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

22

Genomic DNA

HIS4 mutation-

Yeast - genomic integration via homologous recombination

HIS4

gfY

pt Vector DNA

FunctionalHIS4 gene

DefectiveHIS4 gene

Yfg

tp

Genomic DNA

33

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

6

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

9

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:

13

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)

14

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

16

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

17

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

18

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)

21

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

22

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

26

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

27

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

28

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

33

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