of the chloramphenicol acetyl transferase assay
2. Take 40 pL of cell extract, add 2 yL O.lM DTT, 6 pL glycerol and 500 pL of
60 mM Na2HP04, 40 mM NaH*PO,, 2H20, 1 mM MgS04 7H,O, 10 mM KCl,
40 mM P-mercaptoethanol. Vortex and incubate for 5 mm.
3 Add 100 pL (2 mg/mL) ONPG (O-nitrophenyl-P-o-galactopyranoside), and
incubate the reaction at 37Â°C until a visible yellow color 1s achieved. (This can
take from 5 min to 24 h.)
4 Stop the reaction with 250 uL lMNa&!O,.
5. Measure the colorometric change in a spectrophotometer at 420 nm.
18.104.22.168 BRADFORD ASSAY FOR PROTEIN CONCENTRATION (20)
1. Followmg transfectton, the cells are harvested and then lysed as described The
lysed cells are then spun to remove the cell debris and an estimate of protein
concentration can be performed on the supernatant to allow the assay of chloram-
phenicol acetyl transferase or P-galactosidase to be carried out on equal amounts
of total protein.
2. Add 1 mL of dye reagent to 5 pL of each sample.
3. Measure the absorbance of the sample at 595 nm after 15 min.
4. If the absolute concentration of protein is needed, then a standard curve can be
constructed using BSA as standard (draw As95vs [BSA] mg/mL)
Once a region of the promoter that can confer a pattern of regulation on another
gene has been identified, the promoter can be truncated until the effect IS lost
allowing identification of the precise region of the promoter that confers this
effect (see, for example, ref. 21). Subsequently, this region can be cloned into a
vector m which a heterologous promoter drives the CAT gene in order to determme
if rt can confer a specific pattern of regulation on a heterologous promoter (22)
3.2. Identification of Cellular Transcription Factors
The ability of a partrcular region of the gene promoter to produce a specific
pattern of gene expression normally 1s dependent on its abihty to bind one or
more specific transcrrptron factors that are present only in a specific cell type,
that are activated m response to a particular strmulus, or that can interact wtth
a vu-ally encoded regulatory protein (for review, see ref. 23). Hence, once a
region of the promoter that produces a particular pattern of regulatton has been
identified, It is necessary to identify the transcription factors that bind to rt so
they can be characterized and then activity m different cell types and under
different conditrons investigated. To do this, whole-cell or nuclear extracts con-
taunng these factors are prepared and their bmdmg to the specific DNA
sequences investigated by several different techniques.
3 2 7 Preparation of Cellular Extracts
Extracts can be prepared either from whole cells or from isolated nuclei. All
procedures are carried out at 4Â°C and the extracts stored at -70Â°C after preparatton.
3 2.1 .l . PREPARATION OF NUCLEAR EXTRACTS (24)
Harvest 5 x lo7 to 10â€™ cells and wash them with PBS.
