prevent airborne chemical pollutants from being vented
into the air. Bacteria can also remove sulfur from coal be­
fore it is burned and help clean up toxic waste dumps. One
such strain was given genes that allowed it to clean up lev­
els of toxins that would have killed other strains. Further,
these bacteria were given "suicide" genes that caused them
to self-destruct when the job had been accomplished.
Organic chemica ls are often synthesized by having cat­
alysts act on precursor molecules or by using bacteria to
carry out the synthesis. Today, it is possible to go one step
further and manipulate the genes that code for these en­
zymes. For instance, biochemist s discovered a strain of bac­
teria that is especially good at producing phenylalanine, an
organic chemical needed to make aspartame, the dipeptide
sweetener better known as NutraSweet. They isolated, al­
tered, and formed a vector for the appropriate genes so that
various bacteria could be genetically engineered to produce
phenylalanine.
Many major mining companies already use bacteria to
obtain various metals. Genetic engineering can enhance the
ability of bacteria to extract copper, uranium, and gold from
low-grade sources. Some mining companies are testing ge­
netically engineered organisms that have improved bio­
leaching capabilities.
Transgenic Plants
Techniques have been developed to introduce foreign genes
into immature plant embryos or into plant cells called proto­
plasts that have had the cell wall removed. It is possible to
treat protoplasts with an electric current while they are sus­
pended in a liquid containing foreign DNA. The electric cur­
rent makes tiny, self-sealing holes in the plasma membrane
through which genetic material can enter. Protoplasts go on
to develop into mature plants.
Foreign genes transferred to cotton, corn, and potato
strains have made these plants resistan t to pests because
their cells now produce an insect toxin. Similarly, soybeans
have been made resistant to a common herbicide. Some corn
and cotton plants are both pest- and herbicide-resis tant.
These and other genetically engineered crops that are ex­
pected to have increased yield are now sold commercially.
Plants are also being engineered to produce human
proteins, such as hormones, clotting factors, and antibodies,
in their seeds. One type of antibody made by corn can de­
liver radioisotopes to tumor cells, and another made by soy­
beans can be used to treat genital herpes.
Transgenic Animals
Techniques have been developed to insert genes into the eggs
of animals. It is possible to microinject foreign genes into
eggs by hand, but another method uses vortex mixing. The
eggs are placed in an agitator with DNA and silicon-carbide
c h a p t c r 16
Biotechnology and Genomics
271
16-5
needles, and the needles make tiny holes through which the
DNA can enter. When these eggs are fertilized, the resulting
offspring are transgenic animals. Using this technique, many
types of animal eggs have taken up the gene for bovine
growth hormone (bGH). The procedure has been used to
produce larger fishes, cows, pigs, rabbi ts, and sheep.
Gene pharming, the use of transgenic farm anima ls to
produce pharmaceuticals, is being pursued by a number of
firms. Genes that code for therapeutic and diagnostic
proteins are incorporated into an animal's DNA, and the pro­
teins appear in the animal's milk. Plans are underway to pro­
duce drugs for the treatment of cystic fibrosis, cancer, blood
diseases,and other disorders by this method. Figure 16.4 out­
lines the procedure for producing transgenic mammals:
DNA containing the gene of interest is injected into donor
eggs. Following in vitro fertiliza tion, the zygotes are placed
in host females, where they develop. After female offspring
mature, the product is secreted in their milk.
human gene \
....,.
microinject ion of human gene
i
:= de
_v_
elop
<<
"' hoot go"
.. ..
Cloning Transgenic Animals
For many years, it was believed that adult vertebrate ani­
mals could not be cloned. Although each cell contains a
copy of all the genes, certain genes are turned off in mature,
specialized cells. Cloning of an adult vertebrate requires
that all the genes of an adult cell be turned on again if de­
velopment is to proceed normally. This had long been
thought impossible.
