PBIO 427/527: Molecular Genetics Lecture 2 - Revie

1
PBIO 427/527 Molecular GeneticsLecture 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

2
Prokaryotic gene expression
3
Prokaryotic gene expression

• Alternatively, see
• http//www.whfreeman.com/lodish4e/con_index.htm?99
  anm

4
In prokaryotes, RNA polymerase binds to the -10
and -35 regions of the promoter relative to the
start site of transcription (1)
promoter
operator
5
Eukaryotic gene organization
enhancers silencers
6
Eukaryotic gene organization and RNA processing
7
Basic Transcriptional Mechanism and mRNA
Splicing Animations

• MCB Chapter 4-Basic Transcriptional Mechanism
  animation
• http//bcs.whfreeman.com/lodish5e/pages/bcs-main.a
  sp?vcategorys00010n04000i04010.01o00510
  006100052000530005400056000570005900060000
  700007100001000020000300004000050010000200
  00300004000050000600007000080000900010000
  110001200013000140001500016000170001800019
  0002000021000220002300099000ns0
• MCB Chapter 12-mRNA splicing animation
• http//bcs.whfreeman.com/lodish5e/pages/bcs-main.a
  sp?vcategorys00010n12000i12010.02o00510
  006100052000530005400056000570005900060000
  700007100001000020000300004000050010000200
  00300004000050000600007000080000900010000
  110001200013000140001500016000170001800019
  0002000021000220002300099000ns1211

8
Eukaryotic gene expression
9
MCB Chapter 4-Life Cycle of mRNA

• http//bcs.whfreeman.com/lodish5e/pages/bcs-main.a
  sp?vcategorys00010n04000i04010.02o00510
  006100052000530005400056000570005900060000
  700007100001000020000300004000050010000200
  00300004000050000600007000080000900010000
  11000ns0

10
Recombinant DNA cloning procedure
11
Recombinant DNA cloning procedure

• See MCB Chapter 9 Plasmid Cloning
• http//bcs.whfreeman.com/lodish5e/pages/bcs-main.a
  sp?vcategorys00010n09000i09010.05o00510
  006100052000530005400056000570005900060000
  700007100001000020000300004000050010000200
  00300004000050000600007000080000900010000
  11000ns437

12
Restriction enzymes DNA methylation
13
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
14
Mapping of restriction enzyme sites
15
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 chromosome) E. coli 50-300
P1 bacteriophage-derived AC E. coli 100-300
YAC Yeast 100-2,000
Human AC Cultured human cells gt2,000
16
Plasmid cloning vectors

• Three important features
• Cloning site
• Ori-an origin of replication
• A selectable marker (ampr)

17
pGEM-3Z
18
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
19
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
20
Genomic library construction
21
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
22
Production of a (radio-)labeled DNA probe by the
random primer method uses the Klenow fragment of
DNA polymerase
5
3
5
3
5
3
23
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!
24
Complementary DNA or cDNA cloningcDNA library
constructionNote ds cDNAs are typically
placed in a cloning vector such as bacteriophage
lambda (l) or a plasmid
25
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)

26
Screening a cDNA library using DNA hybridization
to a (radio-)labeled DNA probe
27
Screening a cDNA library with a labeled
oligonucleotide probe based on a known peptide
sequence
28
Using polynucleotide kinase andg-32P-labeled ATP
to radiolabel oligonucleotide probes
29
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
125INote see also MCB Chapter 9 for a related
animation http//bcs.whfreeman.com/lodish5e/pages/
bcs-main.asp?vcategorys00010n09000i09010.04
o005100061000520005300054000560005700059
00060000700007100001000020000300004000050
010000200003000040000500006000070000800009
000100001100012000130001400015000160001700
018000190002000021000220002300099000ns58
9
30
Animations for two related uses of expression
vectors

• Expression cloning of receptor proteins-see MCB
  Chapter 9
• http//bcs.whfreeman.com/lodish5e/pages/bcs-main.a
  sp?vcategorys00010n09000i09010.04o00510
  006100052000530005400056000570005900060000
  700007100001000020000300004000050010000200
  00300004000050000600007000080000900010000
  110001200013000140001500016000170001800019
  0002000021000220002300099000ns589
• Looking for protein-protein interactions with the
  yeast two hybrid system-see MCB Chapter 11
• http//bcs.whfreeman.com/lodish5e/pages/bcs-main.a
  sp?s00010n11000i11010.01vcategoryo00510
  006100052000530005400056000570005900060000
  700007100001000020000300004000050010000200
  00300004000050000600007000080000900010000
  110001200013000140001500016000170001800019
  0002000021000220002300099000ns798tuid0
  rau0

31
Plus/min (/-) or differential screening
32
A cosmid cloning systemanother possible cloning
vector which can be used for genomic library but
not for cDNA libraries
33
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