Biotechnology and Genetic Engineering PBIO 4500/5500 Eukaryotic gene organization Restriction enzymes Cloning vectors

Eukaryotic gene organization enhancers silencers

Eukaryotic gene organization & RNA processing

Copyright © 2013 by W. H. Freeman and Company Molecular Cell Biology, 7 th Edition Lodish et al. Figure 4.14 Structure of the 5’ methylated cap.

Basic Transcriptional Mechanisms and mRNA Splicing Animations MCB Chapter 4-Basic Molecular Genetic Mechanisms (animations) Life Cycle of mRNA  Basic Transcriptional Mechanisms  MCB Chapter 8-Post-transcriptional Gene Control (animation) mRNA Splicing 

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 

MCB Chapter 7-Yeast Two Hybrid System (exploiting transcriptional activators) MCB Chapter 7-Transcriptional Control of Gene Expression (animation) Yeast Two-Hybrid System 

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.

Recombinant DNA cloning procedure

MCB Chapter 5 - Molecular Genetic Techniques (animation) Plasmid Cloning 

Restriction enzymes & DNA methylation

Recognition sequences of some REs EnzymeRecognition siteType 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 systemHost cellInsert capacity (kb) PlasmidE. coli Bacteriophage E. coli10-20 CosmidE. coli35-45 Bacteriophage P1E. coli BAC (bacterial artificial chromosome) E. coli P1 bacteriophage- derived AC E. coli YACYeast100-2,000 Human ACCultured human cells>2,000 Cloning vectors and their insert capacities

Plasmid cloning vectors Three important features 1.Cloning site 2.Ori-an origin of replication 3.A selectable marker (amp r )

pBR322 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). ori

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.pBR322M13mp18/19IPTGX-Gal

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 AntibioticDescription Ampicillin (Amp) Inhibits bacterial cell wall synthesis; inactivated by  - lactamase, which cleaves the  -lactam ring of amp Hygromycin B (HygB)Blocks translocation from amino acyl site to peptidyl site 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)Blocks protein initiation complex formation and causes misreading during translation Tetracycline (Tet)Binds to 30S ribosomal subunit and inhibits protein synthesis; tet r gene encodes a protein which prevents transport of tet into the cell