2 Resuspend the cells m 5 vol of hypotomc buffer A and protease mhibitors, 0 5
mM PMSF 1 ug/mL pepstatin A, 1 pg/mL aprotmm, and 10 mA4 P-glycero-
phosphate, and stand them on ice for 10 mm
3 Spm the cells at 1OOOgfor 10 mm and resuspend m 3 vol of buffer A
4 Add NP40 to 0.05% and homogenize the cells wrth 20 strokes m a tight-fittmg
5 Check the samples for release of nuclei by phase-contrast microscopy.
6. Spin at 1OOOgfor 10 mm to pellet the nuclet
7 Resuspend the nuclei m 1 mL buffer C with protease mhrbltors as m step 2
8 Measure the volume of solutton and add NaCl to 400 mM Incubate the solution
on ice for 30 mm
9 Spm at 16,000g for 20 mm at 4â€™C m a refrigerated mtcrofuge
Ahquot the supematant and snap-freeze m hqutd nitrogen. Store ahquots at -70Â°C
22.214.171.124. PREPARATION OF WHOLE-CELL EXTRACTS (25)
1 Resuspend the cells m buffer C.
2 Homogenize the cells wtth 20 strokes in a tight-fitting Dounce homogenizer
3 Add NaCl to a final concentratton of 400 &and incubate the solution on tee for 30 mm.
4 Spin at 16,OOOgfor 20 mm at 4Â°C m a refrigerated mtcrofuge.
5. Snap-freeze supernatant ahquots and store them at -70Â°C
HSV-Cellular Protein Interactions
Protem binds to DNA
Fig. 1. DNA mobility shift assay m which the bmdmg of a protein (B) to a radtoac-
tlvely labeled DNA sequence is detected by its ability to form a slow moving complex
with the DNA
3.2.2. DNA Mobility Shift Assay
Once extracts have been prepared from the cells of interest, they can be used
in a number of ways to study the proteins binding to a particular sequence. The
simplest of these IS the gel retardation or DNA mobility shift assay(26,27) that
relies on the principle that a DNA fragment to which a protein has bound will
move more slowly in gel electrophoresis than the same fragment without bound
protein. Hence, the proteins binding to a particular DNA fragment can be
investigated by radioactively labeling the fragment, incubating it with cell
extract and electrophoresmg on a nondenaturing gel. The retarded DNA-
protein complexes can then be visualized by autoradrography (Fig. 1). More-
over, by carrying out the assayusing protems prepared from different cell types
or from the same cell type under different condrtrons, the nature of the factors
binding to a specrfic piece of DNA in different situattons can be determined.
Once the protems binding to this sequence have been identified in this way,
it 1s possible to investigate the precise sequence specifictty of this binding.
This IS achieved by Including a large excessof an unlabeled ohgonucleotide of
specific sequence m the bmding reaction. If the protein bindmg to the labeled
ohgonucleotide can also bmd to this unlabeled ohgonucleotide it will do so
and the retarded band will disappear. The sequence spectficity of a novel btnd-
ing protein that can be determined m this way, may provide clues about its
relationship to previously characterized transcription factors with identical or
related DNA-binding specificittes. Further information about the relationship
of a novel factor to known factors can also be obtained by including antibody
to a previously characterized factor m the binding reactton. If this antibody
reacts with the protein of interest, it will either prevent its bmdmg to DNA, so
abolishing the complex, or produce a so-called supershift of the complex by
binding to the DNA-bound protein and decreasing the mobihty of the complex.
Similarly, the interaction of a viral protein with the cellular protein can be
followed either by comparing the patterns obtained from infected or uninfected
cells or by adding a small amount of the purified viral protein and mvestigating
whether a super-shifted complex containing the anttbody IS formed (see, for
example, ref. 4).
3 2.2 1. LABELING OF OLIGONUCLEOTIDE PROBES
FOR DNA MOBILITY SHIFT ASSAY
1 Anneal the separately synthesized strands of the ollgonucleotide by heating
eqmmolar amounts of each to 80Â°C for 2 mm, and then coolmg slowly to
2. Incubate 2 pmol of annealed ohgonucleotide with 20 pCr gamma [32P]ATP m 50
mM Tris-HCl, pH 7.6, 10 mM MgCl,, 5 mMDTT, 0.1-d EDTA and 4 U of T4
kmase at 37Â°C for 30 mm.
3. Separate the labeled ohgonucleotrde from the free probe on a Sephadex G25 col-
umn and recover the void volume m 200 pL of STE One microliter of probe
should be sufficient for each band shift incubation.
3 2.2.2. DNA MOBILITY SHIFT ASSAY
1 Set up a 20-p.L binding reaction containing 4% Ficoll, 20 mM HEPES pH 7 9,
1 mM MgCl,, 0.5 mM DTT, 50 mM KCl, 2 pg poly dIdC (Pharmacta, Uppsala,
Sweden), 10 fmol double-stranded end-labeled ohgonucleotide or DNA fragment
probe and approx 2 pg of whole-cell or nuclear protein extract.