In 1997, however, Scottish scientists announced that
they had produced a cloned sheep called Dolly. Since then,
calves and goats have also been cloned, as described in Figure
16.4. After enucleated eggs from a donor are microinjected
with 2n nuclei from the same transgenic animal, they are
coaxed to begin development in vitro. Development contin­
ues in host females until the clones are born. The offspring are
clones because all have the genotype and phenotype of the
adult that donated the 2n nuclei. In a procedure that pro­
duced cloned mice, the 2n nuclei were taken from corona ra­
diata cells. Corona radiata cells are those that cling to an egg
after ovulation occurs. Now that scientists have a way to
clone animals, this procedure will undoubtedly be used rou­
tinely to procure biotechnology products.
egg
milk
transgen ic goat
t/
adult cells
2n nuclei
egg donor
enucleated
eggs
development within host goats
·
Animal Organs as Biotechnology Products
The Health Focus on the next page discusses how it may be
possible for genetically engineered pigs to serve as a source
of organs for human transplant operations. Alternatively,
scientists are learning how to stimulate human cells to con­
struct organs in the laboratory.
.. . . .. .. .. . .
cloned transgenic goats
milk
Genetically engineered bacteria,plants,and animals are
used to make biotechnology products. Procedures have
also been developed to clone these animals. Organs
made in the laboratory are also biotechnology products.
Figure 16.4 Transgenic mammals.
A genetically engineered egg develops in a host to create a
transgenic goat that produces a biotechnology product in its milk.
Nuclei from the transgenic goat are transferred into donor eggs,
which develop into cloned transgenic goats.
@)
Health Focus
Organs for
1 6-6
Transplant
- - -
Genetic Basis of Life
. ,.. ·'· -
lthough it is now possible to trans­
plant various organs, there are not
enough human donors to go around.
Thousands of patients die each year while
waiting for an organ. It's no wonder,then,
that scientists are suggesting we get or­
gans from a source other than other hu­
mans. Xenotransplantation [Gk. xenos,
strange, foreign) is the use of animal or­
gans instead of human organs in trans­
plant patients. You might think that apes,
such as the chimpanzee or the baboon,
would be a scientifically suitable species
for this purpose. But apes are slow breed­
ers and probably cannot be counted on to
supply all the organs needed. Also, many
people might object to using apes for this
purpose. In contrast, animal husbandry
has long included the raising of pigs as a
meat source, and pigs are prolific. A fe­
male pig can become pregnant at six
months of age and can have two litters a
year,each averaging about ten offspring.
Ordinarily, the human body would vi­
olently reject transplanted pig organs. Ge­
netic engineering, however, can make
these organs less antigenic. Scientists have
produced a strain of pigs whose organs
would most likely, even today, survive for
a few months in humans. They could be
used to keep a patient alive until a human
organ was available. The ultimate goal is
to make pig organs as widely accepted by
humans as type 0 blood. (A person with
type 0 blood is called a universal donor
because the red blood cells carry no A or B
antigens.)
As xenotransp lantation draws near,
other concerns have been raised. Some ex­
perts fear that animals might be infected
with viruses, akin to Ebola virus or the
"mad cow" disease virus. After infecting a
transplant patient, these viruses might
spread into the general populace and be­
gin an epidemic. As an indication of this
possibility, scientists believe that HIV was
spread to humans from monkeys when
humans ate monkey meat. Advocates of
using pigs for xenotransplantation point
out that pigs have been around humans
for centuries without infecting them with
any serious diseases.
An alternative to xenotransplantation
also exists. Just a few years ago, scientists
believed that transplant organs had to
272
p a re II
-
come from humans or other animals.
Now, however, tissue engineering is
demonstrating that it is possible to make
some bioartificial organs-hybrids cre­
ated from a combination of living cells
and biodegradable polymers. Presently,
lab-grown hybrid tissues are on the mar­
ket. For example, a product composed of
skin cells growing on a polymer is used to
temporarily cover the wounds of burn pa­
tients. Similarly,damaged cartilage can be
replaced with a hybrid tissue produced af­
ter chondrocytes are harvested from a pa­
tient. Another connective tissue product
made from fibroblasts and collagen is
available to help heal deep wounds with­
out scarring. Soon to come are a host of
other products, including replacement
corneas, heart valves, bladder valves, and
breast tissue.
