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

Prokaryotic gene expression

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

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

Eukaryotic gene organization enhancers silencers

Eukaryotic gene organization and RNA processing

Basic Transcriptional Mechanism and mRNA Splicing Animations MCB Chapter 4-Basic Transcriptional Mechanism animation main.asp?v=category&s=00010&n=04000&i= &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= &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 main.asp?v=category&s=00010&n=12000&i= &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= &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

Eukaryotic gene expression

MCB Chapter 4-Life Cycle of mRNA main.asp?v=category&s=00010&n=04000&i= & 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= & 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

Recombinant DNA cloning procedure

See MCB Chapter 9 – Plasmid Cloning main.asp?v=category&s=00010&n=09000&i= & 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= & 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

Restriction enzymes & DNA methylation

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

Mapping of restriction enzyme sites

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

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

pGEM-3Z

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

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

Genomic library construction

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

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

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!

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

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)

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

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

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

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 main.asp?v=category&s=00010&n=09000&i= &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 main.asp?v=category&s=00010&n=09000&i= &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

Animations for two related uses of expression vectors Expression cloning of receptor proteins-see MCB Chapter 9 main.asp?v=category&s=00010&n=09000&i= &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= &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 main.asp?s=00010&n=11000&i= &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= &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

Plus/min (+/-) or differential screening

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

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