Vocabulary…

1. Vocabulary… • Positive gene control • Negative gene control • Lac Operon • Trp Operon • Plasmid • Transformation • Conjugation • Retrovirus • Lytic cycle • Lysogenic cycle • Operon • Inducible and repressible • Promoter • Terminator • Enhancer • Regulatory Gene • Inducer • Repressor • Regulatory Protein/Sequence

2. operator: “switch” that controls access of RNA polymerase to genes for transcription • repressor: a protein that binds to the operator to block transcription • corepressor: small molecule that binds to repressor to make it active (helps block transcription) • regulatory gene: gene that code for the production of repressors (always onat a low rate) • Regulatory sequence: stretch of DNA that interacts with regulatory proteins to control transcription (promoter, terminator, enhancer) • inducer: small molecule that inactivates the repressor (allows transcription)

3. Gene ExpressionBiotechnologyBacteria and Viruses Chapters 18, 19, and 20 3

4. Genes regulated at the transcription stage (mostly). • feedback inhibition (positive and negative) • Important in embryonic development (eukaryotes) to create differentiated cells • Also important in cancer prevention and regulation of the cell cycle • There are differences in the way prokaryotes and eukaryotes regulate gene expression…why??

5. Prokaryotic Gene Regulation • Express only those genes that are needed by the cell (allows for efficient metabolism) • Adjust activity of enzymes • Adjust production level of enzymes • Genes switched on/off as needed due to environmental conditions • Operon model: basic mechanism for the control of gene expression in bacteria

6. Operons… • Operon: entire stretch of DNA required to produce a protein (enzyme)—under the control of a single promoter • this may include several genes • operator + promoter + genes • Negative Gene Regulation (operon switched off by repressor) • Lac operon (inducible) • Trp operon (repressible)

7. Lac Operon • inducible operon. usually “off” but can be turned on by allolactose (sugar--isomer of lactose) • inactivates repressor to allow for transcription • genes code for 3 enzymes that utilize lactose • Figures 18.4(a) and 18.4(b)

8. Trp Operon • repressible operon. controls synthesis of tryptophan (amino acid) • is usually “on”, but can be switched off by a repressor. (prevents transcription) • controlled by a regulatory gene (trpR). (always expressed at a low rate) • allosteric regulation! • Increased concentration of tryptophan will result in less of it synthesized by bacteria • Figures 18.3(a) and 18.3(b)

9. Positive gene regulation (regulatory protein interacts directly with genome to switch transcription on) • Positive feedback! • Cyclic AMP (cAMP) and Lac operon • bacteria preferentially use glucose for energy (glycolysis) but will use lactose in its absence (lacoperon switches on) • cAMP accumulates when glucose is scarce • enhances the production of enzymes from the lacoperon (directly stimulates gene expression) • Binds to specific region in promoter • also has the ability to enhance the transcription of other genes (not just lac)

10. Bacteria Three shapes (cocci, bacilli, spirilla) • Gram+ and Gram- • Cell wall type • lack complex compartmentalization (prokaryotes) • circular chromosome located in nucleoid (region of cytoplasm)

11. Plasmids (independently replicating DNA carrying few genes) • R plasmid (carries resistance genes for specific antibiotics) -MRSA (methicillin resitance) -Multi-drug resistant strains -Enterococcus sp. -Pseudomonas sp. -Tuberculosis Implications for human and veterinary medicine, as well as agriculture

12. Bacterial Reproduction • No sexual reproduction • asexual (binary fission) • New mutations increase genetic diversity rapidly due to short generation times (rapid evolution)

13. Bacterial Reproduction • Horizontal Gene Transfer… • Transformation (take in foreign DNA from its surroundings) • produces recombinant cells (DNA from two different cells) • Transduction (bacteriophages--viruses carry genes from one cell to another) • recombinant cells produced • Conjugation (DNA transferred between two cells--one cell donates the other receives) • Transposition (DNA segments move with and between molecules) • “jumping genes”—move from chromosome to plasmid

14. Eukaryotic Gene Regulation • No Operons!! • Differential gene expression: different genes expressed by cells within the same genome • Depends on cell function or stage of development • Like prokaryotes, most often regulated at the stage of transcription (but much more complex) • Eukaryotes do have the ability to control gene expression at every step of the process

15. http://www.bozemanscience.com/031-gene-regulation/ How it works… • Transcription factors need to be present for the process to begin • These bind to specific DNA sequences “upstream” of the gene to be expressed (regulatory regions—enhancer/promoter) • Attracts RNA polymerase to attach • Controlling availability of these factors in a cell is a major component in regulating gene expression • Some factors are activators (increase expression) others are repressors (decrease expression) • These determine what (or if) genes will be expressed (and how much) • This changes phenotype of the organism!

16. Applications to Biotech • Plasmids can be used to genetically engineer organisms, also to further study specific genes • Easily moved between organisms, easily manipulated • restriction enzymes (restriction endonucleases) are used to cut DNA at specific sequences (restriction sites) • Produces blunt or sticky ends (depends on cut) • Hundreds have been identified, each recognizes a specific site • EcoR1 cuts at GAATTC

17. DNA (cut with the same enzyme) is then combined with other DNA making a recombinant DNA sequence • Base-pairing occurs, ligase seals break • This can produce some very interesting organisms! It allows for cells to produce proteins they wouldn’t normally. • Insulin, vaccines, oil spill clean up, agriculture, etc.