Announcements
• Reading for today: Genomic equivalence,
cloning, stem cells, pp. 727-730 (704-709)
• Reading for Wednesday: Cell biology of
cancer, pp. 762-784 (562-571)
• Final Exam: Monday, May 1, 8-10 AM, 130 pts.
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30 questions from unit 4, Days 34-43
13 comprehensive questions
Bring a calculator
Any equations you need will be given.
Outline/Learning Objectives
After reading the text, attending
lecture, and reviewing
lecture notes, you should be
able to:
• Describe how proteins turn
over in eukaryotic cells.
• Describe the potential fates
of stem cells in general.
• Distinguish reproductive
cloning from therapeutic
cloning.
• Describe how embryonic
stem cells are obtained, and
how they can be used.
A.
B.
C.
D.
Protein turnover
Control of Eukaryotic gene
expression
Cloning
Embryonic Stem cells
Energetics of Translation
• For 100 amino acid protein:
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–
–
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1 ATP  AMP for AA activation
2 GTP  GDP for elongation
1 GTP  GDP for translocation
Sub-total:
– 1 GTP  GDP for initiation
– 1 GTP  GDP for termination?
– Total:
High energy P bonds
2
2
1
5 X 100 amino acids
500
1
1
502
• X 7.3 kcal/mol per High energy P bond = 3665 kcal/mol
• Indirect costs: mRNA, ribosome synthesis, chaperones,
import
GDP  ATP  GTP  ADP
nucleosidediphosphate kinase
Protein Turnover: Ubiquitin Tags Proteins for
Degradation (Nobel Prize, Chemistry, 2004)
enzyme
Differences between eukaryotic
and prokaryotic gene expression:
“What is true of E. coli is only partly true of elephants”
1. Genome size
•
Much bigger, w/ non-coding DNA, introns
2. Genome compartmentalization
•
Transcription in nucleus, translation in cytoplasm
3. Genome organization
•
Chromosomes w/ nucleosomes, “operons” rare
4. mRNA stability
•
Euk: hours (stable environment), Prok: minutes (variable
environment)
5. Posttranslational modification
•
Allow more specialized functions, additional regulation
6. Protein turnover
•
Euk: proteolytic pathway, Prok: dilution over time
Levels of Control of
Eukaryotic Gene
Expression
It’s more complicated
being multicellular.
Cloning Mammals: Evidence of nuclear
equivalence and reprogramming
Black (ooplasm donor)
Cloned mice (Agouti)
Dolly (July 5, 1996 - Feb. 14, 2003)
and one of her 6 lambs
Wilmut et al. 1997. Nature 385:810-814.
Agouti (nucleus donor)
Albino (nucleus donor)
^ Cloned mice (b. 1998) and
their “parents.” Coat color is
used as a marker. Conclusion:
Some differentiated nuclei can
be completely reprogrammed.
Two ways to make an embryo:
1. Sexual reproduction
Source: Human Cloning and Genetic Modification: The Basic Science You Need
to Know, Association of Reproductive Health Professionals, www. arhp.org
Two ways to make an embryo:
2. Cloning or asexual reproduction
“Nuclear Transplantation”
Source: www. arhp.org
Human Reproductive
vs. Therapeutic Cloning
Reproductive cloning
• Goal is to produce a
clonal embryo to
implant in a
mother’s womb with
intent to carry the
child to birth.
Therapeutic cloning
• Goal is to produce a
clonal embryo to
generate embryonic
stem cells.
• ES cells can be
used to treat clonal
donor without risk of
immune rejection.
Top 10 causes of U.S. deaths (1998)
Cause
1 Heart disease
2 Cancer
3 Stroke
4 Chronic lung disease
5 Accidents
6 Pneumonia/influenza
7 Diabetes
8 Suicide
9 Kidney disease
10 Chronic liver disease
# (1000’s)
724
541
158
158
98
92
65
31
26
25
#/100,000
268.2
200.3
58.6
58.6
36.2
34.0
24.0
11.3
9.7
9.3
%
31.0
23.3
6.8
6.8
4.2
3.9
2.8
1.3
1.1
1.1
Tissue damage, e.g. spinal cord injury, also potentially treatable with stem cells.
What’s a stem cell?
• Stem cells are defined by their ability to:
– Continue to grow and divide
– Given the right signals (e.g. hormones or
growth/differentiation factors), to differentiate into
a specialized cell type
• Stem cells have different potencies:
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–
–
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Unipotent: makes one cell type
Multipotent: makes several cell types
Pluripotent: makes most cell types
Totipotent: makes all possible cell types (zygote)
Where do stem cells come from?
Evidence for ES cell pluripotency
Evidence of adult stem cell pluripotency
Stem Cell Therapeutics
ES cells in the news: The most
spectacular fraud in biomedical research
• What three accomplishments did Dr. Hwang Woo Suk claim?
1. First line of human embryonic stem cells generated from nucleus of
adult cell > first step in therapeutic cloning (2004). FRAUD
2. Improvement in efficiency of above, generating ES cell lines from 9
patients > practical treatment through therapeutic cloning (Science
2005. 308:1777-1783). FRAUD
3. First cloning of a dog (Nature 2005. 436:641). VALID
• What ethical breaches apparently occurred in his laboratory?
1. Junior researchers were compelled to donate eggs for research in his
lab.
2. Payments were made to egg donors through a Seoul hospital.
3. He lied about it this knowledge to the journal Science.
4. Dr. Woo Suk ordered subordinates to falsify data.
Korean Stem Cell Scandal, 2005-2006
(Hwang et al. 2005)
Duplicated
Duplicated
Cropped
Cropped
Cropped
Some Stances on Human
Cloning and Stem Cell Research
• Support any human cloning
– Few biomedical scientists
– Clonaid (Raelians - Bahamas), Severino Antinori (Italy), Panos
Zavos (Kentucky)
• Oppose reproductive cloning, support therapeutic cloning
– Many biomedical scientists
– Hatch-Feinstein-Specter-Kennedy et al. bill S.303 in U.S. Senate
– Nancy Reagan
• Oppose any human cloning
– Right-to-life groups: destruction of embryo, slippery slope
– Brownback-Landrieu et al. bill S.245 in U.S. Senate
– President Bush
Normal and transformed cell
properties
Normal cells
• Regulated growth
• Dependant on GF’s
• Contact inhibited
• Flattened cells, normal
nuclei, normal
chromosome number
• Normal cell surface
receptors
• Normal gene
expression
Transformed (cancer) cells
• Uncontrolled growth
• Independent of GF’s
• Loss of contact inhibition
• Rounded cells, large
nuclei, abnormal
chromosome number
• Change in cell surface
receptors
• Altered gene expression