1. PBIO 427/527: Molecular Genetics Lecture 2 - Review Prokaryotic gene structure, processing & regulation Eukaryotic gene structure, processing & regulation Restriction enzymes & gel electrophoresis DNA cloning & cloning vectors Gene libraries & screening cDNA libraries & screening

2. Prokaryotic gene expression

3. Alternatively, see: http://www.whfreema n.com/lodish4e/con_i ndex.htm?99anmhttp://www.whfreema n.com/lodish4e/con_i ndex.htm?99anm

4. In prokaryotes, RNA polymerase binds to the - 10 and -35 regions of the promoter relative to the start site of transcription (+1) promoteroperator

5. Eukaryotic gene organization enhancers silencers

6. Eukaryotic gene organization and RNA processing

7. Basic Transcriptional Mechanism and mRNA Splicing Animations MCB Chapter 4-Basic Transcriptional Mechanism animation http://bcs.whfreeman.com/lodish5e/pages/bcs- main.asp?v=category&s=00010&n=04000&i=04010.01&o=|00510|0 0610|00520|00530|00540|00560|00570|00590|00600|00700|00710| 00010|00020|00030|00040|00050|01000|02000|03000|04000|05000 |06000|07000|08000|09000|10000|11000|12000|13000|14000|1500 0|16000|17000|18000|19000|20000|21000|22000|23000|99000|&ns =0http://bcs.whfreeman.com/lodish5e/pages/bcs- main.asp?v=category&s=00010&n=04000&i=04010.01&o=|00510|0 0610|00520|00530|00540|00560|00570|00590|00600|00700|00710| 00010|00020|00030|00040|00050|01000|02000|03000|04000|05000 |06000|07000|08000|09000|10000|11000|12000|13000|14000|1500 0|16000|17000|18000|19000|20000|21000|22000|23000|99000|&ns =0 MCB Chapter 12-mRNA splicing animation http://bcs.whfreeman.com/lodish5e/pages/bcs- main.asp?v=category&s=00010&n=12000&i=12010.02&o=|00510|0 0610|00520|00530|00540|00560|00570|00590|00600|00700|00710| 00010|00020|00030|00040|00050|01000|02000|03000|04000|05000 |06000|07000|08000|09000|10000|11000|12000|13000|14000|1500 0|16000|17000|18000|19000|20000|21000|22000|23000|99000|&ns =1211http://bcs.whfreeman.com/lodish5e/pages/bcs- main.asp?v=category&s=00010&n=12000&i=12010.02&o=|00510|0 0610|00520|00530|00540|00560|00570|00590|00600|00700|00710| 00010|00020|00030|00040|00050|01000|02000|03000|04000|05000 |06000|07000|08000|09000|10000|11000|12000|13000|14000|1500 0|16000|17000|18000|19000|20000|21000|22000|23000|99000|&ns =1211

8. Eukaryotic gene expression

9. MCB Chapter 4-Life Cycle of mRNA http://bcs.whfreeman.com/lodish5e/pages/bcs- main.asp?v=category&s=00010&n=04000&i=04010.02& o=|00510|00610|00520|00530|00540|00560|00570|0059 0|00600|00700|00710|00010|00020|00030|00040|00050| 01000|02000|03000|04000|05000|06000|07000|08000|0 9000|10000|11000|&ns=0http://bcs.whfreeman.com/lodish5e/pages/bcs- main.asp?v=category&s=00010&n=04000&i=04010.02& o=|00510|00610|00520|00530|00540|00560|00570|0059 0|00600|00700|00710|00010|00020|00030|00040|00050| 01000|02000|03000|04000|05000|06000|07000|08000|0 9000|10000|11000|&ns=0

