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PBIO 427/527: Molecular GeneticsLecture 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
Prokaryotic gene expression
Regulation lac Operon.mov
• Alternatively, see:• http://www.whfreema
n.com/lodish4e/con_index.htm?99anm
In prokaryotes, RNA polymerase binds to the -10 and -35 regions of the promoter relative to
the start site of transcription (+1)
promoter operator
Eukaryotic gene organization
enhancerssilencers
Eukaryotic gene organization and RNA processing
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|00610|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|15000|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|00610|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|15000|16000|17000|18000|19000|20000|21000|22000|23000|99000|&ns=1211
Eukaryotic gene expression
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|00590|00600|00700|00710|00010|00020|00030|00040|00050|01000|02000|03000|04000|05000|06000|07000|08000|09000|10000|11000|&ns=0
Recombinant DNA cloning procedure
Recombinant DNA cloning procedure
• 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|00590|00600|00700|00710|00010|00020|00030|00040|00050|01000|02000|03000|04000|05000|06000|07000|08000|09000|10000|11000|&ns=437
Restriction enzymes & DNA methylation
Recognition sequences of some REs
Enzyme Recognition site Type 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 system Host cell Insert capacity (kb)
Plasmid E. coli 0.1-10
Bacteriophage E. coli 10-20
Cosmid E. coli 35-45
Bacteriophage P1 E. coli 80-100
BAC (bacterial artificial chromosome)
E. coli 50-300
P1 bacteriophage-derived AC
E. coli 100-300
YAC Yeast 100-2,000
Human AC Cultured human cells
>2,000
Cloning vectors and their insert capacities
Plasmid cloning vectors
Three important features1. Cloning site2. Ori-an origin of replication3. A selectable marker (ampr)
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
Antibiotic Description
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; tetr 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’
5’5’
3’
3’3’
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-32P-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 125I
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=09010.04&o=|00510|00610|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|15000|16000|17000|18000|19000|
20000|21000|22000|23000|99000|&ns=589
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|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|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|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|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