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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
Prokaryotic gene expression
• Alternatively, see:
• http://www.whfreema
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)
promoter operator
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
• http://bcs.whfreeman.com/lodish5e/pages/bcs-
main.asp?v=category&s=00010&n=04000&i=04010.01&o=|00510|0
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• 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
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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|0059
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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|0059
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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
Cloning vectors and their insert capacities
Vector system Host cell Insert capacity (kb)
Plasmid E. coli 0.1-10
Bacteriophage l E. coli 10-20
Cosmid E. coli 35-45
Bacteriophage P1 E. coli 80-100
BAC (bacterial artificial E. coli 50-300
chromosome)
P1 bacteriophage- E. coli 100-300
derived AC
YAC Yeast 100-2,000
Human AC Cultured human cells >2,000
Plasmid cloning vectors
Three important features
1. Cloning site
2. Ori-an origin of replication
3. 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 b-
lactamase, which cleaves the b-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’ 3’
5’ 3’
3’ 5’
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 (l)
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
g-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=090
10.04&o=|00510|00610|00520|00530|00540|0056
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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
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• 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
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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 (l), and cosmids
