MCB 7200 Molecular Biology Eukaryotic gene organization Restriction

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, 7 th Edition Lodish

Figure 4. 14 Structure of the 5’ methylated cap. Molecular Cell Biology, 7 th

Basic Transcriptional Mechanisms and m. RNA Splicing Animations MCB Chapter 4 -Basic Molecular Genetic Mechanisms (animations) • Life Cycle of m. RNA u http: //bcs. whfreeman. com/lodish 7 e/#800911__812036__ • Basic Transcriptional Mechanisms u http: //bcs. whfreeman. com/lodish 7 e/#800911__812037__ MCB Chapter 8 -Post-transcriptional Gene Control (animation) • m. RNA Splicing u http: //bcs. whfreeman. com/lodish 7 e/#800911__812057__

Figure 4. 11 Three stages in transcription. Molecular Cell Biology, 7 th Edition Lodish

Prokaryotic vs. eukaryotic gene organization

Alternative splicing of eukaryotic 1° RNA transcripts

Eukaryotic gene expression

MCB Chapter 4 -Life Cycle of m. RNA MCB Chapter 4 -Basic Molecular Genetic Mechanisms (animation) • Life Cycle of m. RNA u http: //bcs. whfreeman. com/lodish 7 e/#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 u http: //bcs. whfreeman. com/lodish 7 e/#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, 7 th 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 u http: //bcs. whfreeman. com/lodish 7 e/#800911__812047__

Restriction enzymes & DNA methylation

Recognition sequences of some REs Enzyme Eco. RI Bam. HI Pst. I Sau 3 A 1 Pvu. II Hpa. I Hae. III Not. I 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 3’ P extension 5’ P extension 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 P 1 E. coli 80 -100 BAC (bacterial artificial E. coli chromosome) 50 -300 P 1 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, 7 th Edition Lodish et al. Copyright © 2013 by W. H. Freeman and Company

p. BR 322 ori The plasmid p. BR 322 is one of the most commonly used E. coli cloning vectors. p. BR 322 is 4361 bp in length and contains: (1) the replicon rep responsible for the replication of plasmid (source – plasmid p. MB 1); (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 p. MB 1); (3) bla gene, coding for beta-lactamase that confers resistance to ampicillin (source – transposon Tn 3); (4) tet gene, encoding tetracycline resistance protein (source – plasmid p. SC 101).

p. UC 18/19 p. UC 18 and p. UC 19 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. p. UC 18/19 plasmids contain: (1) the p. MB 1 replicon rep responsible for the replication of plasmid (source – plasmid p. BR 322). The high copy number of p. UC plasmids is a result of the lack of the rop gene and a single point mutation in rep of p. MB 1; (2) bla gene, coding for beta-lactamase that confers resistance to ampicillin (source – plasmid p. BR 322); (3) region of E. coli operon lac containing CAP protein binding site, promoter Plac, lac repressor binding site and 5’-terminal part of the lac. Z gene encoding the N-terminal fragment of beta-galactosidase (source – M 13 mp 18/19). 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 lac. ZDM 15). 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 lac. Z gene (codons 6 -7 of lac. Z 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.

p. GEM-3 Z

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 T 4 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 (Hyg. B) Kanamycin (Kan) Binds to 30 S ribosomal subunit and inhibits protein synthesis; inactivated by a phosphotransferase Neomycin (Neo) Binds to 30 S ribosomal subunit and inhibits protein synthesis; inactivated by a phosphotransferase Streptomycin (Str) Tetracycline (Tet) Binds to 30 S ribosomal subunit and inhibits protein synthesis; tetr gene encodes a protein which prevents transport of tet into the cell