Transcript Document

Updated 11/15/10 4:51 PM
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Put a DNA regulatory region upstream of a reporter gene to analyze its elements
Space for res. enz. to bind
(“elbow room”)
PCR
Reporter
gene
Transfect
Typical reporter vector features
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Popular reporters to study promoter/enhancers
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Beta-galactosidase (β-gal) – detection by several different assays
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Chloramphenicol acetyl transferase (CAT) – detected by a sensitive radioactive
assay
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Luciferase (firefly, Renilla [jellyfish]) – detection, easy dual, sensitive luminescent
assay
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Green fluorescent protein (GFP, BFP, YFP)) – cytological, visible in living cells,
fusion proteins, FACS
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Neomycin phosphotransferase (neo)–selectable drug resistance (geneticin or
G418 resistance)
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similarly: resistance to hygromycin, puromycin, histidinol, bleomycin, zeostin
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Dihydrofolate reductase (DHFR) – selectable in dhfr- cells, amplifiable, fusion
proteins work
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Suicide selection: Herpes simplex virus thymidine kinase (HSVTK)
FACS = fluorescence-activated cell sorter
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Gangcyclovir selection AGAINST the presence of enzyme activity
(compare to 5-fluoro-orotic acid (FOA) resistance in yeast, URA3-)
Gancilovir, ATP
(non-toxic)
Gangcylovir, ATP
HSVTK
Gancilovir-PO4
Mammalian TK
toxicity, death
(Ganciclovir itself is not toxic)
lox
lox
Use example: Site-directed recombination
Engineered chromosome:
Replacement plasmid:
WT protein of interest
HSVTK
CRE recombinase
(cassette exchange)
Mut. protein of interest
gangcylovir
Mut. protein of interest
Select recombinants as HSVTK-, ganciclovir-resistant
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Testing for a cell-specific promoter:
chloramphenicol acetyl transferase (CAT) reporter assay
CAT cDNA is from a
prokaryotic source.
CAT is not found
in mammalian cells.
Therefore low backgrounds.
A
Thin layer
chromatography (TLC)
B
diacetylated
14C-chloramphenicol
monoacetylated
Positive control
Negative control
unacetylated
Applied Molec. Genet., U. Ariz
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Reporter enzyme substrates for different purposes
Substrates for beta-galactosidase, for example:
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ONPG (ortho-nitrophenyl-beta-galactoside) – spectrophotometric
measurement (420 nm – blue color – simplest)
•
X-gal (5-Bromo-4-chloro-3-indolyl-ß-D-galactoside) – blue precipitate - for
cytology or colony detection
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Umbelliferyl–galactoside (-> umbelliferone, fluorescent, reading in a
fluorimeter allows more sensitive quantification than spectrophotometry)
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Galacton-STAR or some such (-> chemiluminescent product = emission of
light, so lower background than fluorescence)
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Lactose (glucose-beta-galactose disaccharide) – allows growth if
hydrolyzed; growth phenotype. For microbial cells usually.
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Mapping transcriptional
elements upstream of a
promoter:
Light units of
luciferase in
hepatocytes
Mapping with restriction
enzyme mediated deletions
luciferase reporter
cDNA gene
Conclusion:
Applied Molec. Genet., U. Ariz
Footprinting: detects sites on DNA to which protein are bound
DNA + DNA-binding protein
Population of
molecules
Naked DNA
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32P end-label
(e.g., by phosphorylation
of the 5’ OH with
polynucleotide kinase and
gamma 32P-ATP)
Population of
molecules
Partial DNase
missing
Gel
electrophoresis.
autoradiography
Footprint
Many unlabeled fragments are
present but not seen
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DNA footprint data
Note uneven cleavage
of naked DNA by DNase
Note enhanced
cleavage (sensitization)
as well as protection
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Protein-DNA binding: EMSA or gel shift
(EMSA = electrophoretic mobility shift assay)
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2
3
4
5
competitor
(supershift)
(shift)
DNA element
(Even though the hexagon looks like a protein here)
Applied Molec. Genet. U. Arizona
Protein-DNA binding: EMSA or gel shift
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(EMSA = electrophoretic mobility shift assay)
http://brc.se.fju.edu.tw/protein/interact/binding.htm
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Protein DNA complexes
migrate more slowly than
naked DNA
Gel shifts (EMSA
(competed only by specific probe)
(two molecules
of protein bound)
Supershift
(surpershifted
complex is not
competed by NONspecific probe)
SELEX
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for protein binding sites on nucleic acids
Systematic Evolution of Ligands by Exponential Enrichment
Synthesize ~1014 oligomers with a random central section (e.g., 20-mer=1013)
( = 1 microgram =~ several $100)
Incubate with a
protein of interest
Reiterate the cycle
5 to 10 times.