10. Recombinant DNA cloning procedure

11. See MCB Chapter 9 – Plasmid Cloning http://bcs.whfreeman.com/lodish5e/pages/bcs- main.asp?v=category&s=00010&n=09000&i=09010.05& o=|00510|00610|00520|00530|00540|00560|00570|0059 0|00600|00700|00710|00010|00020|00030|00040|00050| 01000|02000|03000|04000|05000|06000|07000|08000|0 9000|10000|11000|&ns=437http://bcs.whfreeman.com/lodish5e/pages/bcs- main.asp?v=category&s=00010&n=09000&i=09010.05& o=|00510|00610|00520|00530|00540|00560|00570|0059 0|00600|00700|00710|00010|00020|00030|00040|00050| 01000|02000|03000|04000|05000|06000|07000|08000|0 9000|10000|11000|&ns=437

12. Restriction enzymes & DNA methylation

13. Recognition sequences of some REs EnzymeRecognition siteType of cut end EcoRI G ￬ A-A-T-T-C 5’ P extension BamHI G ￬ G-A-T-C-C 5’ P extension PstI C-T-G-C-A ￬ G 3’ P extension Sau3A1 ￬ G-A-T-C 5’ P extension PvuII C-A-G ￬ C-T-G Blunt end HpaI G-T-T ￬ A-A-C Blunt end HaeIII G-G ￬ C-C Blunt end NotI G ￬ C-G-G-C-C-G-C 5’ P extension

14. Mapping of restriction enzyme sites

15. Vector systemHost cellInsert capacity (kb) PlasmidE. coli0.1-10 Bacteriophage E. coli10-20 CosmidE. coli35-45 Bacteriophage P1E. coli80-100 BAC (bacterial artificial chromosome) E. coli50-300 P1 bacteriophage- derived AC E. coli100-300 YACYeast100-2,000 Human ACCultured human cells>2,000 Cloning vectors and their insert capacities

16. Plasmid cloning vectors Three important features 1.Cloning site 2.Ori-an origin of replication 3.A selectable marker (amp r )

17. pGEM-3Z

18. Cloning foreign DNA into a plasmid vector Alkaline phosphatase-removes 5’ phosphate (P) groups of DNA molecules; BAP is more stable but less active than CIP T4 DNA ligase –joins 5’ phosphate (P) groups of DNA molecules to 3’ hydroxyl (OH) groups of DNA

19. Some antibiotics commonly used as selective agents AntibioticDescription Ampicillin (Amp) Inhibits bacterial cell wall synthesis; inactivated by  - lactamase, which cleaves the  -lactam ring of amp Hygromycin B (HygB) Kanamycin (Kan)Binds to 30S ribosomal subunit and inhibits protein synthesis; inactivated by a phosphotransferase Neomycin (Neo)Binds to 30S ribosomal subunit and inhibits protein synthesis; inactivated by a phosphotransferase Streptomycin (Str) Tetracycline (Tet)Binds to 30S ribosomal subunit and inhibits protein synthesis; tet r gene encodes a protein which prevents transport of tet into the cell

20. Genomic library construction

21. Screening a genomic library using DNA hybridization to a (radio-)labeled DNA probe Note: a cDNA is commonly (radio-)labeled and used as a DNA probe to screen a genomic library

22. Production of a (radio-)labeled DNA probe by the random primer method [uses the Klenow fragment of DNA polymerase] 5’ 3’

23. The first step in making a cDNA library: Purification of polyadenylated mRNA using oligo(dT)- cellulose Note: selection of the proper source (organ, tissue) of the RNA is critical here!

