Announcements Reading for today: Genomic equivalence, cloning, stem cells, pp. 727-730 (704-709) Reading for Wednesday: Cell biology of cancer, pp. 762-784.

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.– 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. Protein turnoverB. Control of Eukaryotic gene

expressionC. CloningD. Embryonic Stem cells

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: 502

• X 7.3 kcal/mol per High energy P bond = 3665 kcal/mol

• Indirect costs: mRNA, ribosome synthesis, chaperones, import

ADPGTPATPGDP kinase ediphosphat nucleoside

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

enzyme

Differences between eukaryoticand 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 ofEukaryotic Gene

Expression

It’s more complicatedbeing multicellular.

Cloning Mammals: Evidence of nuclear equivalence and reprogramming

^ 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, 1996 - Feb. 14, 2003)and one of her 6 lambs

Wilmut et al. 1997. Nature 385:810-814.

Black (ooplasm donor) Agouti (nucleus donor)

Albino (nucleus donor)Cloned mice (Agouti)

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

Source: www. arhp.org

“Nuclear Transplantation”

Human Reproductivevs. 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 # (1000’s) #/100,000 %1 Heart disease 724 268.2 31.02 Cancer 541 200.3 23.33 Stroke 158 58.6 6.84 Chronic lung disease 158 58.6 6.85 Accidents 98 36.2 4.26 Pneumonia/influenza 92 34.0 3.97 Diabetes 65 24.0 2.88 Suicide 31 11.3 1.39 Kidney disease 26 9.7 1.110 Chronic liver disease 25 9.3 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:– 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 mostspectacular 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). FRAUD2. 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

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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