Wednesday, April 28, 1999
Announcements: Review in class on Friday.

Lecture notes:

Retinoblastoma: rare eye tumor which occurs in children before age 4. It is a dominant inherited trait.

A missing "anticancer gene" which, if present, keeps an oncogene turned off, is the cause of
retinoblastoma. This is called a tumor supressor gene.

Xeroderma pigmentosum: missing enzymes in DNA repair. Autosomal recessive inheritance. These people develop
multiple skin cancers as a result of UV light damage to skin cell DNA.

Colon Cancer

Lung Cancer

Breast Cancer

Hot of the presses, there is another tumor supressor gene involved here. It is called the p53 gene. When it is missing or mutated there is a chance of getting these 3 cancers.

These seem to be "multifactoral," because something else is involved besides the missing p53 gene.

New research suggests that an altered p53 gene is involved in 50% of the cancers.

Many genes have been found for rare familial diseases that also cause cancers.

1. It decreases the rate at which viruses, normal cells and tumor cells multiply.
2. It alters metabolism of cancer cells.
3. It increases the number of antigens on the cell surface so they can be recognized and destroyed
4. It stimulates natural killer cells into greater activity.

Alpha Interferon is used in the treatment of "hairy cell" leukemia.

Interferons are being tested on other cancers: Kaposi's sarcoma, malignant melanoma, myeloma, some kidney cancers.

Research is also being done on interleukins and cancer.

1. Change in bowel or bladder habits.
2. A sore that does not heal.
3. Unusual bleeding or discharge.
4. Thickening or lump in breast or elsewhere.
5. Indigestion of difficulty swallowing.
6. Obvious change in wart or mole.
7. Nagging cough or hoarseness.

Definitions:

Plasmid: small circular molecular molecule of double stranded DNA that is found in bacteria and yeast.

They replicate as independent units when the bacteria or yeast divide.

They often carry important genes- such as those that confer antibiotic resistance.

**Recombinant DNA technology exploits these features :1. Small size circular DNA
2. Antibiotic resistance

Restriction nucleases (also called restriction endonucleases)

These enzymes cut DNA at sites of specific base sequences.

"Endonucleases" cut in the middle of the DNA rather than at the ends.

The ones used in DNA technology make staggered cuts.

Annealing process: complementary base pairs automatically form H-bonds.

1. Extract and cut plasmid with a restriction endonuclease.
2. Cut DNA to be studied with the same endonuclease.
3. Put them together and their "sticky ends" will anneal (pairing or complementary bases)
4. DNA ligase is used to seal the breaks in the "backbone" of the DNA.
5. Put the plasmids back into the yeast or bacteria.
6. Find the plasmid with the gene that you need. (This is the hard part)
7. "Grow it up" in fementation tanks.
8. Extract: a. desired protein/hormone.
   b. DNA fragment to be studied.

What Have Gene Jockeys Done so far?

Insulin: for diabetes - replaces pig or cow insulin (allergic reactions)

Growth Hormone: (human somatotropin) for pituitary dwarfism and burns.

TPA: (tissue plasminogen activator) dissolves clots during a heart attack

Alpha Interferon: treatment for "hairy cell" leukemia

Factor VIII: for hemphiliacs

Vaccines: recombinant vaccines - "vector and purified antigen" rabies, hepatitis B

This technique was developed to make large quantities of pure, single-type antibodies.

Hybridomas make monoclonal antibodies.

Hybridomas are hybrid cells which result from fusion of an antibody producing B-cell and a B-lymphocyte tumor cell.

Monoclonal Antibodies are the antibodies produced by these hybridomas. Mononuclear means one clone.

1. Inject antigen "X" into an animal.
2. Animal makes antibodies to "X".
3. Extract antibody producing cells form animal (these cells are all different, only some of which are making the "right" antibody).
4. Myeloma tumor cells (immortal) are fused with all the B cells extracted from the animal.
5. The cells are grown in a special medium. Only the hybridomas survive.
6. Now you have to find the antibody you're looking for.
7. Make colonies of cells from each hybridoma
8. Test each one with antigen "X".
9. The ones that react are the ones you are looking for.

Pregnancy tests: HCG

Orthoclone: used to reverse kidney transplant rejection. This is the first monoclonal antibody approved for use inside the
body.

Diagnostics: take tissue or fluid form the body and expose it to monoclonal antibody against "antigen" you are looking for.
It there is "antigen" present, there will be a reaction.
Test for sickle hemoglobin
Colon, breast and ovarian cancers make unique antigens.

Cancer Imaging: radioactive antibodies find cancer and lock on (guided missiles) and doctors can now see where they all are and how large they are. Treatment can be more successful.

Field testing for agricultural products is under the "White House Office of Science and Technology Policy"

Further regulation depends on the nature of the products.

The Environmental protection Agency governs microbial pesticides.

Agriculture Department governs genetically altered plants.

FDA governs animal and human recombinant products.

OSHA oversees safety standards for the workplace.

Companies must have an Institutional Biosafety Committee. Two of the members must be from outside the company from health and environmental fields.