Manipulating Proteins, DNA, and RNA

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Chapter 8

• Manipulating Proteins, DNA, and RNA

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• WHY STUDY CELLS IN CULTURE?
• More homogeneous population
• Controlled experimental conditions
• Clonal isolates - a genetically homogeneous
  population of cells arising from a single cell
• Assumption that response reflects what occurs at
  the unicellular level
• WHY STUDY MICROORGANISMS LIKE BACTERIA, YEAST,
  AND VIRUSES?
• Easy and fast - will grow well on minimal medium
  (carbon source glucose nitrogen source
  ammonium chloride salts)
• When grown on a semisolid surface (eg. agar) can
  easily generate clonal isolates
• viruses have small genomes

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CLONAL GROWTH
Mixed bacterial culture

• Clonal bacterial culture
• all bacteria are genetically identical

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PREPARING A PRIMARY CULTURE OF ANIMAL CELLS

• isolate a fragment of tissue of choice (eg.
  skin, muscle
• dissect away undesirable tissues and membranes
• mince and digest the extracellular matrix (ECM)
  with one or more proteinases (eg. trypsin,
  collagenase)
• isolate free cells (eg. by filtration or
  centrifugation) and plate onto petri dishes under
  appropriate growth medium
• very rich media- 9 essential amino acids can not
  be synthesized by adult vertebrates
  H,I,L,K,M,F,A,T,W,V. Medium must also contain
  C,Q and Y because these aa are made by
  specialized cells in the body and
  vitamins-SERUM-non cellular part of blood

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ADVANTAGES and DISADVANTAGES of growing animal
cells in culture

• ADVANTAGES
• allows specific cell types to be studied free of
  the influence of surrounding tissues in the
  intact animal
• provides more control over experimental
  conditions
• can mimic cell-cell and cell-ECM interactions
  seen in tissues
• clonal colonies can be generated in 2 weeks
• defined, serum-free medium formulations are
  available for some cell types

• DISADVANTAGES
• question of cell behaviour in culture vs. in
  tissues
• can be difficult to grow or to maintain
  consistent growth conditions from one experiment
  to another
• growth medium is more complex - requires
  essential amino acids, vitamins, serum (hormones,
  growth factors, etc.)

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ANIMAL CELLS IN CULTURE
Tissue culture flask
Growth medium
Cells
Gelatin or collagen substratum
37oC
5 CO2
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Two classes of animal cell cultures

• PRIMARY CULTURES
• best representation of cell behaviour in normal
  tissues
• have a finite lifespan (Hayflick limit - undergo
  replicative senescence after 50-60 generations
  (doublings divisions)
• cell types commonly prepared include fibroblasts
  (skin), myoblasts (skeletal muscle),
  cardiomyocytes (heart)

• TRANSFORMED CELLS
• can grow indefinitely in culture (have acquired
  one or more genetic mutations that allow them to
  escape senescence
• often these cells are less phenotypically
  related to the source tissue
• some can retain the ability to differentiate
  (eg. rodent muscle cell lines)
• examples include tumour cell lines (eg. HeLa
  cervical cancer cells established in 1952)

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NORMAL AND TRANSFORMED CELLS
EARLY MITOTIC
SENESCENCE
TRANSFORMATION
CARCINOMA
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HYBRID CELL LINES (HETEROKARYONS)

• prepared by fusion of primary cells (human or
  mouse) with a transformed rodent (eg. hamster or
  mouse) cell line
• accomplished by co-incubating the two cell types
  with agents that promote cell membrane fusion
  (eg. polyethylene glycol (PEG), enveloped
  viruses) followed by some form of metabolic
  selection provided by the primary cells (eg. HAT
  medium hypoxanthine (purine substrate for
  salvage pathway to produce guanylate)
  aminopterin (an antifolate that blocks the de
  novo purine synthetic pathway) thymidine (to
  provide for thymidylate synthesis))
• in human-rodent fusions, tendency is for the
  cells to lose human chromosomes - growth in
  selective medium that requires maintenance of a
  particular human chromosome can lead to the
  production of somatic cell hybrid panels
  containing defined human chromosomes for genetic
  mapping
• hybridoma immortal cell line that produces a
  monoclonal (monospecific) antibody - produced
  from fusion of B-lymphocytes isolated from mouse
  spleens or lymph nodes (which together produce
  polyclonal antibodies following challenge with an
  antigen of interest), followed by clonal
  expansion and analysis of individual colonies for
  the production of the monoclonal antibody of
  interest

