Biotechnology and Genetic EngineeringPBIO 450/550

•Eukaryotic gene organization•Restriction enzymes•Cloning vectors

Eukaryotic gene organization

enhancerssilencers

Eukaryotic gene organization & RNA processing

Basic Transcriptional Mechanism and mRNA Splicing Animations

• MCB Chapter 4-Basic Transcriptional Mechanism animation• http://bcs.whfreeman.com/lodish5e/pages/bcs-main.asp?v=category&s=

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• MCB Chapter 12-mRNA splicing animation• http://bcs.whfreeman.com/lodish5e/pages/bcs-main.asp?v=category&s=

00010&n=12000&i=12010.02&o=|00510|00610|00520|00530|00540|00560|00570|00590|00600|00700|00710|00010|00020|00030|00040|00050|01000|02000|03000|04000|05000|06000|07000|08000|09000|10000|11000|12000|13000|14000|15000|16000|17000|18000|19000|20000|21000|22000|23000|99000|&ns=1211

Prokaryotic vs. eukaryotic gene organization

Alternative splicing of eukaryotic 1° RNA transcripts

Eukaryotic gene expression

MCB Chapter 4-Life Cycle of mRNA

• http://bcs.whfreeman.com/lodish5e/pages/bcs-main.asp?v=category&s=00010&n=04000&i=04010.02&o=|00510|00610|00520|00530|00540|00560|00570|00590|00600|00700|00710|00010|00020|00030|00040|00050|01000|02000|03000|04000|05000|06000|07000|08000|09000|10000|11000|&ns=0

Recombinant DNA cloning procedure

Recombinant DNA cloning procedure

• See MCB Chapter 9 – Plasmid Cloning• http://bcs.whfreeman.com/lodish5e/pages/bcs-main.asp?

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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

Vector system Host cell Insert capacity (kb)

Plasmid E. coli 0.1-10

Bacteriophage E. coli 10-20

Cosmid E. coli 35-45

Bacteriophage P1 E. coli 80-100

BAC (bacterial artificial chromosome)

E. coli 50-300

P1 bacteriophage-derived AC

E. coli 100-300

YAC Yeast 100-2,000

Human AC Cultured human cells

>2,000

Cloning vectors and their insert capacities

Plasmid cloning vectors

Three important features1. Cloning site2. Ori-an origin of replication3. A selectable marker (ampr)

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

pUC18/19pUC18 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

Some antibiotics commonly used as selective agents

Antibiotic Description

Ampicillin (Amp) Inhibits bacterial cell wall synthesis; inactivated by -lactamase, which cleaves the -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