Transcript Regulation of Gene Activity in Eukaryotes

Kuang-Hui Lu:
Chapter 16
Molecular Biology is
Expanding Its Reach
Uses of Recombinant DNA
Technology in Research
• Creating bacteria and other organisms capable of
synthesizing both useful and economically
important molecules.
• Mapping the genomes of humans and of
organisms utilized in research.
• Supplying DNA and RNA sequences as research
tools.
• Altering the genotype of organisms (both plants
and animals) (transgenesis)
• Potentially correcting genetic defects in animals
(gene therapy)
Mammals Made to Order
• It is difficult to isolate an experimentally
induced change in both copies of a given
gene in eukaryotic genome (diploid).
• One current approach to overcoming this
difficulty is the use of targeted mutagenesis
via homologous recombination.
• Using this technique, it is possible to replace
an endogenous allele (copy) of a gene with
one that has been genetically engineered.
Mammals Made to Order
• In some instances, new, and sometimes novel,
gene have been introduced into the organism.
• In other cases, a specific gene has been
inactivated, then introduced into the organism
in order to learn more about the normal role of
the gene in embryonic development, tissue
differentiation, the development of cancer, or in
the functioning of the immune system. ---knockout animals.
ES cells: embryonic stem cells (they are totipotent)
* c-abl: the cellular homolog of an oncogene
from the Abelson murine leukemia virus
Fig. Mammals made to order: production of transgenic mice.
Oncogenes
• Oncogene – a gene that, in its
nonmutant “cellular” form, is involved in
the normal regulation of cell division.
– When disrupted or altered, it is associated
with development of certain forms of
cancer.
a neomycin like
substance
Fig. (Continued) Mammals made to order: production of transgenic mice.
Concerns and Problems of
Transgenic Animals
• If the engineered gene is essential to
embryonic development, homozygous
offspring may die in uterus.
• The recipient (or host) organism plays an
important role, not yet fully understood, in
the successful incorporation of the donor
cells into the developing embryo and
subsequent germ-line transmission.
Concerns and Problems of
Transgenic Animals
• In the current methodology nonhomologous
recombination is far more frequent than
homologous recombination, and rigorous
selection and careful analysis of cloned,
transformed ES cells are necessary before
they can be used as donors.
Gene Therapy
• The cells to be genetically altered can
first be removed from the body.
• The genetic alteration is carried out in
vitro.
• Altered cells having the desired genotype
can be selected in the laboratory.
• Reintroduced the cells into the original
donor or other suitable recipient
Uses of Recombinant DNA
Technology in Medicine
• Detection of a genetic disease
– Enzymatic assays
– Gel electrophoresis of gene products
– Immunological methods
– Chromosomal analysis
– Restriction analysis
Restriction Fragment Length Polymorphisms
(RFLPs) Associated with Sickle Cell Anemia
Fig. Simplified example of a DNA fingerprint resulting from the presence
of variable numbers of tandem repeats (VNTRs).
Use of DNA Fingerprints in Forensics
Use of DNA Fingerprints in
Paternity Determination
Identification of Codling Moth with
Specific DNA Markers (1)
Identification of Codling Moth with
Specific DNA Markers (2)
Uses of Recombinant DNA
Technology in Pharmaceutics
• A gene that codes for a particular peptide, along with an
appropriate promoter and a sequence that instructs the
cell to secrete the gene product, are inserted into an
expression vector (e.g., phage l gt11)
• Examples
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Somatostatin
Insulin
Human growth hormone
Interferons
Opioid peptides
Thymosin
Tissue plasminogen activator
Urokinase
Hemophilia factor VIII
Uses of Recombinant DNA
Technology in Pharmaceutics
• If the gene producing the therapeutic
product is of eukaryotic origin, special
problems must be taken into consideration:
– Eukaryotic promoters are not usually
recognized by bacterial RNA polymerases;
hence the eukaryotic gene must be linked to a
bacterial promoter.
– The mRNA transcribed from the eukaryotic gene
may not be translatable on bacterial ribosomes.
Uses of Recombinant DNA
Technology in Pharmaceutics
– Introns may be present, and bacteria are
unable to excise eukaryotic introns.
– The protein itself often must be processed
and bacteria cannot recognize processing
signals in eukaryotic gene products.
– Eukaryotic proteins may be recognized as
foreign material by bacterial proteases and
degraded.
Uses of Recombinant DNA
Technology in Pharmaceutics
• To solve the problems that may
encounter by using prokaryotic cells
– Use of a yeast artificial chromosome (YAC)
– Use of transgenic animals (primarily sheep
and cows)
• Vaccine production, e.g. gp120 of HIV surface
antigen
• Hemophilia factor IX
• a-1-antitrypsin (causes emphysema)
“Dolly”- the First Cloned Animal
• The first successful cloning of
a lamb from a cell (a
fibroblast) taken from the
udder of an adult sheep.