Tissue engineers have also created cel­
lular implants-cells producing a useful
product encapsulated w ithin a narrow
plastic tube or a capsule the size of a dime
or quarter. The pores of the container are
large enough to allow the product to dif­
fuse out but too small for immune cells to
enter and destroy the cells. An implant
whose cells secrete natural pain killers will
survive for months in the spinal cord and
can be easily withdrawn when desired. A
"bridge to a liver transplant" is a bedside
vascular apparatus. The patient's blood
passes through porous tubes surrounded
by pig liver cells. These cells convert toxins
in the blood to nonpoisonous substances.
The goal of tissue engineering is to
produce fully function ing organs for
transplant. After nine years, a Harvard
Medical School team headed by Anthony
Ata la has produced a working urinary
bladder in the laboratory (Fig. 16A). After
testing the bladder in laboratory animals,
the Harvard group is ready to test it in hu­
mans whose own bladders have been
damaged by accident or disease, or will
not function properly due to a congenital
birth defect. Another group of scientists
has been able to grow arterial blood ves­
sels in the laboratory. Tissue engineers
hope that they will one day produce
larger internal organs such as the liver or
kidney.
Figure 16A Tissue engineering.
This urinary bladder was made in the laboratory by tissue engineering.
ch apr e r
16
Biotechnology and Genomics
16.3 The Human
Genome Project
A genome is all the genetic information
of an individ ual or a species. The Human
Genome Project has two goals: (1) to con­
struct a map that shows the sequence of
base pairs along our chromosomes,
and (2) to construct a map that shows
the sequence of genes a long the human
chromosomes.
The Base Sequence Map
Researchers have now completed the first
goal. They know the sequence of the three
billion base pairs, one af ter the other, along
the length of our chromosomes. It took
some fif teen years for researchers to com­
plete this monumental task. Two rival
groups have been at work on the project.
The International Human Genome Se­
quencing Consortium, which consists of
laboratories in many different countries,
depends on the support of public funds, in­
cluding substantial contributions from the
United States government. On the other
hand, Celera Genomics, a private company
that is supported by a pharmaceutica l firm,
has been sequencing the genome for only a
few years. These competing groups used
slightly different techniques, but their data
match.
Even though we now know the se­
quence of bases in the human genome,
much work still needs to be done to make
sense out of what we have discovered. We
have found that there is little difference be­
tween the sequence of our bases and other
organisms whose DNA sequences are also
known. From this we can conclude that we
share a large number of genes with much
simpler organisms, including bacteria! It's
possible that eventua lly we will discover
that our uniqueness is due to the regulation
of these genes.
The Genetic Map
The genetic ma p tells the location of genes
along each chromosome. Figure 16.5shows
the loci of significan t mu ta nt genes on hu­
man chromosome 17. Many genes have
had their loci determined. Still, we do not
273
16-i
know the sequence of all the genes on any
particular chromosome.
Completing the chromosomal genetic
map should accelerate now that the base­
pair sequence map is complete. Research­
ers need only know a short sequence of
bases in a gene of interest in order for the
computer to search the genome for a
match. Then, the computer will tell the re­
searcher where this gene is located.
A question still being hotly debated is
the total number of human genes. Much of
our DNA consists of nucleotide repeats that
do not code for a protein. So far, researchers
have found only 30,000 genes that code for
proteins. This number seems terribly low;
that is, a round worm has 20,000 genes, so a
human, which is certainly more complex
than a roundworm, should have many
more genes. Some researchers think more
genes are yet to be identified. Others, be­
lieving they have found most of our genes,
speculate that each of these genes could
code for about three proteins,simply by us­
ing different combinations of exons.
