Applications of the DNA Story - How can we use what we know?

Recombinant DNA

A. What about the possibility of tansfering genes among organisms?

1. We could change mutant genes to good ones

2. We could program bacteria to make difficult proteins

a. Human pituitary hormone - only available from cadavers

b. Insulin - only available from cattle carcases

c. Interferon - tiny amounts available from human blood donations

d. Bovine Growth Hormone - could double milk production

B. Natural gene transfers

1. Bacterial conjugations

a. Plasmids - little pieces o DNA not on bacterial chromosomes

b. Some plasmids can cause bacteria to transfer genes from one to another -fertility factor

c. Other plasmids become part of bacterial chromosome

1. Can transfer part of bacterial genome

2. Transduction

a. Bacterial viruses - most set up infection - not so neat

b. Some viruses form plasmids and even some become part of bacterial genome - called prophage

1. They will return to virus form with Xrays, UV or chemicals

2. Some viruses take a bit of bacterial DNA with them

3. New genes become active in bacterium

3. Can put plasmids into new bacteria mechanically

a. Brute force microporation

b. Electroporation

c. Chemicals

C. Gene Splicing

1. Manipulation of natural transformation

2. Two kinds of enzymes

a. Restriction endonucleases - They break DNA at a particular location - scissors

1. EcoR1 breaks DNA at AATT locations

b. Ligase mends the breaks (glue)

3. These allow us to cut and splice DNA - scissors and glue

4. Example - tetracycline resistant plasmid - put new gene into it

a. Take plasmid out of cell - cut with endonuclease

b. Add your own DNA sequence into the plasmid

c. Re-attach with ligase

d. You have made a new plasmid

e. Put the plasmid back into the cell - you get the new gene product

f. Bonus - your gene product will appear in every tetracycline- resistant cell - so you can find it

g. Now you can grow up a big batch of your new bacterium and you get lots of your gene product!!!!

D. What you can do

1. You can make lots of otherwise tough to make proteins

2. What has been done?

a. Insulin - nearly all is recombinant

b. Interferon - we have it - I have a good friend with malignant melanoma whose life was saved by recombinant interferon

c. Bovine Growth hormone

1. Doubles the milk production of a cow

2. Ethical problem - should we do this?

E. What about adding genes to eukaryotes?

1. Transgenic plants

1. Agrobacterium tumefaciens - bacterium

a. The bacterium injects a plasmid into a plant cell

b. Normally produces a tumor

c. Bacterium lives in the tumor and is fed by the plant

2. When Agrobacterium affects a plant, it puts in a plasmid that sits down in the plant genome

3. If we put a new gene into the plasmid - it puts a new gene into the plant

4. What genes would we add?

a. Bacillus thuringensis toxin - kills insects

1. Put the insecticide into the plant

2. Vertebrates are immune

3. Problem of non-target organisms - does transgenic pollen affect monarch butterflies?

b. Resistance to roundup (herbicide)

1. This would allow farmers to apply more herbicide without killing their crop

c. High lysine corn

1. It would be helpful to humans if we could design a corn plant that puts more of the essential amino acids in its seeds

2. Transgenic Animals

a. You could put a gene into a fertilized egg

b. Get an animal that produces a new gene product

c. Put clotting factor gene into sheep and make it in milk