1. RESTRICTION ENDONUCLEASES RESTRICTION ENDONUCLEASES CUT AT SPECIFIC SITES & LEAVE STICKY ENDS EcoR1EcoR1 animation Leave “sticky ends” that can be used to join DNA from different organisms

2. PLASMIDS Small circular self replicating DNA molecule in bacteria separate from bacterial chromosome 2-30 genes Often carry genes for antibiotic resistance or genetic recombination Can be exchanged between bacteria Bacterial “sex” = conjugation (facilitated by F plasmids) Role in rapid evolution Method for spreading “antibiotic resistance” R PLASMIDS

3. PLASMIDS PLASMID MOVIE Part 2PLASMID MOVIE Part 2 LAB 6: Cells can be made “competent” by using calcium chloride and “heat shock” to change their cell walls - makes them better able to pick up plasmids; rapidly growing cells are made competent more easily

4. Can be cut with restriction enzymes and used to incorporate foreign DNA into bacteria Bacteria reproduce, copying the inserted gene along with plasmid PLASMIDS & RECOMBINANT DNA Ti plasmid movie

5. Green Fluorescent Protein (GFP) Genetic tool Originally from jellyfish Way to tell if gene has been incorporated http://www.vet.upenn.edu/schoolresources/communications/publications/bellwether/61/stem_cells.html http://mabryonline.org/blogs/larkin/GFP%5CGFP_aequorea_victoria-1.jpeg

6. REVERSE TRANSCRIPTASE Found in RETROVIRUSES (EX: HIV) Uses RNA message to make a DNA copy Info flows in reverse RNA → DNA Can take eukaryotic RNA message after introns have been removed and change it into a DNA sequence to be read by bacteria (no RNA processing in prokaryotes)

7. REVERSE TRANSCRIPTASE http://biology200.gsu.edu/houghton/4564%20'04/figures/lecture%204/AAAreverse.jpg

8. GENE CLONING

9. http://biology200.gsu.edu/houghton/4564%20'04/figures/lecture%204/pcranimatie.gif PCR movie

10. Genetics of Viruses & Bacteria Chapter 18 http://www.awesomebackgrounds.com/s-energy-and-power.htm Control of gene expression in Eukaryotes

11. Bacteria Bacteria review –one-celled organisms –prokaryotes –reproduce by mitosis binary fission –rapid growth generation every ~20 minutes 10 8 (100 million) colony overnight! –dominant form of life on Earth –incredibly diverse

12. Bacterial genome Single circular chromosome –haploid –naked DNA no histone proteins –~4 million base pairs ~4300 genes 1/1000 DNA in eukaryote

13. No nucleus! No nuclear membrane –chromosome in cytoplasm –transcription & translation are coupled together no processing of mRNA –no introns –but Central Dogma still applies use same genetic code

14. Binary fission Replication of bacterial chromosome Asexual reproduction –offspring genetically identical to parent –where does variation come from?

15. Variation in bacteria Sources of variation –spontaneous mutation –transformation plasmids DNA fragments –transduction –conjugation –transposons bacteria shedding DNA

16. Spontaneous mutation Spontaneous mutation is a significant source of variation in rapidly reproducing species Example: E. coli –human colon (large intestines) –2 x 10 10 (billion) new E. coli each day! –spontaneous mutations for 1 gene, only ~1 mutation in 10 million replications each day, ~2,000 bacteria develop mutation in that gene but consider all 4300 genes, then: 4300 x 2000 = 9 million mutations per day per human host!

17. TRANSFORMATION in bacteria Bacteria are opportunists –pick up naked foreign DNA wherever it may be hanging out have surface transport proteins that are specialized for the uptake of naked DNA –import bits of chromosomes from other bacteria –incorporate the DNA bits into their own chromosome express new gene form of recombination

18. Swapping DNA Genetic recombination by trading DNA 132 arg+ trp- arg- trp+ minimal media

19. Plasmids –small supplemental circles of DNA 5000 - 20,000 base pairs self-replicating –carry extra genes 2-30 genes –can be exchanged between bacteria bacterial sex!! rapid evolution antibiotic resistance –can be imported from environment

20. Plasmids This will be important!

21. Plasmids & antibiotic resistance Resistance is futile? –1 st recognized in 1950s in Japan –bacterial dysentery not responding to antibiotics –worldwide problem now resistant genes are on plasmids that are swapped between bacteria

22. Biotechnology Used to insert new genes into bacteria –example: pUC18 engineered plasmid used in biotech antibiotic resistance gene on plasmid is used as a selective agent

23. TRANSDUCTION with viruses Phage viruses carry bacterial genes from one host to another

24. CONJUGATIONCONJUGATION - Bacteria “sex” movie Conjugation movieConjugation Direct transfer of DNA between 2 bacterial cells that are temporarily joined –results from presence of F plasmid with F factor F for “fertility” DNA –E. coli “male” extends sex pilli, attaches to female bacterium –cytoplasmic bridge allows transfer of DNA

25. TRANSFORMATION in bacteria TRANSDUCTION with viruses CONJUGATION - Bacteria “sex”CONJUGATION moviemovie ConjugationConjugation

26. TRANSPOSONS How Alu jumps