Transcript Class 17 - Columbia University

Nov. 8, 2011 1:00 AM
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Yeast Expression Vector (example)
Saccharomyces cerevisiae
2 mu seq features:
(baker’s yeast)
yeast ori
oriE = bacterial ori
Ampr = bacterial selection
LEU2, e.g. = Leu biosynthesis
for yeast selection
Complementation of
an auxotrophy can
be used instead of
drug-resistance
2μ = 2 micron plasmid
GAPD term’n
Your
favorite
gene
(Yfg)
LEU2
Auxotrophy = state of a mutant
in a biosynthetic pathway
resulting in a requirement for a
nutrient
For growth in E. coli
Ampr
GAPD prom
oriE
GAPD = the enzyme glyceraldehyde-3 phosphate dehydrogenase
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Yeast - genomic integration via homologous
recombination
t
p
Vector
DNA
gfY
HIS4
Genomic
DNA
Genomic
DNA
HIS4 mutation-
t
p
Yfg
Functional
HIS4 gene
Defective
HIS4 gene
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Double recombination Yeast (integration in Pichia pastoris)
HIS4
P. pastoris
-tight control
-methanol induced (AOX1)
-large scale production
(gram quantities)
Vector
DNA
AOX1t
Yfg
3’AOX1
AOX1p
Genomic
DNA
Alcohol oxidase gene
AOX1 gene (~ 30% of total protein)
Genomic
DNA
Yfg
AOX1p
AOX1t
HIS4
3’AOX1
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Primary cells cultured with a limited lifetime.
E.g.,
MEF = mouse embryonic fibroblasts, HDF = Human diploid fibroblasts
Mammalian cell lines (lines implies immortal)
Primary culture: human cells  < 50 generations (doublings). Then senescence.
Low frequency of survivors, increased by mutagens (carcinogens)
Mouse cells  earlier senescence, higher frequency of survivors
Human cells + 3 exogenous genes  tumor cells (ras, SV40 T, telomerase)
(Hahn et al., Creation of human tumour cells with defined genetic elements. Nature. 1999. 400:464-8)
Cell lines are typically aneuploid (abnormal number of and rearranged
chromosomes).
Often sub-tetraploid in number (human diploid chroosome number = 46, HeLa
cells ~69, or 82 etc. variable).
Expression in mammalian cells
Lab examples of immortal cell lines:
HEK293 Human embyonic kidney (high transfection efficiency)
HeLa
Human cervical carcinoma (historical, low RNase)
CHO
Chinese hamster ovary (hardy, diploid DNA content, mutants)
Cos
Monkey cells with SV40 replication proteins (-> high transgene copies)
3T3
Mouse or human exhibiting ~regulated (normal-like) growth
+ various others,
many differentiated to different degrees, e.g.:
BHK
Baby hamster kidney
HepG2 Human hepatoma
GH3
Rat pituitary cells
PC12
Mouse neuronal-like tumor cells
MCF7 Human breast cancer
HT1080 Human fibroblastic cells with near diploid karyotype
IPS
induced pluripotent stem cells
and:
Common in industry for production:
NS1
mAbs
Vero
vaccines
CHO
mAbs, other therapeutic proteins
PER6 mAbs, other therapeutic proteins
Mouse plasma cell tumor cells
African greem monkey cells
Chinese hamster ovary cells
Human retinal cells
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Mammalian cell expression
Generalized gene structure for mammalian expression:
polyA site
Mam.prom.
intron
5’UTR
Intron is
optional but
a good idea
cDNA gene
3’UTR
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Popular mammalian cell promoters
•
•
•
•
•
•
•
•
•
SV40 LargeT Ag (Simian Virus 40)
RSV LTR (Rous sarcoma virus)
MMTV (steroid inducible) (Mouse mammary tumor virus)
HSV TK (low expression) (Herpes simplex virus)
Metallothionein (metal inducible, Cd++)
CMV early (Cytomegalovirus)
Actin
EIF2alpha (EIF = eukaryotic initiation [of translation] factor)
Engineered inducible / repressible:
tet, ecdysone, glucocorticoid (tet = tetracycline)
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Engineered regulated expression:
Tetracycline-reponsive promoters
Tet-OFF (add tet  shut off)
Tet-OFF
tTA = tet activator fusion protein:
tetR = tet repressor (original role)
tetR
domain
VP16 transcription
activation domain
active
No tet.
Binds tet operator (multiple copies)
(if tet not also bound)
Tet-OFF
VP16 transcription
tetR
activation domain
domain
Allosteric
change in
conformation
Tetracycline (tet), or,
better, doxicyclin (dox)
not active
tTA gene must be in cell (permanent transfection, integrated):
polyA site
CMV
prom.
tTA cDNA
(Bujold et al.)
polyA site
Tet-OFF, cont.
