1. Announcements
Reading for today: Genomic equivalence, cloning, stem cells, pp ( )
Reading for Wednesday: Cell biology of cancer, pp ( )
Final Exam: Monday, May 1, 8-10 AM, 130 pts.
30 questions from unit 4, Days 34-43
13 comprehensive questions
Bring a calculator
Any equations you need will be given.

2. 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.
Protein turnover
Control of Eukaryotic gene expression
Cloning
Embryonic Stem cells

3. Energetics of Translation
For 100 amino acid protein: High energy P bonds
1 ATP  AMP for AA activation 2
2 GTP  GDP for elongation 2
1 GTP  GDP for translocation 1
Sub-total: 5 X 100 amino acids
500
1 GTP  GDP for initiation 1
1 GTP  GDP for termination? 1
Total:
X 7.3 kcal/mol per High energy P bond = 3665 kcal/mol
Indirect costs: mRNA, ribosome synthesis, chaperones, import

4. Protein Turnover: Ubiquitin Tags Proteins for Degradation (Nobel Prize, Chemistry, 2004)
enzyme

5. 2. Genome compartmentalization 3. Genome organization
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

6. Levels of Control of Eukaryotic Gene Expression It’s more complicated
being multicellular.

7. Cloning Mammals: Evidence of nuclear equivalence and reprogramming
Black (ooplasm donor)
Agouti (nucleus donor)
Cloned mice (Agouti)
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.
Dolly (July 5, Feb. 14, 2003)
and one of her 6 lambs
Wilmut et al Nature 385:

8. 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

9. Two ways to make an embryo: 2. Cloning or asexual reproduction
“Nuclear Transplantation”
Source: www. arhp.org

10. 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.

11. Top 10 causes of U.S. deaths (1998)
Heart disease
Cancer
Stroke
Chronic lung disease
Accidents
Pneumonia/influenza
Diabetes
Suicide
Kidney disease
Chronic liver disease
Tissue damage, e.g. spinal cord injury, also potentially treatable with stem cells.

12. 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:
Unipotent: makes one cell type
Multipotent: makes several cell types
Pluripotent: makes most cell types
Totipotent: makes all possible cell types (zygote)

13. Where do stem cells come from?

14. Evidence for ES cell pluripotency

15. Evidence of adult stem cell pluripotency

16. Stem Cell Therapeutics
Figures\Chapter21\DevBio7e21232.jpg

17. ES cells in the news: The most spectacular fraud in biomedical research
What three accomplishments did Dr. Hwang Woo Suk claim?
First line of human embryonic stem cells generated from nucleus of adult cell > first step in therapeutic cloning (2004). FRAUD
Improvement in efficiency of above, generating ES cell lines from 9 patients > practical treatment through therapeutic cloning (Science : ). FRAUD
First cloning of a dog (Nature :641). VALID
What ethical breaches apparently occurred in his laboratory?
Junior researchers were compelled to donate eggs for research in his lab.
Payments were made to egg donors through a Seoul hospital.
He lied about it this knowledge to the journal Science.
Dr. Woo Suk ordered subordinates to falsify data.

18. Korean Stem Cell Scandal, 2005-2006 (Hwang et al. 2005)
Duplicated
Duplicated
Cropped
Cropped
Cropped

19. 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

20. 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