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