RESTRICTION ENDONUCLEASES CUT AT SPECIFIC SITES & LEAVE STICKY ENDS

1. RESTRICTION ENDONUCLEASES CUT AT SPECIFIC SITES & LEAVE STICKY ENDS EcoR1 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 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. PLASMIDS & RECOMBINANT DNA Can be cut with restriction enzymes and used to incorporate foreign DNA into bacteria Bacteria reproduce, copying the inserted gene along with plasmid • Ti plasmid movie

5. http://mabryonline.org/blogs/larkin/GFP%5CGFP_aequorea_victoria-1.jpeghttp://mabryonline.org/blogs/larkin/GFP%5CGFP_aequorea_victoria-1.jpeg 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

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

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

11. Bacteria • Bacteria review • one-celled organisms • prokaryotes • reproduce by mitosis • binary fission • rapid growth • generation every ~20 minutes • 108 (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 1010 (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 1 3 2 arg+ trp- arg- trp+ minimal media

19. Plasmids • 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. This will beimportant! Plasmids

21. Plasmids & antibiotic resistance • Resistance is futile? • 1st 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. CONJUGATION - Bacteria “sex”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” movieConjugation

26. TRANSPOSONS • How Alu jumps