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1. Nov. 8, 2011 1:00 AM. 1. GAPD term’n. LEU2. GAPD prom. Amp r. ori E. Yeast Expression Vector (example). Saccharomyces cerevisiae (baker’s yeast). 2 mu seq features: yeast ori ori E = bacterial ori Amp r = bacterial selection LEU2, e.g. = Leu biosynthesis for yeast selection. - PowerPoint PPT Presentation
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111Yeast Expression Vector (example)
2μ = 2 micron plasmid
2 mu seq features:yeast orioriE = bacterial oriAmpr = bacterial selectionLEU2, e.g. = Leu biosynthesisfor yeast selection
Saccharomyces cerevisiae(baker’s yeast)
oriE
Your favorite
gene(Yfg)
LEU2
Ampr
GAPD term’n
GAPD prom
Complementation of an auxotrophy can be used instead of drug-resistance
Auxotrophy = state of a mutant in a biosynthetic pathway resulting in a requirement for a nutrient
GAPD = the enzyme glyceraldehyde-3 phosphate dehydrogenase
For growth in E. coli
Nov. 8, 2011 1:00 AM
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Genomic DNA
HIS4 mutation-
Yeast - genomic integration via homologous recombination
HIS4
gfY
pt Vector DNA
FunctionalHIS4 gene
DefectiveHIS4 gene
Yfg
tp
Genomic DNA
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Double recombination Yeast (integration in Pichia pastoris)
AOX1 gene (~ 30% of total protein)
Genomic DNA
AOX1p
Yfg
AOX1t HIS4 3’AOX1
Genomic DNA
HIS4
Yfg
AOX1p
AOX1t
3’AOX1
Vector DNA
P. pastoris-tight control-methanol induced (AOX1)-large scale production (gram quantities)
Alcohol oxidase gene
4
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).
5Expression in mammalian cellsLab 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 hepatomaGH3 Rat pituitary cellsPC12 Mouse neuronal-like tumor cellsMCF7 Human breast cancerHT1080 Human fibroblastic cells with near diploid karyotypeIPS induced pluripotent stem cells and:
Common in industry for production:NS1 mAbs Mouse plasma cell tumor cellsVero vaccines African greem monkey cellsCHO mAbs, other therapeutic proteins Chinese hamster ovary cellsPER6 mAbs, other therapeutic proteins Human retinal cells
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Mammalian cell expression
Generalized gene structure for mammalian expression:
cDNA geneMam.prom.
polyA site
intron
5’UTR3’UTR
Intron is optional but a good idea
7
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)
8Engineered regulated expression:Tetracycline-reponsive promotersTet-OFF (add tet shut off)
tTA cDNA
tTA = tet activator fusion protein:tetR = tet repressor (original role)
tetRdomain
VP16 transcriptionactivation domain
No tet.Binds tet operator (multiple copies)(if tet not also bound)
tetRdomain
Tetracycline (tet), or,better, doxicyclin (dox)
active
not active
CMV prom.
polyA sitetTA gene must be in cell (permanent transfection, integrated):
Tet-OFF
Tet-OFF
(Bujold et al.)
Allosteric change in conformation
VP16 transcriptionactivation domain
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MIN. CMV prom. your favorite gene
polyA site
Mutliple tet operator elements
MIN. CMV prom. your favorite gene
polyA site
tetRdomain
VP16 tc’nact’n domain
not activelittle transcripton (2%?, bkgd)
Doxicyclin present:
MIN. CMV prom. your favorite gene
polyA siteactivePlenty of transcripton
No doxicyclin:
tetRdomain
VP16 tc’nact’n domain
RNA po l
Tet-OFF, cont.
10
Tetracycline-reponsive promotersTet-ON (add tet turn on gene
tTA cDNA
tetRdomain
VP16 tc’nact’n domain
tetRdomain
VP16 tc’nact’n domain
Tetracycline (tet), or,better, doxicyclin (dox)
active
not active
Full CMV prom.
polyA site
Different fusion protein: Does NOT bind tet operator(if tet not bound)
Tet-ON
Must be in cell (permanent transfection, integrated): commercially available (293, CHO) or do-it-yourself
11
MIN. CMV prom. your favorite gene
polyA site
Mutliple tet operator elements
MIN. CMV prom. your favorite gene
polyA site
active
Doxicyclin absent:
MIN. CMV prom. your favorite gene
polyA siteactivePlenty of transcripton (> 50X)
Add dox:
tetRdomain
VP16 tc’nact’n domain
RNA pol II
Tet-ON
tetRdomain
VP16 tc’nact’n domain
not active little transcription (bkgd.)
doxicyclin
12
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
TF… transcription factorTBP: TATA binding proteinTAF: TBP associated proteinBRE: TFIIB response element
INR: transcription initiator elementDPE: downstream promoter element
The transcription complex either recruits RNA Pol II or activates a bound RNA Pol II
Core transcriptional elements
-28-35
For review see Smale and Katonga, Ann. Rev. Biochem. 72: 449-479 (2003)
GGGCGCC; CCACGCC
TATA(AT)AA(GA) YYAN(TA)YY
Y = C or T (pyrimidine)
(AG)G(AT)(CT)(GAC)
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Many transcriptional enhancer elements often lie upstream of promoters,allowing for many combinations of TF binding
15
Put a DNA regulatory region upstream of a reporter gene to analyze its elements
PCR
Space for res. enz. to bind
Reportergene
Transfect
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Popular reporters to study promoter/enhancers
• Beta-galactosidase (β-gal) – detection by several different assays
• Chloramphenicol acetyl transferase (CAT) – detection, sensitive radioactive assay
• Luciferase (firefly, Renilla [jellyfish]) – detection, easy dual, sensitive luminescent assay
• 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)
• Dihydrofolate reductase (DHFR) – selectable in dhfr- cells, amplifiable, fusion proteins work
• Suicide selection: Herpes simplex virus thymidine kinase (HSVTK)
FACS = fluorescence-activated cell sorter
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diacetylated
monoacetylated
Testing for a cell-specific promoter: chloramphenicol acetyl transferase (CAT) reporter assay (www.biochem.arizona.edu/classes/bioc471/pages/Lecture15/Lecture15.html)
Thin layer chromatography (TLC)
CAT cDNA is from a prokaryotic source. CAT is not foundin mammalian cells.Therefore low backgrounds
A B
14C-chloramphenicol
unacetylatedPositive controlNegative control
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Reporter enzyme substrates for different purposes
• 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
• 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.