24. Complementary DNA or cDNA cloning: cDNA library construction Note: ds cDNAs are typically placed in a cloning vector such as bacteriophage lambda ( ) or a plasmid

25. There are several possible ways to screen a cDNA library Using a DNA probe with a homologous sequence (e.g., a homologous cDNA or gene clone from a related species) Using an oligonucleotide probe based on a known amino acid sequence (requires purification of the protein and some peptide sequencing) Using an antibody against the protein of interest (note: this requires use of an expression vector) Plus/minus or differential screening (the least specific way)

26. Screening a cDNA library using DNA hybridization to a (radio-)labeled DNA probe

27. Screening a cDNA library with a labeled oligonucleotide probe based on a known peptide sequence

28. Using polynucleotide kinase and  - 32 P-labeled ATP to radiolabel oligonucleotide probes

29. Immunological screening of an expression cDNA library with a primary antibody and labeled secondary antibody; note the label is often an enzyme label like alkaline phosphatase or horseradish peroxidase, but it can also be 125 I Note: see also MCB Chapter 9 for a related animation http://bcs.whfreeman.com/lodish5e/pages/bcs- main.asp?v=category&s=00010&n=09000&i=090 10.04&o=|00510|00610|00520|00530|00540|0056 0|00570|00590|00600|00700|00710|00010|00020 |00030|00040|00050|01000|02000|03000|04000| 05000|06000|07000|08000|09000|10000|11000|1 2000|13000|14000|15000|16000|17000|18000|19 000|20000|21000|22000|23000|99000|&ns=589 http://bcs.whfreeman.com/lodish5e/pages/bcs- main.asp?v=category&s=00010&n=09000&i=090 10.04&o=|00510|00610|00520|00530|00540|0056 0|00570|00590|00600|00700|00710|00010|00020 |00030|00040|00050|01000|02000|03000|04000| 05000|06000|07000|08000|09000|10000|11000|1 2000|13000|14000|15000|16000|17000|18000|19 000|20000|21000|22000|23000|99000|&ns=589

30. Animations for two related uses of expression vectors Expression cloning of receptor proteins-see MCB Chapter 9 http://bcs.whfreeman.com/lodish5e/pages/bcs- main.asp?v=category&s=00010&n=09000&i=09010.04&o=|00510|00610|00520|00530|005 40|00560|00570|00590|00600|00700|00710|00010|00020|00030|00040|00050|01000|0200 0|03000|04000|05000|06000|07000|08000|09000|10000|11000|12000|13000|14000|15000 |16000|17000|18000|19000|20000|21000|22000|23000|99000|&ns=589http://bcs.whfreeman.com/lodish5e/pages/bcs- main.asp?v=category&s=00010&n=09000&i=09010.04&o=|00510|00610|00520|00530|005 40|00560|00570|00590|00600|00700|00710|00010|00020|00030|00040|00050|01000|0200 0|03000|04000|05000|06000|07000|08000|09000|10000|11000|12000|13000|14000|15000 |16000|17000|18000|19000|20000|21000|22000|23000|99000|&ns=589 Looking for protein-protein interactions with the yeast two hybrid system-see MCB Chapter 11 http://bcs.whfreeman.com/lodish5e/pages/bcs- main.asp?s=00010&n=11000&i=11010.01&v=category&o=|00510|00610|00520|00530|005 40|00560|00570|00590|00600|00700|00710|00010|00020|00030|00040|00050|01000|0200 0|03000|04000|05000|06000|07000|08000|09000|10000|11000|12000|13000|14000|15000 |16000|17000|18000|19000|20000|21000|22000|23000|99000|&ns=798&t=&uid=0&rau=0http://bcs.whfreeman.com/lodish5e/pages/bcs- main.asp?s=00010&n=11000&i=11010.01&v=category&o=|00510|00610|00520|00530|005 40|00560|00570|00590|00600|00700|00710|00010|00020|00030|00040|00050|01000|0200 0|03000|04000|05000|06000|07000|08000|09000|10000|11000|12000|13000|14000|15000 |16000|17000|18000|19000|20000|21000|22000|23000|99000|&ns=798&t=&uid=0&rau=0

31. Plus/min (+/-) or differential screening

32. A cosmid cloning system: another possible cloning vector which can be used for genomic library but not for cDNA libraries

33. In summary, you have seen: How to make and screen gene libraries How to make and screen cDNA libraries Several different cloning vectors including plasmids, bacteriophage lambda ( ), and cosmids