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Common Cell Types
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Production of Hybrid Cells
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Generation of Monoclonal Antibodies
MOVIE
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Fractionation of Cells
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Velocity and Equilibrium Sedimentation
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Chromatography
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Matrices Used for Chromatography
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Elution Profiles from different matrices
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SDS-PAGE
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SDS-PAGE
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MOVIE
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Isoelectric Focusing
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Peptide Mapping of Proteins
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CENTRAL DOGMA and GENE CLONING
chromosome
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3
gene
Untranslated region (UTR)
Untranslated region (UTR)
Coding region
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AAAAAAA
3
mRNA
FUNCTION
protein
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GENE CLONING DNA to PROTEIN
chromosome
MUTATION
DNA
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3
gene
cDNA
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AAAAAAA
3
mRNA
FUNCTION
protein
PROTEIN
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DNA CLONING
A method for identifying and purifying a
particular DNA fragment (clone) of interest from
a complex mixture of DNA fragments, and then
producing large numbers of the fragment (clone)
of interest.
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DNA CLONING TOOLS
RESTRICTION ENZYMES VECTORS DNA LIGASE COMPETENT
BACTERIAL CELLS ANTIBIOTICS
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DNA CLONING RESTRICTION ENZYMES

• RESTRICTION ENZYMES Bacterial proteins (enzymes)
  that cut DNA molecules at specific sequences
  (endonucleases).
• restriction site a specific 4- to 8-bp DNA
  sequences identified by a restriction enzyme
• restriction sites are typically short inverted
  repeat sequences
• restriction fragment a piece of DNA that is
  released from a larger piece of DNA (eg. genomic
  DNA) following digestion with one or more
  restriction enzymes
• several hundred different restriction enzymes
  are known, each with its own unique restriction
  site

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DNA CLONING RESTRICTION ENZYMES
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HindIII
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3
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DNA CLONING RESTRICTION ENZYMES OVERHANGS
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HindIII
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SmaI
KpnI
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DNA CLONING RESTRICTION MAPS
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DNA CLONING DNA LIGASE
-OH
P-
-P
OH-
2 ATP
DNA ligase ATP
2 AMP 2PPi
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DNA CLONING plasmid vectors
bacterial plasmid
E. coli
origin of replication (ori)
multiple cloning site (MCS) - HindIII - EcoRI -
KpnI - SmaI - BamHI - XbaI
ampicillin resistance gene (amp)
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DNA CLONING TRANSFORMATION
VECTOR


COMPETENT CELLS Chemically treated to enhance
DNA uptake
E. coli
TRANSFORMED BACTERIA
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DNA CLONING SELECTION

Luria Broth Agar Ampicillin
ONLY AMPICILLIN-RESISTANT (PLASMID-CONTAINING)
BACTERIA CAN GROW
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DNA CLONING LARGE SCALE GROWTH
millions of copies of the recombinant plasmid
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DNA CLONING PLASMIDS

• PLASMID A circular double-stranded DNA molecule
  that replicates in bacteria and is separate from
  the bacterial genome
• engineered to contain only sequences needed to
  function as a DNA cloning vector
• a bacterial origin of replication (ori)
• an antibiotic resistance gene (eg. B-lactamase
  confers resistance to ampicillin (amp))
• one or more unique restriction enzyme cutting
  sites which can be used to insert a piece of
  foreign DNA (MCS)
• may contain a B-galactosidase gene that is
  interrupted when DNA is inserted into the MCS
• may also contain promoters that drive expression
  of a foreign gene in either prokaryotic or
  eukaryotic cells

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Movie cloning
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cDNAs
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Clone Libraries
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Detection of specific RNA or DNA molecules by
gel-transfer hybridizationslide 1
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Detection of specific RNA or DNA molecules by
gel-transfer hybridizationslide 2
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DNA Sequencing
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Dideoxy-Sequencing (Sanger)
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Dideoxy-Sequencing (Sanger) contd
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Dideoxy-Sequencing (Sanger) contd

• MOVIE

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Reading Frames (6)
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Genes are found on either DNA strand
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Polymerase Chain Reaction (PCR) slide 1
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Polymerase Chain Reaction (PCR) slide 2
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Polymerase Chain Reaction (PCR) slide 3
MOVIE
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PCR Genomic or cDNA
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Technology allows you to move from protein to
gene and from gene to protein
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Fusion Proteins for Analysis of Function
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Fluorescence Energy Transfer (FRET)
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Affinity Coupled with Immunoprecipitation Tags
Facilitates the ID of Associated Proteins
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Yeast-Two-Hybrid Assay is used to discover
protein-protein interactions
MOVIE
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To study the function of proteins in vivo one
needs to identify mutants within the gene that
encodes your protein and evaluate the outcome.
Temperature Sensitive (TS) Mutants in Bacteria or
Yeast
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The use of TS-mutants in yeast identified
proteins that played critical roles in the export
of proteins
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Mutations introduce a phenotype

• MOVIE

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Single Nucleotide Polymorphisms (SNP) can be used
in Linkage Analysis to identify genes or
Chromosomal regions that are responsible for
inherited disorders
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DNA Microarrays monitor the expression of
thousands of genes in one experiment
MOVIE
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Cluster Analysis used to identify sets of genes
that are coordinately regulated
The expression of 8600 genes (Columns) were
analyzed under 12 time points. Red represents
increase in expression green a decrease relative
to untreated cells.
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Cells can be genetically engineered to carry
different types of mutations
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Embryonic Stem (ES) cells can be genetically
engineered and used to make a new animal
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The genetically engineered ES cells are used to
generate a chimeric animal, which is then used to
make completely ES-derived animals