Transcript Lecture 2

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
Lodish et al.
Copyright © 2013 by W. H. Freeman and Company
Figure 4.14 Structure of the 5’ methylated cap.
Molecular Cell Biology, 7th Edition
Lodish et al.
Copyright © 2013 by W. H. Freeman and Company
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
Lodish et al.
Copyright © 2013 by W. H. Freeman and Company
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
Lodish et al.
Copyright © 2013 by W. H. Freeman and Company
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
EcoRI
BamHI
PstI
Sau3A1
PvuII
HpaI
HaeIII
NotI
Recognition site
G￬A-A-T-T-C
G￬G-A-T-C-C
C-T-G-C-A￬G
￬G-A-T-C
C-A-G￬C-T-G
G-T-T￬A-A-C
G-G￬C-C
G￬C-G-G-C-C-G-C
Type of cut end
5’ P extension
5’ P extension
3’ P extension
5’ P extension
Blunt end
Blunt end
Blunt end
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
chromosome)
50-300
P1 bacteriophagederived AC
E. coli
100-300
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
Lodish et al.
Copyright © 2013 by W. H. Freeman and Company
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 betagalactosidase 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 blactamase, 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