PBIO 427527: Molecular Genetics Lecture 2 Review

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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

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Prokaryotic gene expression
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Prokaryotic gene expression

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

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In prokaryotes, RNA polymerase binds to the -10
and -35 regions of the promoter relative to the
start site of transcription (1)
promoter
operator
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Eukaryotic gene organization
enhancers silencers
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Eukaryotic gene organization and RNA processing
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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
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  00300004000050000600007000080000900010000
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  0002000021000220002300099000ns0
• MCB Chapter 12-mRNA splicing animation
• http//bcs.whfreeman.com/lodish5e/pages/bcs-main.a
  sp?vcategorys00010n12000i12010.02o00510
  006100052000530005400056000570005900060000
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  00300004000050000600007000080000900010000
  110001200013000140001500016000170001800019
  0002000021000220002300099000ns1211

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Eukaryotic gene expression
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MCB Chapter 4-Life Cycle of mRNA

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

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Recombinant DNA cloning procedure
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Recombinant DNA cloning procedure

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

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Restriction enzymes DNA methylation
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Recognition sequences of some REs
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Mapping of restriction enzyme sites
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Cloning vectors and their insert capacities
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Plasmid cloning vectors

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

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pGEM-3Z
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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
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Some antibiotics commonly used as selective agents
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Genomic library construction
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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
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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
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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!
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Complementary DNA or cDNA cloningcDNA library
constructionNote ds cDNAs are typically
placed in a cloning vector such as bacteriophage
lambda (l) or a plasmid
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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)

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Screening a cDNA library using DNA hybridization
to a (radio-)labeled DNA probe
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Screening a cDNA library with a labeled
oligonucleotide probe based on a known peptide
sequence
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Using polynucleotide kinase andg-32P-labeled ATP
to radiolabel oligonucleotide probes
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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
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018000190002000021000220002300099000ns58
9
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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-main.a
  sp?vcategorys00010n09000i09010.04o00510
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  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
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  110001200013000140001500016000170001800019
  0002000021000220002300099000ns798tuid0
  rau0

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