Substrates for beta-galactosidase, for example:
19
Mapping transcriptional elements upstream of a promoter:
Mapping with restrictionenzyme mediated deletions
Conclusion:
Light units of luciferase in hepatocytes
20
HSVTKGancilovir, ATP Gancilovir-PO4
Mammalian TKGancylovir, ATP
toxicity, death
Use example: Site-directed recombination
Engineered chromosome: WT protein of interest HSVTK
lox
lox
Replacement plasmid:
Mut. protein of interest
gancylovir
Mut. protein of interest
Select recombinants as HSVTK-, gancilovir-resistant
Gancyclovir selection AGAINST the presence of enzyme activity
CRE recombinase(cassette excnahge)
(Ganciclovir itself is not toxic)
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Footprinting: detects sites on DNA to which protein are bound
Naked DNA DNA + DNA-binding protein
Pop
ulat
ion
of m
olec
ules
missing
Pop
ulat
ion
of m
olec
ules
Partial DNase
Gel electrophoresis.autoradiography
Footprint
Further promoter characterization: binding speicificity
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Note uneven cleavage of naked DNA by DNase
23
(EMSA = electrophoretic mobility shift assay)
(shift)
(supershift)
1 2 3 4 5
DNA element
U. Arizona
Protein-DNA binding: EMSA or gel shift
(Even though the hexagon looks like a protein here)
competitor
24
(surpershifted complex is not competed by NON-specific probe)
(competed only by specific probe)
(two molecules of protein bound)
Protein DNA complexes migrate more slowly than naked DNA
Gel shifts (EMSA
Super-shift
25
SELEX
Binding to Protein,e.g.
sequences consensus
by PCR
Synthetic, range usually 6 to 40-mers (huge number)
Separate using nitrocellulose binding, gel electrophoresis, etc.
(re-iterate 3-10 times)
(usually a protein)
(T7 RNA Pol from an embedded T7Pol promoter
;
for protein binding sites
Systematic Evolution of Ligands by Exponential Enrichment
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Binding to protein of interest
RThttp://www.molmed.uni-luebeck.de/T.%20Restle/Bilder/SELEX.jpg
Practical capacity:
1014 random sequences
(random ~21-mer = 421)Re-adding the T7 promoter sequence on the PCR primer
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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.
Consensus:
Binding site for a “puf “ protein, implicated in mRNA degradation
Code
Integer
Base Name Meaning
Complement
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 11 Not-A (B follows A) G|T|C V
D 12 Not-C (D follows C) G|A|T H
H 13 Not-G (H follows G) A|T|C D
V 14 Not-T (or U) (V follows U) G|A|C B
N,X 15 ANy nucleotide G|A|T|C N
- 16 Gap of indeterminate length Gap -
Description
Nucleic acid degenerate base abbreviations20-mer
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Got this far
29
Measuring gene expression via RNA
• Northern blot• RNase protection• Primer extension
• RT-PCR• Q-RT-PCR
• Microarray• RNAseq
30
http://www.gene-quantification.de/mrna.html#northern
Alternative polyadenylation sites 2 dhfr mRNAs
Northern blotting
Denaturing gel for true MW (urea, formamide)
31
RNase protection (RPA)
1-2-3-4-5-61-2-4-5-6
Mutant-exon3 Wild type
dhfr mRNA
32
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Primer extension: map the 5’ end of an mRNA
1
3 major start
minor start
34
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):
35
Full length
Truncated
Magnetic avidin beads
Next:
Use an XhoI-tailed adapter-primer to copy the RNA into cDNA
Use RNaseI to digest SS RNA. Biotinylated 3’ end cleaved. 5’ incomplete cDNAs lose their cap-biotin.
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.
36“Nanostrings” to quantify mRNA levels by single molecule counting
900 nt m13 segmentslabeled with one of 4fluorescent dyes. Make a unique color-code, ligate to 30-50- nt mRNA-specific seq and to a 5’ universal repeat.Can make up to 800 of these.
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]
Strech out via electrophoresis and then anchor far end.
Avidin coated surface.B=biotinylated
Geiss et al. Nat. Biotech. 26:317, 2008
37
Digital droplet PCR, or digital PCT, dPCRQuantalife (Bio-Rad)
PCR in droplets
Read + or – in instrument
Data
λ=average no. of occurrencesf= probability of k occurrencesFor k=0, f0=e-λ
Observe f, calculate λ
Poisson distribution:
Aqueous microspheres in water-in-oil emulsion
Positive (green, here) microspheres had >= 1 templates. All positives have same intensity, as PCR plateau.
38
Protein-protein interactions
Yeast 2-hybrid systemYeast 3-hybrid and 1 hybrid systemsCo-immunoprecipitationPull-downsFar western blotsBiacore (surface plasmon resonance, SPR)Fragment complementation
39
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.
40Example of a result of a pull-down experiment
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.
Also identfy by MW (or mass spec)
Western blot Total protein