Transcript Chapter08_MBP1022H

Chapter 8
 Manipulating
Proteins,
DNA, and RNA
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
CLONAL GROWTH
Mixed bacterial culture
Bacterial colonies on
agar plate
(each colony contains
~107 cells after ~12 h)
Clonal bacterial culture
• all bacteria are
genetically identical
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
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 cellECM 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.)
ANIMAL CELLS IN CULTURE
Tissue culture flask
Growth medium
Cells
Gelatin or collagen
substratum
37oC
5% CO2
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)
NORMAL AND TRANSFORMED CELLS
EARLY MITOTIC
SENESCENCE
CARCINOMA
TRANSFORMATION
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
Common Cell Types
Production of Hybrid Cells
Generation of Monoclonal Antibodies
MOVIE
Fractionation of Cells
Velocity and Equilibrium Sedimentation
Chromatography
Matrices Used for Chromatography
Elution Profiles
from different
matrices
SDS-PAGE
SDS-PAGE
MOVIE
Isoelectric Focusing
Peptide Mapping of Proteins
CENTRAL DOGMA and GENE CLONING
chromosome
enhancer promoter exon intron
5’
DNA
3’
gene
Untranslated
region (UTR)
RNA
5’
Coding region
Untranslated
region (UTR)
AAAAAAA 3’
mRNA
PROTEIN
protein
FUNCTION
GENE CLONING: DNA to PROTEIN
chromosome
MUTATION
5’
DNA
3’
gene
cDNA
5’
AAAAAAA 3’
mRNA
PROTEIN
protein
FUNCTION
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.
DNA CLONING TOOLS
RESTRICTION ENZYMES
VECTORS
DNA LIGASE
COMPETENT BACTERIAL
CELLS
ANTIBIOTICS
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
DNA CLONING: RESTRICTION ENZYMES
5’
AAGCTT
3’
TTCGAA
5’
A
3’
TTCGA
3’
HindIII
5’
AGCTT
3’
A
5’
DNA CLONING: RESTRICTION ENZYMES
OVERHANGS
5’
AAGCTT
3’
TTCGAA
5’
5’
CCCGGG
3’
3’
HindIII
3’
GGGCCC
5’
5’
GGTACC
3’
3’
CCATGG
5’
SmaI
KpnI
DNA CLONING: RESTRICTION MAPS
H
H KS
H K SS
H
S KK
HK S
HindIII digestion
H
H
H KS
H K SS
H
H
H
S KK
H
DNA CLONING: DNA LIGASE
5’
A
3’
TTCGA
AGCTT
3’
A
5’
2 ATP
DNA ligase
+
ATP
2 AMP +2PPi
5’
A AGCTT
3’
3’
TTCGA A
5’
DNA CLONING: plasmid vectors
bacterial
plasmid
E. coli
multiple
cloning
site (MCS)
origin of
replication
(ori)
- HindIII
- EcoRI
- KpnI
- SmaI
- BamHI
- XbaI
ampicillin resistance gene (amp)
DNA CLONING: TRANSFORMATION
“VECTOR”
+
+
“COMPETENT CELLS”
E. coli
Chemically treated to enhance
DNA uptake
“TRANSFORMED”
BACTERIA
DNA CLONING: SELECTION
+
Luria Broth Agar
+
Ampicillin
ONLY AMPICILLIN-RESISTANT (PLASMIDCONTAINING) BACTERIA CAN GROW
DNA CLONING: LARGE SCALE
GROWTH
millions of
copies of the
recombinant
plasmid
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
Movie cloning
cDNAs
Clone Libraries
Detection of specific RNA or DNA molecules
by gel-transfer hybridization
slide 1
Detection of
specific RNA or
DNA molecules
by gel-transfer
hybridization
slide 2
DNA Sequencing
Dideoxy-Sequencing (Sanger)
Dideoxy-Sequencing (Sanger) cont’d
Dideoxy-Sequencing (Sanger)
cont’d

MOVIE
Reading Frames (6)
Genes are found on either DNA
strand
Polymerase Chain Reaction
(PCR) slide 1
Polymerase Chain Reaction
(PCR) slide 2
Polymerase Chain Reaction
(PCR) slide 3
MOVIE
PCR: Genomic or cDNA
Technology allows you to move
from protein to gene and from gene
to protein
Fusion Proteins for Analysis of Function
Fluorescence Energy Transfer (FRET)
Affinity Coupled with Immunoprecipitation Tags
Facilitates the ID of Associated Proteins
Yeast-Two-Hybrid Assay
is used to discover protein-protein interactions
MOVIE
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
The use of TS-mutants in yeast identified
proteins that played critical roles in the
export of proteins
Mutations introduce a phenotype

MOVIE
Single Nucleotide Polymorphisms (SNP) can be used in
Linkage Analysis to identify genes or Chromosomal
regions that are responsible for inherited disorders
DNA
Microarrays
monitor the
expression of
thousands of
genes in one
experiment
MOVIE
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.
Cells can be genetically engineered to
carry different types of mutations
Embryonic
Stem (ES)
cells can be
genetically
engineered
and used to
make a new
animal
The genetically
engineered ES
cells are used to
generate a
chimeric animal,
which is then
used to make
completely ESderived animals