Biotechnology and Genetic Engineering-PBIO 450/550 - Get as PowerPoint by HC120727062721

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									   PBIO 427/527: Molecular Genetics
          Lecture 2 - Review
•Prokaryotic gene structure, processing & regulation
•Eukaryotic gene structure, processing & regulation
    •Restriction enzymes & gel electrophoresis
          •DNA cloning & cloning vectors
            •Gene libraries & screening
            •cDNA libraries & screening
Prokaryotic gene expression
Prokaryotic gene expression

              • Alternatively, see:
              • http://www.whfreema
                n.com/lodish4e/con_i
                ndex.htm?99anm
In prokaryotes, RNA polymerase binds to the -
 10 and -35 regions of the promoter relative to
       the start site of transcription (+1)




                    promoter     operator
            Eukaryotic gene organization




enhancers
silencers
Eukaryotic gene organization and RNA processing
    Basic Transcriptional Mechanism and
         mRNA Splicing Animations
• MCB Chapter 4-Basic Transcriptional Mechanism animation
• http://bcs.whfreeman.com/lodish5e/pages/bcs-
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• MCB Chapter 12-mRNA splicing animation
• http://bcs.whfreeman.com/lodish5e/pages/bcs-
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Eukaryotic gene expression
  MCB Chapter 4-Life Cycle of mRNA

• http://bcs.whfreeman.com/lodish5e/pages/bcs-
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Recombinant DNA cloning procedure
 Recombinant DNA cloning procedure

• See MCB Chapter 9 – Plasmid Cloning
• http://bcs.whfreeman.com/lodish5e/pages/bcs-
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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
      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                50-300
chromosome)
P1 bacteriophage-         E. coli                100-300
derived AC
YAC                       Yeast                  100-2,000
Human AC                  Cultured human cells   >2,000
               Plasmid cloning vectors




Three important features
1. Cloning site
2. Ori-an origin of replication
3. A selectable marker (ampr)
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 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)        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
 Genomic
  library
construction
Screening a genomic
  library using DNA
  hybridization to a
(radio-)labeled DNA
         probe

    Note: a cDNA is
commonly (radio-)labeled
  and used as a DNA
   probe to screen a
    genomic library
Production of a (radio-)labeled DNA probe by the random primer
    method [uses the Klenow fragment of DNA polymerase]

                   5’                                    3’




                   5’                                    3’
                         3’      5’
The first step in making
    a cDNA library:
     Purification of
polyadenylated mRNA
   using oligo(dT)-
        cellulose

Note: selection of the
proper source (organ,
tissue) of the RNA is
     critical here!
Complementary DNA or
   cDNA cloning:
    cDNA library
    construction


    Note: ds cDNAs are
typically placed in a cloning
       vector such as
 bacteriophage lambda (l)
         or a plasmid
   There are several possible ways to
         screen a cDNA library
• Using a DNA probe with a homologous
  sequence (e.g., a homologous cDNA or gene
  clone from a related species)
• Using an oligonucleotide probe based on a
  known amino acid sequence (requires
  purification of the protein and some peptide
  sequencing)
• Using an antibody against the protein of interest
  (note: this requires use of an expression vector)
• Plus/minus or differential screening (the least
  specific way)
  Screening a
  cDNA library
   using DNA
 hybridization to
a (radio-)labeled
   DNA probe
Screening a cDNA library with a labeled oligonucleotide
     probe based on a known peptide sequence
           Using polynucleotide kinase and
g-32P-labeled ATP to radiolabel oligonucleotide probes
 Immunological screening of
 an expression cDNA library
 with a primary antibody and
labeled secondary antibody;
   note the label is often an
  enzyme label like alkaline
phosphatase or horseradish
  peroxidase, but it can also
             be 125I
   Note: see also MCB Chapter 9 for a related
                   animation
 http://bcs.whfreeman.com/lodish5e/pages/bcs-
main.asp?v=category&s=00010&n=09000&i=090
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        Animations for two related uses of
               expression vectors
• Expression cloning of receptor proteins-see MCB Chapter 9
•   http://bcs.whfreeman.com/lodish5e/pages/bcs-
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• Looking for protein-protein interactions with the yeast two
  hybrid system-see MCB Chapter 11
•   http://bcs.whfreeman.com/lodish5e/pages/bcs-
    main.asp?s=00010&n=11000&i=11010.01&v=category&o=|00510|00610|00520|00530|005
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Plus/min (+/-)
or differential
  screening
   A cosmid cloning
        system:
another possible cloning
  vector which can be
used for genomic library
   but not for cDNA
        libraries
   In summary, you have seen:
• How to make and screen gene libraries
• How to make and screen cDNA libraries
• Several different cloning vectors
  including plasmids, bacteriophage
  lambda (l), and cosmids

								
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