As discussed in the Health Focus on
page 274, researchers are hopeful that map­
ping the human chromosomes will help
them not only discover mutant genes for
many more human disorders but also de­
velop medicines to treat these disorders. In
addition, it may be possible to locate genes
suitable for gene therapy to cure human ill­
nesses or enhance a phenotype. Such genes
might be inserted into the egg before it is
fertilized.
There are many ethical questions re­
garding how our knowled ge of the human
genome should be used. Therefore, it is
imperative that everyone be educated
about the human genome because in the
end it is the public that will have to decide
these issues.
retinitis
pigmentosa
cataract
diabetes
susceptibility
cancer
deafness
Charcot-Marie­
Tooth neuropathy
osteogenesis
imperfecta
osteoporosis
anxiety-related
personality traits
Alzheimer disease
susceptibility
neurofibromatosis
leukemia
dementia
muscular dystrophy
breast cancer
ovarian cancer
pituitary tumor
yeast infection
susceptibility
growth hormone
deficiency
myocardial infarction
susceptibility
One goal of the Human Genome Project
has been met: to sequence the DNA
bases of each chromosome.
Geneticists are continuing to work on
the second goal:to map the loci of
genes on each chromosome. This
information should contribute to the
health of human beings.
small-cell
lung cancer
Figure 16.5 Genetic map
of chromosome 17.
This map shows the sequence of
mutant genes that cause the
diseases or conditions noted.
@
16-8
Health Focus
New Cures on the Horizon
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ack in the 1980s, Leroy Hood couldn't
get funding for the DNA sequencer
he was developing. Biologists didn't like
the idea of just "collecting facts," and it
took several years before an entrepreneur
decided to fund the project. Without
ever-better DNA sequencers, the Human
Genome Project would never have com­
pleted its monumental task of determin­
ing the sequence of bases inour DNA.
Now that we know the sequence of all
the bases in the DNA of all our chromo­
somes, biologists all over the world be­
lieve that this knowledge will result in
rapid medical advances for ourselves and
our children. At least four categories of
improvement are expected: (1) Many
more medicines will be available to keep
us healthy; (2) medicines will be safer due
to genome scans; (3) a longer life span,
even to over 100 years, may become com­
monplace; and (4) we will be able to
shape the genotypes of our offspring.
First prediction: Many new
medicines will be available.
Genome sequence data will allow scien­
tists to determine all the proteins that are
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p a rt
II
Genetic Basis of Life
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--1.c:.
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active only during development plus all
those that are still active in adults.
Most drugs are either proteins or
small chemicals that are able to interact
with proteins. Many of these small chemi­
cals target proteins that act as signals be­
tween cells or within the cytoplasm of
cells. Today's drugs were usually discov­
ered in a hit-or-miss fashion, but now we
can take a more systematic approach to
finding effective drugs. For example, it is
known that all receptor and signaling pro­
teins start with the same ten-amino-acid
sequence. Now, it is possible to scan the
human genome for all genes that code for
this sequence of ten amino acids, and
thereby find all the signaling proteins.
Thereafter,they must be tested.
In a recent search for a protein that
makes wounds heal, researchers cultured
skin cells with fourteen proteins (found by
chance) that can cause skin cells to grow.
Only one of these proteins made skin cells
grow and did nothing else. They expect
this protein to become an effective drug
for conditions such as venous ulcers,
which are skin lesions that affect many
thousands of people in the United States.
Such tests, leading to effective results, can
be carried out with all the signaling pro­
teins scientists will discover by scanning
the human genome.
People's genotypes differ. We all have
mutations that account for our various ill­
nesses. Knowing each patient's mutations
will allow physicians to match the right
drugs to the particular patient.
three bases, repeated over and over
again, interrupt a gene and affect its ex­
pression. It is not yet clear how many SNPs
will be medically significant, but the pres­
ent estimate is on the order of 300,000.
How will a physician be able to deter­
mine which of the 300,000 SNPs and other
types of mutations are in your genome?
