3A1 Continued

1. 3A1 Continued Phenotypes are determined through protein activities

2. PROTEINS DICTATE PHENOTYPE Proteins dictate virtually every reaction in the cell • directly responsible for observable characteristicsHow do the proteins work to direct phenotype? • Structural functions (for example, in the maintenance of cell and/or tissue shape and rigidity) • Transport of molecules • Communication between cells. • Substantial proportion of proteins are enzymes , catalyzing chemical reactions for the synthesis and transformation of virtually all biological molecules.

3. EXAMPLES Varying types and quantities of all biological molecules in the cells and tissues of an individual is what ultimately leads to phenotypic variation. • Slight variation in the activity of an enzyme for pigment synthesis in a plant may result in white flowers rather than red • Slight difference in a protein responsible for cell communication during the development of leaf tissue might result in variation of leaf shape Understanding the path from genotype to phenotype is a major concern of modern molecular biology and one of the ultimate goals of the human genome project

4. GENETIC ENGINEERING

5. Gel Electrophoresis • Separate DNA fragments by size • Gel made of agarose or polyacrylamide • Submersed in buffer that can carry current • Subjected to an electrical field • Negatively-charged DNA migrates towards the positive pole • Larger fragments move slower, smaller move faster • DNA is visualized using fluorescent dyes

8. DNA Analysis

9. Polymerase chain reaction (PCR) • Developed by Kary Mullis • Awarded Nobel Prize • Allows the amplification of a small DNA fragment using primers that flank the region • Each PCR cycle involves three steps: • Denaturation (high temperature) • Annealing of primers (low temperature) • DNA synthesis (intermediate temperature) • Taq polymerase

10. After 20 cycles, a single fragment produces over one million (220) copies!

11. Genetic Engineering • Has generated excitement and controversy • Expression vectors contain the sequences necessary to express inserted DNA in a specific cell type • Transgenic animals contain genes that have been inserted without the use of conventional breeding

12. In vitro mutagenesis • Ability to create mutations at any site in a cloned gene • Has been used to produce knockout mice • A known gene is inactivated • The effect of loss of this function is then assessed on the entire organism • An example of reverse genetics

16. Medical Applications • Medically important proteins can be produced in bacteria • Human insulin • Interferon • Atrial peptides • Tissue plasminogen activator • Human growth hormone • Problem has been purification of desired proteins from other bacterial proteins

17. Genetically engineered mouse with human growth hormone

18. Vaccines • Subunit vaccines • Genes encoding a part of the protein coat are spliced into a fragment of the vaccinia (cowpox) genome • Injection of harmless recombinant virus leads to immunity • DNA vaccines • Depend on the cellular immune response (not antibodies)

20. Gene therapy • Adding a functional copy of a gene to correct a hereditary disorder • Severe combined immunodeficiency disease (SCID) illustrates both the potential and the problems • On the positive side, 15 children treated successfully are still alive • On the negative side, three other children treated have developed leukemia (due to therapy)

21. Agricultural Applications • Ti (tumor-inducing) plasmid • Most used vector for plant genetic engineering • Obtained from Agrobacterium tumefaciens, which normally infects broadleaf plants • Part of the Ti plasmid integrates into the plant DNA and other genes can be attached to it • However, bacterium does not infect cereals such as corn, rice, and wheat

25. Applications of PCR • Allows the investigation of minute samples of DNA • Forensics – drop of blood, cells at base of a hair • Detection of genetic defects in embryos by analyzing a single cell • Analysis of mitochondrial DNA from early human species