2. Incubate on ice for 40 min.
3. Load on to 4% polyacrylamrde:bu-acrylamide (29: 1) gel in 0.25X TBE and run
in 0.25X TBE at 150 V for approx 2â€™/2 h
4 Dry the gel under vacuum onto 3MM paper (Whatmann, Mamstone, UK) and
HS V-Cellular Protein Interactions 265
DNA fragment labelled at one end
10 20 30 40
* ! I I
DNase I dIgestIon
bound between 10
and 20 bases from
Fig. 2. DNase I footprinting assay in which the region bound by a protein 1sldentl-
fied by Its resistance to digestion by DNase I.
3.2.3. DNase I Footprinting Assay
Having identified a protein binding to a specific DNA sequence, the area of
contact between the DNA and protein can be localized by using the same
extracts to carry out a DNase I footprinting assay(28,29). To do this, a double-
stranded DNA fragment labeled at only one end is incubated wtth the protem
extract and then digested with a small amount of DNase I. Each molecule
will be cut only once or a very few times by the enzyme, giving rise to a ladder
of bands when the sample is run on a denaturing gel. Regions where protein
has bound to the DNA, however, will be protected from digestion and hence
will appear as a blank area or footprint on the gel (Fig. 2).
1 Set up 100˜pL bindmg reactions as for band-shift assay Incubate on ice for 40 mm.
2 Dilute a 2-ug/uL stock of DNase I 1.100 immediately before use in buffer
Add 1 pL to each sample and incubateat room temperaturefor a carefully timed
3 Stop the reaction by adding 100pL 50 mM Trts-HCI, pH 8 0, 2% SDS, 10 mA4
EDTA, 10 pg glycogen, 0.4 mg/mL protemaseK Incubate at 37Â°C for 30 mm,
and then at 70Â°C for 2 mm
4 Extract the reaction with phenol chloroform (1 1) and then with chloroform And
15 uL 5M LiCl and 600 uL ethanol and leave the samples overnight at -20Â°C to
precrprtate the DNA
5 Spin down the DNA and resuspend it m 5-10 uL of sample loading buffer. Load
20-50 counts per second in each well and run on a 6% denaturmg poly-
acrylamide:bu-acrylamide (19: 1) gel containing 1X TBE and 42% urea (w/v)
6. Dry the gel under vacuum, and autoradiograph
3.2.4. Methylation Interference Assays
The mteraction between a DNA-bmdmg protein and its specific DNA-bmd-
ing site can be more precisely studied using the methylation interference assay
m which the effect on the binding of the protein of methylatmg specific G
residues m its bmdmg site 1s assessed(30). This method allows the precise
assessmentof the mteraction of the DNA bmding protein with mdividual nucle-
otides within its binding site. To do this, the DNA is partially methylated so
that on average only one G residue per DNA molecule is methylated, and used
m a standard DNA mobility shift assay. Followmg electrophoresis, the DNA
that has bound protein and that which does not are both excised from the gel,
and their level of methylation at specific G restdues compared by cleaving
methylated Gs wtth ptperidme (31). A lack of methylated G residues at a par-
ticular site tn the protein-bound DNA mdicates that methylatton at this G blocks
protem binding, and that it therefore plays a critical role in protein bmdmg (Fig. 3).
Methylation interference can therefore be used as a supplement to DNase I
footprmting by identifymg the precise protein: DNA mteractions within the
1, To prepare parttally methylated probe, add end-labeled DNA to 200 uL 50 mM
sodium cacodylate pH 8 0, 1 mM EDTA
2. Chill on ice and add 1 nL dimethyl sulfate (DMS) Incubate at 20Â°C for 3 min
3. Add 2 5 uL 3M sodium acetate pH 7 0 and 600 uL ethanol and incubate at -2Oâ€™C
overnight to precipitate the DNA.
4 Followmg centrifugatron, dtscard the DMS-contammg supernatant mto a 5M
NaOH solution. Wash the pellet with 70% ethanol, dry tt, and resuspend tt m
10 pL of water.
5 Carry out a 120-200 pL DNA mobility shift reaction using 50-100 fmol of par-
tially methylated probe (approx 4 x lo5 cpm).
6. Incubate the samples for 1 h on Ice and load them onto three to five wells of a
HS VLCellular Protem Interactions