1

Put a DNA regulatory region upstream of a reporter gene to analyze its elements

PCR

Space for res. enz. to bind(“elbow room”)

Reportergene

Transfect

Typical reporter vector features

Updated 11/15/10 4:51 PM

2

Popular reporters to study promoter/enhancers

• Beta-galactosidase (β-gal) – detection by several different assays

• Chloramphenicol acetyl transferase (CAT) – detected by a 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 (geneticin or G418

resistance)

• similarly: resistance to hygromycin, puromycin, histidinol, bleomycin, zeostin

• Dihydrofolate reductase (DHFR) – selectable in dhfr- cells, amplifiable, fusion proteins work

• Suicide selection: Herpes simplex virus thymidine kinase (HSVTK)

FACS = fluorescence-activated cell sorter

3

HSVTKGancilovir, ATP(non-toxic)

Gancilovir-PO4

Mammalian TKGangcylovir, ATP

toxicity, death

Use example: Site-directed recombination

Engineered chromosome: WT protein of interest HSVTK

lox

lox

Replacement plasmid:

Mut. protein of interest

gangcylovir

Mut. protein of interest

Select recombinants as HSVTK-, ganciclovir-resistant

Gangcyclovir selection AGAINST the presence of enzyme activity

(compare to 5-fluoro-orotic acid (FOA) resistance in yeast, URA3-)

CRE recombinase(cassette exchange)

(Ganciclovir itself is not toxic)

4

diacetylated

monoacetylated

Testing for a cell-specific promoter: chloramphenicol acetyl transferase (CAT) reporter assay

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

Applied Molec. Genet., U. Ariz

5

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:

6

Mapping transcriptional elements upstream of a promoter:

Mapping with restrictionenzyme mediated deletions

Conclusion:

Light units of luciferase in hepatocytes

luciferase reporter cDNA gene

Applied Molec. Genet., U. Ariz

7Footprinting: detects sites on DNA to which protein are bound

Naked DNA DNA + DNA-binding proteinP

opul

atio

n of

mol

ecul

es

missing

Pop

ulat

ion

of m

olec

ules

Partial DNase

Gel electrophoresis.autoradiography

Footprint

32P end-label(e.g., by phosphorylation of the 5’ OH with polynucleotide kinase and

gamma 32P-ATP)

Many unlabeled fragments are present but not seen

8

Note uneven cleavage of naked DNA by DNase

DNA footprint data

Note enhanced cleavage (sensitization) as well as protection

9

(EMSA = electrophoretic mobility shift assay)

(shift)

(supershift)

1 2 3 4 5

DNA element

Applied Molec. Genet. U. Arizona

Protein-DNA binding: EMSA or gel shift

(Even though the hexagon looks like a protein here)

competitor

10

http://brc.se.fju.edu.tw/protein/interact/binding.htm

(EMSA = electrophoretic mobility shift assay)

Protein-DNA binding: EMSA or gel shift

11

(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

12

SELEX 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)

Incubate with a protein of interest

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

Reiterate the cycle 5 to 10 times.If 99.9% efficient:enough

( = 1 microgram =~ several $100)

Last step: clone and sequence the “winners”

13

Binding to protein of interest

RThttp://www.molmed.uni-luebeck.de/T.%20Restle/Bilder/SELEX.jpg

Practical capacity ($700):

1014 random sequences

(random ~21-mer = 421)

14

= 3 x 1014 molecules, or enough for 30-fold coverage

10 ug

molecules= 3E+13

X 6.00E+17 molecules per umole

= 0.00005 umoles of the 60-mer

~20000 daltons19800 =X MW of a nucleotide=@330 ug/umole

60 nt

20 nt variable region + two 20 primer templates = 60 nt total length

420 = ~1013 unique sequences

15PUM2, a novel murine puf protein, and its consensus RNA-binding siteWhite 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 abbreviations (FYI)20-mer

16

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.

17

18Goal: to improve tissue plasminogen activator as a therapeutic “clot-busting” treatmentMeans: 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.

19

hepatocyte

mAb

mAb competes with hepatocyte for binding

tPA

tPAtPA

Kringle domain (~100 Aas)

K

K

K

20

Got this far(two topics through next graphic)

21

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 domainbut low binding to fluorescent mAb vs. kringle domain

22

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.

23

For reiteration of the process

Sort the cells with low fluorescence

,

24

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.

25

MAb to protease domain

MAb to kringle-1 domain

enriched

Low kringle-1 reactivity

FITC = fluorescein reagent. PE = phycoerythrin (fluorescent protein)

No good

good good good good

Collect these

26

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

27

WT

Compete. So still bind.

But still haveprotease activity

Hepatoma cell binding assay:measure competition for binding of fluorescently labeled WT TPA

Can’t compete (good)No competitor

Binding assay, initial condition

28

29