1. MCB 720: Molecular Biology
Eukaryotic gene organization
Restriction enzymes
Cloning vectors

2. Eukaryotic gene organization
Enhancers
Silencers
Insulators

3. Eukaryotic gene organization & RNA processing

4. Basic Transcriptional Mechanism and mRNA Splicing Animations
MCB Chapter 4-Basic Transcriptional Mechanism animation
MCB Chapter 12-mRNA splicing animation

5. Prokaryotic vs. eukaryotic gene organization

6. Alternative splicing of eukaryotic 1° RNA transcripts

7. Eukaryotic gene expression

8. MCB Chapter 4-Life Cycle of mRNA

9. MCB Chapter 11-Yeast Two Hybrid System (exploiting transcriptional activators)

10. 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.

11. Recombinant DNA cloning procedure

12. Recombinant DNA cloning procedure
See MCB Chapter 9 – Plasmid Cloning

13. Restriction enzymes & DNA methylation

14. 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
PstI
C-T-G-C-A￬G
3’ P extension
Sau3A1
￬G-A-T-C
PvuII
C-A-G￬C-T-G
Blunt end
HpaI
G-T-T￬A-A-C
HaeIII
G-G￬C-C
NotI
G￬C-G-G-C-C-G-C

15. Mapping of restriction enzyme sites

16. Cloning vectors and their insert capacities
Vector system
Host cell
Insert capacity (kb)
Plasmid
E. coli
0.1-10
Bacteriophage l
10-20
Cosmid
35-45
Bacteriophage P1
80-100
BAC (bacterial artificial chromosome)
50-300
P1 bacteriophage-derived AC
YAC
Yeast
100-2,000
Human AC
Cultured human cells
>2,000

17. Plasmid cloning vectors
Three important features
Cloning site
Ori-an origin of replication
A selectable marker (ampr)

18. 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).

19. 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.

20. pGEM-3Z

21. 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

22. 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.

23. Some antibiotics commonly used as selective agents
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)
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

24. Homework (2 pages max., send to showalte@ohio.edu by Mon., 1/31)
Explain how Invitrogene’s Gateway cloning system works.
What are insulators and how do they work?
How do hygromycin B and streptomycin work?