– growth the fibroblast cells in
culture
– remove the nucleus of the egg
– transplant the nucleus of the
fibroblast cell into the
enucleated sheep eggs
– Implant into the uterus of a
female sheep
Problems Associated with
Cloning Animals
• Many of the cloned animals have been
– grossly abnormal
– failed to survive
– exhibited significant growth abnormalities
– aged prematurely
– developed degenerative diseases such as
arthritis at an unusually age
Transgenic Pigs
• Cloned pigs were genetically engineered as
potential organ donors to humans
• The alpha 1,3 galactosyl transferase gene
(GATA1), a gene causes the human immune
system to reject pig organs, was “knocked out”
in these animals
• Knocked out the GATA1 gene is not likely to
eliminate the problem of tissue and organ
rejection
To Produce Other Medical
Important Substances
• Recombinant vaccines
– Hepatitis B virus
– AIDs – HIV virus
• Recombinant tissue plasminogen activator
(TPA) – for treating heart attack
• Interleukin-2 and g-interferon – for cancer
treatment
• Human growth hormone (hGH) – for treating
hypopituitary dwarfism
Supportive Therapy and
Gene Therapy
• Supportive (replacement) therapy: the
malfunctioning cells are replaced with
normal cells.
e.g. bone-marrow transplantation
• Gene therapy: restitution of the normal
gene in vivo.
e.g. genetic diseases (i.e. cancer); severe
combined immunodeficiency (SCID),
familial hypercholesterolemia
A Gene Therapy Trial
• Severe combined immunodeficiency (SCID) is caused by
a defect in the gene which produces the enzyme
adenosine deaminase (ADA).
• In individuals who lack this enzyme, the nucleotide 2’deoxyadnosine accumulates in the bloodstream, killing
both the B and T lymphocytes of the immune system.
A Gene Therapy Trial
• Gene therapy
– Removed T lymphocytes from the SCID victim
– Mixed with a genetically engineered retrovirus
containing a normal copy of the ADA gene
– Verified the ADA gene was introduced into the
genome of the T cells and they were functioning
– Transfused the cells into the patient
– Serum ADA levels rose
• Since T cells are relatively short-lived, this
gene therapy procedure must be repeated
periodically.
Other Examples of Gene Therapy
• Familial hypercholesterolemia
– A defect in the gene that codes for the low density
lipoprotein (LDL) receptor.
– A functional copy of the LDL receptor gene was
introduced into the separated liver cell by modified
virus.
– Reintroduce the cells to the liver
• Vascular endothelial growth factor (vegf)
stimulates growth of collateral blood vessels
around the area of an arterial blockage.
The Problems to Be Solved
before Gene Therapy
• It is not possible to remove certain types of cells
from the body in order to genetically to modify
them, nor can all types of cell be grown
successfully in culture.
• There are major problems inherent in attempting
to introduce foreign DNA into specific types of
human cells in vivo.
– viral vector infect only certain type of cells in vivo
– lack selectivity of infection
– inset their DNA at random locations within the human
genome
A Successful Gene Therapy
• The normal gene must reach the tissue
which the gene is normally expressed,
enter the cell.
• The introduced normal gene undergo
homologous recombination with the
defective gene
• The gene is expressed at an appropriate
time, and at appropriate level.
Uses of Recombinant DNA
Technology in Agriculture
• Plants
– Fixing nitrogen
– Improving photosynthesis
– Providing resistance to pests, pathogens and
herbicides
• e.g. Bacillus thuringiensis endotoxin gene
– Increasing resistance to frost, drought and
increased salinity
• Animals
– Reach maturity more rapidly
– Produce more milk or leaner meat
– Produce pharmaceuticals
Agrobacterium tumefaciens
Tumor inducing (Ti) plasmid
Fig. A transgenic tobacco plant which has been transformed with a firefly
lucieferase gene.
Effect of an extra growth
hormone gene in mice
• Transgenic mouse (left)
carries a gene for rat
growth hormone
• Normal mouse (right)
Other Commercial and
Industrial Applications
• Fermentation of cellulose or even plastic
waste
• Fermentation at high temperature
• Oil-metabolizing genes insert into a marine
bacterium
• Degradation of other toxic products in the
environment
• Conversion waste into usable food for
animals or humans
Social and Ethical Issues
• Whether scientists are “playing God” by
altering the genomes of living organisms,
thereby altering their evolutionary future?
• What would happen if some of these
recombinant plants cross-pollinate with their
wild relatives?
• Are certain genetically engineered foods safe?
• Are genetic engineering legal?
Genetic Testing
• Who should have access to the information
derived from genetic tests?
– Only the individual?
– His or her spouse?
– Parents?
– Children?
– Siblings?
– His or her physician, employer, or insurance
company?
Gene Therapy
• What types of traits should be altered,
who should decide?
• Do we really know what is best for the
future of our species?
• Who will access to gene therapy?
– Will this be reserved for the wealthy?
– Will it be covered by our health insurance?
What about human cloning or
transgenic humans ?