If 99.9% efficient:
enough
Separate the protein-DNA
complexes from the free DNA:
e.g., using EMSA, IP,
nitrocellulose, protein on beads
in a column
Dissociate complex,
PCR amplify the bound
DNA fraction
Last step: clone and sequence the “winners”
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Practical capacity ($700):
1014 random sequences
(random ~21-mer = 421)
Binding to
protein of
interest
http://www.molmed.uniluebeck.de/T.%20Restle/
Bilder/SELEX.jpg
RT
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20 nt variable region
total length
420
=
+ two
20 primer templates = 60 nt
~1013 unique sequences
60 nt X MW of a nucleotide=
@330 ug/umole
19800 =
~20000 daltons
= 0.00005 umoles of the 60-mer
X 6.00E+17 molecules per umole
= 3E+13 molecules
10 ug = 3 x 1014 molecules, or
enough for 30-fold coverage
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PUM2, a novel murine puf protein, and its
consensus RNA-binding site
White EK, Moore-Jarrett T, Ruley HE. RNA.
2001 Dec;7(12):1855-66.
20-mer
Consensus:
Description
Binding site for a “puf “ protein,
implicated in mRNA degradation
Nucleic acid degenerate base abbreviations (FYI)
Cod
e
Intege
r
Base Name
Meanin
g
Complemen
t
A
1
Adenine
A
T
C
2
Cytosine
C
G
G
3
Guanine
G
C
T
4
Thymine
T
A
U
4
Uracil
U
A
R
5
(PuRine)
G|A
Y
Y
6
(PYrimidine)
T|C
R
K
7
(Keto)
G|T
M
M
8
(AMino)
A|C
K
S
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Strong interaction (3 H bonds)
G|C
S
W
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Weak interaction (2 H bonds)
A|T
W
B
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Not-A (B follows A)
G|T|C
V
D
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Not-C (D follows C)
G|A|T
H
H
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Not-G (H follows G)
A|T|C
D
V
14
Not-T (or U) (V follows U)
G|A|C
B
N,X
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ANy nucleotide
G|A|T|C
N
-
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Gap of indeterminate length
Gap
-
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TPA = Tissue plasminogen activator, dissolves clots
Problem: Cleared quickly from bloodstream by liver
Bind to hepatocytes in liver via TPA’s kringle domain
Want to isolate a TPA mutant protein with less affinity for hepatocytes
Must be still enzymatically active of course.
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Goal: to improve tissue plasminogen activator as a therapeutic “clot-busting” treatment
Means:
Reduce or eiminate the binding of tPA to liver cells, as this clears it from the blood
Authors here use a mammalian cells as the carrier of the DNA
and the cell surface as a display site.
Display was via a fusion protein to a membrane anchor protein, DAF (peptide, really).
DAF = “decay accelerating factor”
What did they do?
Cassette mutagenesis.
What region?
333 bp K1 (kringle-1), known to bind the MAb387, which competes for hepatocyte
binding (so assuming it is the same target epitope).
How did they get kringle mutated?
Error-prone PCR
How did they isolate just the kringle 1 region?
PCR fragment.
How did they get the mutagenized fragment back in?
Introduced restriction sites at the ends, w/o affecting the coding.
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mAb competes with hepatocyte for binding
mAb
K
tPA
tPA
hepatocyte
K
K
tPA
Kringle domain (~100 Aas)
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Got this far
(two topics through next graphic)
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What did they put the mutagenized fragment into?
DAF – TPA fusion protein gene
How did they get it into into cells?
Electroporation
What cells did they use as hosts?
293 carrying SV40 large T antigen
How many copies per cell. And why is that important?
One, by electroporation at low DNA concentration.
[In a transient transfection!]
Binding is dominant. Lack of binding (what they are after) is recessive.
How did they select cells making MAb387-non-binding TPA?
FACS:
Recover cells that bind fluorescent mAb vs. protease domain
but low binding to fluorescent mAb vs. kringle domain
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Tracked down vector: contains SV40 ori and is transfected into 293 cells making SV40
T-antigen. So plasmid replicates during the transient transfection  higher signal.
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,
Sort the cells with
low fluorescence
For
reiteration
of the
process
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How did they recover the plasmid carrying the mutant TPA gene
from the selected cells?
Hirt extraction: Like a plasmid prep, lyse cells gently, high MW DNA entangles and
forms a “clot”.
Centrifuge. Chromosomal DNA  soft pellet; plasmid DNA circles stay in supernatant.
Then re-transfect, re-sort in FACS.
After 2 sorting rounds, test individual E. coli clones: 60% are binding-negative.
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MAb to protease domain
enriched
Collect these
No good
good
good
good
good
Low kringle-1 reactivity
MAb to kringle-1 domain
FITC = fluorescein reagent.
PE = phycoerythrin (fluorescent protein)
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Hepatoma cell binding. How?
Clone mutated regions into regular TPA gene for testing
(no DAF, protein now secreted)
Label WT TPA with fluorescein (FITC, conjugated chemically)
Mix with hepatoma cells and analyze on a flow cytometer (FACS w/o the
sorter part).
See specific and non-specific binding. Subtract non-specific binding:
the amount not competed by excess un-labeled wt TPA.
FITC = fluorescein isothiocyanate
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Hepatoma cell binding assay:
measure competition for
binding of fluorescently
labeled WT TPA
Binding assay,
initial condition
Can’t compete (good)
No competitor
WT
Compete.
So still bind.
But still have
protease activity
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