MIN. CMV prom.
your favorite gene
Mutliple tet operator elements
No doxicyclin:
VP16 tc’n
tetR
domain act’n domain
active
Plenty of transcripton
MIN. CMV prom.
polyA site
your favorite gene
tetR
VP16 tc’n
domain act’n domain
Doxicyclin present:
MIN. CMV prom.
not active
little transcripton (2%?, bkgd)
polyA site
your favorite gene
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Tet-ON
Tetracycline-reponsive promoters
Tet-ON (add tet  turn on gene
Different fusion protein:
Does NOT bind tet operator
(if tet not bound)
tetR
VP16 tc’n
domain act’n domain
not active
tetR
VP16 tc’n
domain act’n domain
active
Tetracycline (tet), or,
better, doxicyclin (dox)
polyA site
Full CMV prom.
tTA cDNA
Must be in cell (permanent transfection, integrated):
commercially available (293, CHO) or do-it-yourself
Tet-ON
polyA site
MIN. CMV prom.
your favorite gene
Mutliple tet operator elements
tetR
VP16 tc’n
domain act’n domain
Doxicyclin absent:
not active
little transcription (bkgd.)
polyA site
MIN. CMV prom.
your favorite gene
Add dox:
VP16 tc’n
doxicyclin tetR
domain act’n domain
active
active
Plenty of transcripton (> 50X)
MIN. CMV prom.
your favorite gene
polyA site
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Biotechnology methods to study transcriptional regulation in cells
Mainly, use of reporter proteins whose cDNA sequence is linked to the promoter.
First, a synopsis of promoter structure:
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General model for transcriptional regulation in higher eukaryotes
Core transcriptional elements
TF… transcription factor
TBP: TATA binding protein
TAF: TBP associated protein
BRE: TFIIB response element
-35
INR: transcription initiator element
DPE: downstream promoter element
-28
GGGCGCC;
TATA(AT)AA(GA)
CCACGCC
YYAN(TA)YY
(AG)G(AT)(CT)(GAC)
Y = C or T (pyrimidine)
The transcription complex either recruits RNA Pol II or activates a bound RNA Pol II
For review see Smale and Katonga, Ann. Rev. Biochem. 72: 449-479 (2003)
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Many transcriptional enhancer elements often lie upstream of promoters,
allowing for many combinations of TF binding
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Put a DNA regulatory region upstream of a reporter gene to analyze its elements
Space for res. enz. to bind
PCR
Reporter
gene
Transfect
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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) – detection, sensitive radioactive assay
•
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
•
•
Neomycin phosphotransferase (neo)–selectable drug resistance (G418R)
(similarly: resistance to hygromycin, puromycin, histidinol, zeocin)
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Dihydrofolate reductase (DHFR) – selectable in dhfr- cells, amplifiable, fusion
proteins work
•
Suicide selection: Herpes simplex virus thymidine kinase (HSVTK)
FACS = fluorescence-activated cell sorter
Testing for a cell-specific promoter: chloramphenicol acetyl transferase (CAT)
reporter assay (www.biochem.arizona.edu/classes/bioc471/pages/Lecture15/Lecture15.html)
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
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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)
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X-gal (5-Bromo-4-chloro-3-indolyl-ß-D-galactoside) – blue precipitate - for
cytology or colony detection
•
Umbelliferyl–galactoside (-> umbelliferone, fluorescent, reading in a
fluorimeter allows more sensitive quantification than spectrophotometry)
•
Galacton-STAR or some such (-> chemiluminescent product = emission of
light, so lower background than fluorescence)
•
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:
Mapping with restriction
enzyme mediated deletions
Conclusion:
Light units of
luciferase in
hepatocytes
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Gancyclovir selection AGAINST the presence of enzyme activity
HSVTK
Gancilovir, ATP
Gancylovir, ATP
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 excnahge)
Mut. protein of interest
gancylovir
Mut. protein of interest
Select recombinants as HSVTK-, gancilovir-resistant
Further promoter characterization: binding speicificity
Footprinting: detects sites on DNA to which protein are bound
Population of
molecules
Population of
molecules
Naked DNA
DNA + DNA-binding protein
Partial DNase
missing
Gel
electrophoresis.
autoradiography
Footprint
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Note uneven cleavage
of naked DNA by DNase
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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)
U. Arizona
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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)
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SELEX
for protein binding sites
(T7 RNA Pol from an embedded
T7Pol promoter
Systematic Evolution of
Ligands by Exponential
Enrichment
(huge number)
Synthetic, range usually 6 to 40-mers
(usually a protein)
;
by PCR
(re-iterate 3-10 times)
Binding to Protein,
e.g.
