Download Freeman 1e: How we got there

Genetic Engineering:
Recombinant DNA Technology
 The discovery of enzymes that cut DNA at
specific locations, along with enzymes that piece
DNA segments back together, gave biologists the
ability to move genes from one location to
another.
 Biologists can determine a gene’s base sequence
once they have obtained many copies of the
gene—by inserting it into loops of DNA called
plasmids in bacterial cells and then allowing the
cells to grow or by performing a polymerase chain
reaction.
• Pituitary dwarfism results from the abnormal production of
growth hormone, encoded by the GH1 gene.
•Humans affected by pituitary dwarfism grow slowly, reaching a
maximum adult height of about 4 feet.
•Early trials showed that people with pituitary dwarfism could
be treated successfully with growth hormone therapy, but only
if the protein came from humans. Growth hormones isolated
from pigs, cows, or other animals were ineffective.
•Growth hormone purified from the pituitary glands of human
cadavers is expensive and has been banned from human
treatment due to contamination with prions—protein
particles that have been implicated as the cause of various
neurodegenerative disorders.
• The recombinant DNA strategy for producing
human growth hormone involved cloning the
human gene, introducing the gene into bacteria (or
yeast), and having these microbes synthesize the
hormone.
•Reverse transcriptase was used to make
complementary DNA (cDNA) from mRNA isolated
from pituitary cells
How Are Plasmids
Used in Cloning?
• Plasmids are small, circular DNA
molecules that replicate
independently of the chromosome
and can be used to carry recombinant
genes in bacteria.
Using Restriction Endonucleases
to Cut DNA
• Restriction endonucleases are
enzymes that cut DNA at specific
base sequences called recognition
sites. Most recognition sites are
palindromic sequences (Figure 19.3).
• Plasmids can be introduced into
bacteria by transformation—the
process of taking up DNA from the
environment and incorporating it into
the genome.
• Cells transformed with a plasmid
carrying an antibiotic resistance gene
will be able to grow in the presence
of the antibiotic.
• The resulting transformed cells
make up a cDNA library—a
collection of bacterial cells, each
containing a vector with one cDNA
(Figure 19.1, step 6).
Using Nucleic Acid
Hybridization to Find
a Target Gene
• A probe is a single-stranded
fragment of a labeled, known gene
that binds to a complementary
sequence in the sample being
analyzed.
• A DNA probe was constructed
based on the approximate growth
hormone amino acid sequence. The
probe was labeled and hybridized to
screen for bacterial colonies
containing a plasmid with the growth
hormone gene (Figure 19.4).
• The U.S Food and Drug Administration has approved use
of the hormone only for children projected to reach adult
heights of less than 5'3" for males and less than 4'11" for
females.
•Growth hormone has also become a popular performanceenhancing drug for athletes. Should athletes be able to
enhance their physical skills by taking hormones or other
types of drugs? Is the drug safe at the dosages being used by
athletes? These kinds of questions form the basis for ethical
concerns about genetic engineering.
Di-deoxy sequencing of DNA – Sanger Technique:
How Was the Huntington’s Disease Gene Found?
• Huntington's disease is a rare but devastating
genetic illness.
•An analysis of pedigrees from families affected
by the disease suggested that the trait results from
a single, autosomal dominant allele. Researchers
set out to identify the gene or genes involved and
to document that one or more genes are altered in
affected individuals.
•A genetic map (or linkage map) was used to
localize the Huntington's gene relative to other
genetic markers (a gene that has been mapped
previously).
• One gene within the isolated
chromosomal region that was
abnormal in people with Huntington's
disease had an unusual number of
CAG codons at the 5' end of the
coding region. Healthy individuals
have about 11–25 of these repeats,
whereas affected individuals have 40
or more.
Genetic Testing
• A genetic test for Huntington's
disease uses PCR to determine the
number of CAG repeats.
Ethical Concerns
over Genetic Testing
• Genetic testing raises many ethical
issues, including whether a pregnancy
should be terminated if a debilitating
disease is found in the fetus and
whether health insurance companies
can deny coverage for individuals
with a genetic disease.
Can Gene Therapy Cure Inherited Diseases in Humans?
Research on Severe Immune Disorders
Using Gene Therapy to Treat XLinked Immune Deficiency
• Gene therapy has been used to treat a type of severe
combined immunodeficiency (SCID), a fatal genetic
disease whose sufferers have a profoundly weakened
immune system.
•The gene responsible for SCID-X1 encodes gc
polypeptide, a key component of several plasma
membrane proteins. When these proteins do not function
correctly, T cells do not mature properly and infants are
helpless to ward off bacterial and viral infections.
•The gene responsible for SCID-X1 encodes gc polypeptide, a key component of
several plasma membrane proteins. When these proteins do not function
correctly, T cells do not mature properly and infants are helpless to ward off
bacterial and viral infections.
•Within four months after treatment, nine of the ten boys had normal levels of
functioning T cells; but 30 months later, two had developed a type of cancer
characterized by unchecked growth of T cells.
19.5 Biotechnology in
Agriculture: The Development of
Golden Rice
• Most strategies for genetic
engineering in agriculture focus on
reducing herbivore damage, making
the crop more resistant to herbicides,
and improving the quality of the food
product.
Rice as a Target Crop
• Although half the world's population
depends on rice as a staple food, it
contains no vitamin A. Lack of
vitamin A in the diet leads to
blindness, diarrhea, respiratory
infections, and childhood diseases
such as measles.
Synthesizing b-Carotene in Rice
• b-carotene is a precursor of vitamin
A. Scientists set out to develop rice
enriched in b-carotene.
• The synthetic pathway for bcarotene has three enzymes (Figure
19.15).
The Agrobacterium
Transformation System
• Agrobacterium tumefaciens is often
used for genetic transformation of
plants through transfer of its Ti
(tumor-inducing) plasmid (Figure
19.16).
Golden Rice
• Agrobacterium was used to transfer
the b-carotene synthetic genes into
cells from a rice plant, eventually
producing a transgenic plant now
called golden rice because it is yellow
from the high concentration of bcarotene (Figure 19.17).
• Enthusiasm about the potential
benefits of golden rice and other
genetically modified foods is
tempered by concern about the risks
of releasing large numbers and types
of recombinant crop plants into the
environment.