The use of a gene chip will quickly and ef­
ficiently provide knowledge of your geno­
type. A gene chip is an array of thousands
of genes on one or several glass slides
packaged together. After the gene chip is
exposed to an individual's DNA, a techni­
cian can note any mutant sequences pres­
ent in the individual's genes. Soon a chip
will be able to hold all the genes carried
within the human genome.
Some disorders, such as sickle-cell dis­
ease, are caused by a single SNP,but many
disorders seem to require more than one,
and possibly in different combinations. In
one study, researchers found that a series
of SNPs, numbered 1-12, were associated
with the development of asthma. A partic­
ular drug, called albuterol, was effective
for patients with certain combinations and
not others. This example and others show
Second prediction:
Medicines will be safer due
to genome scans.
First prediction:
Many new medicines will be coming.
274
Genome scans will allow us to discover
genetically different subgroups of the
population. Physicians will be looking for
two types of mutations in particular. One
type is called single nucleotide polymor­
phisms (SNPs, pronounced "snips"), in
which individua ls differ by only one nu­
cleotide. The other type of mutation is
nucleotide repeats, in which the same
Second prediction:
Genotype testing will be standard.
c h J p r e r 16
Biotechnology and Genomics
275
16-9
...
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that many diseases are polygenic, and that
only a genome scan isable to detect which
mutations are causing an individual to
have the disease, and how it should be
properly treated.
Genome scans are also expected to
make drugs safer to take. A.s you know,
many drugs potentially have unwanted
side effects. Why do some people and not
others have one or more of the side ef­
fects? Most likely, because people have
different genotypes. It is expected that a
physician will be able to match patients to
drugs that are safe for them on the basis
of their genotypes.
Biologists also hope that gene chips
will single out the specific oncogenes and
mutated tumor-suppressor genes that
cause the various types of cancer.The pro­
tein products of these genes will become
targets against which chemists can try to
develop drugs. If so, the current methods
of treating cancer-surgery, radiation,
and chemotherapy to kill all dividing
cells-will no longer be necessary.
Third prediction:
A longer and healthier life will be yours .
...,
-
Third prediction: A longer
and healthier life will be
yours.
Genome sequence data may allow scien­
tists to determine which genes enable
people to live longer. Investigators have
already found evidence for genes that ex­
tend the life span of animals such as
roundworms and fruit flies. The sequenc­
ing of the human genome makes it possi­
ble for scientists to find such genes in hu­
mans also.
For example, we know that the pres­
ence of free radicals causes cellular mole­
cules to become unstable and cells to die.
Certain genes are believed to code for an­
tioxidant enzymes that detoxify free radi­
cals. It could be that human beings with
particular forms of these genes have more
efficient antioxidant enzymes, and there­
fore live longer. If so, researchers will no
doubt be able to locate these genes and
also others that promote a longer life.
Consider, too, that natural selection
favors phenotypes that result in the great­
est number of fertile offspring in the next
generation. Since children are usually
born to younger individuals, natural selec­
tion is indifferent to genes that protect
the body from the deleterious effects of
aging. Researchers can possibly find such
genes, however,in individuals who have a
long life span. Use of these genes would
possibly oppose a destiny, determined so
far only by evolution.
Possible stem cell therapy has gener­
ated much interest of late. Stem cells are
embryonic cells and also some adult cells,
such as those in red bone marrow,that are
nondifferentiated. These cells have the
potential to become any type of tissue,
depending on which signaling molecules
are used. Genome sequence data will
eventually give scient ists knowledge of all
the signaling molecules humans possess.
Stem cells could also be subjected to gene
therapy in order to correct any defective
genes before scientists use them to create
the tissues or organs of the body. Use of
these tissues and organs to repair and/or
'
._
replace worn-out structures could no
doubt expand the human life span.
Fourth prediction: You will
be able to design your
children.