Separate using nitrocellulose binding,
gel electrophoresis, etc.
sequences
 consensus
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Practical capacity:
1014 random sequences
(random ~21-mer = 421)
Re-adding the T7 promoter
sequence on the PCR primer
Binding to
protein of
interest
http://www.molmed.uniluebeck.de/T.%20Restle/
Bilder/SELEX.jpg
RT
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Binding site for a “puf “
protein, implicated in mRNA
degradation
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
Nucleic acid degenerate base abbreviations
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
9
Strong interaction (3 H bonds)
G|C
S
W
10
Weak interaction (2 H bonds)
A|T
W
B
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Not-A (B follows A)
G|T|C
V
D
12
Not-C (D follows C)
G|A|T
H
H
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Not-G (H follows G)
A|T|C
D
V
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Not-T (or U) (V follows U)
G|A|C
B
N,X
15
ANy nucleotide
G|A|T|C
N
-
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Gap of indeterminate length
Gap
-
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Got this far
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Measuring gene expression via RNA
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Northern blot
RNase protection
Primer extension
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RT-PCR
Q-RT-PCR
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Microarray
RNAseq
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Northern blotting
Denaturing gel for
true MW (urea,
formamide)
Alternative
polyadenylation
sites  2 dhfr
mRNAs
http://www.gene-quantification.de/mrna.html#northern
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RNase protection (RPA)
dhfr mRNA
Mutant-exon3
1-2-4-5-6
Wild type
1-2-3-4-5-6
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Primer extension: map the 5’ end of an mRNA
1
minor start
3
major start
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Cap trapping to isolate cDNAs that go to the 5’ end of the mRNA
First biotinylate the ribose residues that carry adjacent ring hydroxyls (diols):
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Next:
Use an XhoI-tailed
adapter-primer to copy
the RNA into cDNA
Full length
Truncated
Magnetic
avidin
beads
Use RNaseI to digest SS
RNA. Biotinylated 3’ end
cleaved. 5’ incomplete
cDNAs lose their capbiotin.
Isolate the surviving
capped DS molecules
with avidin beads.
Get rid of the RNA with
RNase A.
dG tail.
Make second strand with
SacI-tailed oligo dC
Cut with SacI and XhoI
and clone.
“Nanostrings” to quantify mRNA levels by single molecule counting
Geiss et al. Nat. Biotech. 26:317, 2008
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900 nt m13 segments
labeled with one of 4
fluorescent dyes.
Make a unique colorcode, ligate to 30-50- nt
mRNA-specific seq and
to a 5’ universal repeat.
Can make up to 800 of
these.
Strech out via electrophoresis
and then anchor far end.
Avidin coated
surface.
B=biotinylated
Ligate a universal 3’
repeat to the 3’ end
of an mRNA-specific
sequence (35-50 nt)
Fluorescent RNA: T7 promoted
transcription of m13 segment PCR
product using amino-allyl-UTP; then
conjugate to dye.
4 colors, 7 positions, 37=2100 [diff.
neighbors]
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Digital droplet PCR, or digital PCT, dPCR
Quantalife (Bio-Rad)
λ=average no. of occurrences
f= probability of k occurrences
For k=0, f0=e-λ
Observe f, calculate λ
Poisson
distribution:
PCR in droplets
Aqueous
microspheres
in water-in-oil
emulsion
Read + or –
in instrument
Data
Positive (green, here)
microspheres had >= 1 templates.
All positives have same intensity,
as PCR  plateau.
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Protein-protein interactions
Yeast 2-hybrid system
Yeast 3-hybrid and 1 hybrid systems
Co-immunoprecipitation
Pull-downs
Far western blots
Biacore (surface plasmon resonance, SPR)
Fragment complementation
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Measuring protein-protein interactions in vitro
X=one protein
Y= another protein
Pull-downs:
Binding between defined purified proteins, at least one being purified.
Tag each protein differently by making the appropriate cDNA clone.
Examples:
His6-X+HA-Y; bind to nickel or cobalt ion column via X, elute (imidazole), Western via
anti-HA Ab for Y
GST-X + HA-Y; bind to glutathione column, elute (glutathione), Western with anti-HA Ab
His6-X +
35S-Y
(made in vitro); bind Ni column, elute (imid.),  gel + autoradiography.
No antibody needed.
(HA = flu hemagglutinin)
glutathione = gamma-glutamyl-cysteinyl-glycine.
Example of a result of a pull-down experiment
Western blot
Total protein
Also identfy by MW
(or mass spec)
Antibody used in Western
Total protein: no antibody/Western
(stained with Coomassie Blue
or silver stain)
Compare pulled down fraction (eluted)
with loaded. Loaded sample usually only a fraction.
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