MCB 7200: Molecular Biology

•Eukaryotic gene organization

•Restriction enzymes
•Cloning vectors
 Eukaryotic gene organization

Enhancers
Silencers
Insulators
Eukaryotic gene organization & RNA processing
 Figure 4.15 Overview of RNA processing.

Molecular Cell Biology, 7th Edition

Copyright © 2013 by W. H. Freeman and Company
Lodish et al.
 Figure 4.14 Structure of the 5’ methylated cap.

Molecular Cell Biology, 7th Edition

Copyright © 2013 by W. H. Freeman and Company
Lodish et al.
 Basic Transcriptional Mechanisms and
mRNA Splicing Animations
MCB Chapter 4-Basic Molecular Genetic Mechanisms (animations)
• Life Cycle of mRNA
 http://bcs.whfreeman.com/lodish7e/#800911__812036__
• Basic Transcriptional Mechanisms
 http://bcs.whfreeman.com/lodish7e/#800911__812037__

MCB Chapter 8-Post-transcriptional Gene Control (animation)

• mRNA Splicing
 http://bcs.whfreeman.com/lodish7e/#800911__812057__
 Figure 4.11 Three stages in transcription.

Molecular Cell Biology, 7th Edition

Copyright © 2013 by W. H. Freeman and Company
Lodish et al.
Prokaryotic vs. eukaryotic gene organization
Alternative splicing of eukaryotic 1° RNA transcripts
Eukaryotic gene expression
 MCB Chapter 4-Life Cycle of mRNA
MCB Chapter 4-Basic Molecular Genetic Mechanisms (animation)
• Life Cycle of mRNA
 http://bcs.whfreeman.com/lodish7e/#800911__812036__
 MCB Chapter 7-Yeast Two Hybrid System
(exploiting transcriptional activators)
MCB Chapter 7-Transcriptional Control of Gene Expression (animation)
• Yeast Two-Hybrid System
 http://bcs.whfreeman.com/lodish7e/#800911__812055__
 Insulators

Two kinds of insulator functions. (A) Some insulators may function as barriers against
the encroachment of adjacent genomic condensed chromatin. (B) Some insulators may
serve as positional enhancer-blocking elements that prevent enhancer action when
placed between enhancer and promoter, but not otherwise.
 Experimental Figure 5.14 DNA cloning in a plasmid vector permits amplification of a DNA fragment.

Recombinant DNA cloning

procedure

Molecular Cell Biology, 7th Edition

Copyright © 2013 by W. H. Freeman and Company
Lodish et al.
 Recombinant DNA cloning procedure

MCB Chapter 5 - Molecular Genetic Techniques (animation)

• Plasmid Cloning
 http://bcs.whfreeman.com/lodish7e/#800911__812047__
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
 Figure 5.13 Basic components of a plasmid cloning vector that can replicate within an E. coli cell.

3 important features: Cloning site, Ori-an origin

of replication, A selectable marker (ampr)

Molecular Cell Biology, 7th Edition

Copyright © 2013 by W. H. Freeman and Company
Lodish et al.
 pBR322

ori

The plasmid pBR322 is one of the most commonly used E.coli cloning vectors. pBR322 is 4361 bp in
length and contains: (1) the replicon rep responsible for the replication of plasmid (source – plasmid
pMB1); (2) rop gene coding for the Rop protein, which promotes conversion of the unstable RNA I –
RNA II complex to a stable complex and serves to decrease copy number (source – plasmid pMB1); (3)
bla gene, coding for beta-lactamase that confers resistance to ampicillin (source – transposon Tn3); (4)
tet gene, encoding tetracycline resistance protein (source – plasmid pSC101).
pUC18/19
pUC18 and pUC19 vectors are small, high copy number, E.coli plasmids,
2686 bp in length. They are identical except that they contain multiple
cloning sites (MCS) arranged in opposite orientations. pUC18/19 plasmids
contain: (1) the pMB1 replicon rep responsible for the replication of
plasmid (source – plasmid pBR322). The high copy number of pUC
plasmids is a result of the lack of the rop gene and a single point mutation
in rep of pMB1; (2) bla gene, coding for beta-lactamase that confers
resistance to ampicillin (source – plasmid pBR322); (3) region of E.coli
operon lac containing CAP protein binding site, promoter Plac, lac repressor
binding site and 5’-terminal part of the lacZ gene encoding the N-terminal
fragment of beta-galactosidase (source – M13mp18/19). This fragment,
whose synthesis can be induced by IPTG, is capable of intra-allelic (alfa)
complementation with a defective form of beta-galactosidase encoded by
host (mutation lacZDM15). In the presence of IPTG, bacteria synthesize
both fragments of the enzyme and form blue colonies on media with X-Gal.
Insertion of DNA into the MCS located within the lacZ gene (codons 6-7 of
lacZ are replaced by MCS) inactivates the N-terminal fragment of beta-
galactosidase and abolishes alfa-complementation. Bacteria carrying
recombinant plasmids therefore give rise to white colonies.
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
Invitrogen’s Gateway® technology facilitates cloning of genes, into and out of,
multiple vectors via site-specific recombination. Once a gene is cloned into an Entry
clone you can then move the DNA fragment into one or more destination vectors
simultaneously.
 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