Genome sequence data will be used to
identify many more mutant genes that
cause genetic disorders than are presently
known. In the future,it may be possible to
cure genetic disorders before the child is
born by adding a normal gene to any egg
that carries a mutant gene. Or an artificial
chromosome,constructed to carry a large
number of corrective genes, could auto­
matically be placed in eggs. In vitro fertil­
ization would have to be utilized in order
to take advantage of such measures for
curing genetic disorders before birth.
Genome sequence data can also be
used to identify polygenic genes for traits
such as height, intelligence, or behavioral
characteristics. A. couple could decide on
their own which genes they wish to use to
enhance a child's phenotype. In other
words, the sequencing of the human
genome may bring about a genet ically
just society, in which all types of genes
would be accessible to all parents.
Fourth prediction:
You will be able to design your children.
(@
276
part
16-10
reverse
transcription
3. Recombinant
DNA carries
normal gene
into genome.
II
Genetic Basis of Life
4. Return genetically
engineered cells
to patient.
recombinant
DNA
recombinant
RNA
gene
1. Remove bone marrow
stem cells.
2. Use retroviruses to
infect bone marrow
stem cells with
normal gene.
defective
gene
Figure 16.6 Ex vivo gene therapy in humans.
Bone marrow stem cells are withdrawn from the body, an RNA retrovirus is used to insert a normal gene into them, and they are returned to the
body.
16.4 Gene Therapy
Gene therapy is the insertion of genetic material into human
cells for the treatment of a disorder. It includes procedures
that give a patient healthy genes to make up for faulty
genes, as well as the use of genes to treat various other hu­
man illnesses, such as cardiovascular disease and cancer.
Gene therapy includes both ex vivo (outside the body) and
in vivo (inside the body) methods.
Ex Vivo Gene Therapy
Figure 16.6 describes the methodology for treating children
who have SCID (severe combined immunodeficiency).
These children lack the enzyme ADA (adenosine deami­
nase), which is involved in the maturation of T and B cells.
In order to carry out gene therapy, bone marrow stem cells
are removed from the blood and infected with an RNA
retrovirus that carries a normal gene for the enzyme. Then
the cells are returned to the patient. Bone marrow stem cells
are preferred for this procedure because they divide to pro­
duce more cells with the same genes. Patients who have
undergone this procedure show significantly improved im­
mune function associa ted with a sustained rise in the level
of ADA enzyme activity in the blood.
Among the many gene therapy trials, one is for the
treatment of familial hypercholesterolemia , a condition that
develops when liver cells lack a receptor protein for remov­
ing cholesterol from the blood. The high levels of blood cho­
lesterol make the patient subject to fatal heart attacks at a
young age. A small portion of the liver is surgically excised
and then infected with a retrovirus containing a normal
gene for the receptor before being returned to the patient.
Several patients have experienced lowered serum choles­
terol levels following this procedure.
In Vivo Gene Therapy
Cystic fibrosis patients lack a gene that codes for the trans­
membrane carrier of the chloride ion. They often die due to
numerous infections of the respiratory tract. In gene therapy
trials, the gene needed to cure cystic fibrosis is sprayed into
the nose or delivered to the lower respiratory tract by adeno­
viruses or by the use of liposomes, microscopic vesicles that
spontaneously form when lipoproteins are put into a solu­
tion. Investigators are trying to improve uptake, and are also
hypothesizing tha t a combination of all three vectors might
be more successful.
Genes are being used to treat medical conditions such
as poor coronary circulation. It has been known for some
time that VEGF (vascular endothelial growth factor) can
cause the growth of new blood vessels. The gene that codes
for this growth factor can be injected alone or within a virus
into the heart to stimula te branching of coronary blood ves­
sels. Patients report that they have less chest pain and can
run longer on a treadmill.
Gene therapy is increasingly used as a part of cancer
therapy. Genes are being used to make healthy cells more
tolerant of chemotherapy, and to make tumors more vulner­
able to chemotherapy. The gene p53 brings about apoptosis,
and there is much interest in introducing it into cancer cells,
and in that way killing them off.
Both ex vivo and in vivo methods of gene therapy are
playing a role in curing illnesses.