NOTES & TIPS 127

GSK-3 activity. Inclusion of 20 nM wortmannin 11. VandenHeede, J. R., Yang, S. D., Goris, J., and Merlevede, W.
blocked the effects of insulin and gave the same kinase (1980) J. Biol. Chem. 255, 11768 –11774.
activity as the untreated control. Treatment with 800 12. Hemmings, B. A., Yellowlees, D., Kernohan, J. C., and Cohen, P.
(1981) Eur. J. Biochem. 119, 443– 451.
nM PMA, a PKC activator, also caused a 50% decrease
13. Park, I. K., and DePaoli-Roach, A. A. (1994) J. Biol. Chem. 269,
in kinase activity. These results are as expected from 28919 –28928.
previous experiments (27) and validate our assay. 14. Hanger, D. P., Hughes, K., Woodgett, J. R., Brion, J. P., and
Anderton, B. H. (1992) Neurosci. Lett. 147, 58 – 62.
SUMMARY 15. Boyle, W. J., Smeal, T., Defize, L. H. K., Angel, P., Woodgett,
J. R., Karin, M., and Hunter, T. (1991) Cell 64, 573–584.
We have developed a specific and sensitive GSK-3
16. Fiol, C. J., Williams, J. S., Chou, C. H., Wang, Q. M., Roach, P. J.,
kinase assay which does not require prior purification and Andrisani, O. M. (1994) J. Biol. Chem. 269, 32187–32193.
of GSK-3. Its simplicity allows the rapid analysis of 17. Welsh, G. I., and Proud, C. G. (1993) Biochem. J. 294, 625– 629.
large numbers of samples. We have shown that it can 18. Cross, D. A. E., Alessi, D. R., Cohen, P., Andjelkovic, M., and
detect GSK-3 activity in both Dictyostelium and mouse Hemmings, B. A. (1995) Nature 378, 785–789.
cells, and when used to examine the insulin response of 19. Sutherland, C., Leighton, A., and Cohen, P. (1993) Biochem. J.
10T1/2 cells our assay method gives results comparable 296, 15–19.
to those previously reported. The evolutionary distance 20. Stambolic, V., and Woodgett, J. R. (1994) Biochem. J. 303, 701–
between Dictyostelium and mouse GSK-3 kinases sug- 704.
gests that our assay can be applied to all species. In 21. Moule, S. M., Welsh, G. I., Edgell, N. J., Foulstone, E. J., Proud,
both of the cell lines investigated in this report, there is C. G., and Denton, R. M. (1997) J. Biol. Chem. 272, 7713–7719.
no evidence for a lithium-sensitive background of ki- 22. Klein, P. S., and Melton, D. A. (1996) Proc. Natl. Acad. Sci. USA
93, 8455– 8459.
nase activity which acts on the GSM substrate. This,
23. Stambolic, V., Ruel, L., and Woodgett, J. R. (1996) Curr. Biol. 6,
however, may not be the case for other applications and 1664 –1668.
should be tested using an unphosphorylated substrate 24. Hedgepeth, C. M., Conrad, L., Huang, H.-C., Leek, V., and Klein,
control, as in our experiments and in previous assays P. S. (1997) Dev. Biol. 185, 82–91.
(16,17). In the experiments described here we have 25. Fiol, C., Mahrenholz, A. M., Wang, Y., Roeske, R. W., and Roach,
used a novel peptide substrate, GSM; however, we see P. (1987) J. Biol. Chem. 262, 14042–14048.
no reason why this assay should not work with all 26. Woodgett, J. R. (1989) Anal. Biochem. 180, 237–241.
other GSK-3 peptide substrates and we have success- 27. Cook, D., Fry, M. J., Hughes, K., Sumathipala, R., Woodgett,
fully carried out assays using both the original GS-2 J. R., and Dale, T. C. (1996) EMBO J. 15, 4526 – 4536.
substrate and the CREB peptide substrate (unpub- 28. Goode, N., Hughes, K., Woodgett, J. R., and Parker, P. J. (1992)
J. Biol. Chem. 267, 16878 –16882.
lished results).
Acknowledgments. A.J.H. is a Wellcome Trust Senior Biomedical
Research Fellow and this project was supported by a Wellcome trust
grant to W.J.R. L.F. and T.D. are supported by the Cancer Research
Campaign.
Elution of Unmodified Oligodeoxynucleotides
REFERENCES from Zinc-Imidazole Negatively Stained
1. Embi, N., Rylatt, D. B., and Cohen, P. (1980) Eur. J. Biochem. Polyacrylamide Gels
107, 519 –527.
2. Cohen, P., Yellowlees, D., Aitken, A., Donella-Deana, A., Hem-
mings, B. A., and Parker, P. J. (1982) Eur. J. Biochem. 124,
Eugenio Hardy,1 Elder Pupo, José A. Silva,
21–35. Ricardo Silva, Edelgis Coizeau, and
3. Ahmad, Z., Camici, M., DePaoli-Roach, A. A., and Roach, P. J. Lila Castellanos-Serra
(1984) J. Biol. Chem. 259, 3420 –3428. Center for Genetic Engineering and Biotechnology,
4. Woodgett, J. R. (1990) EMBO J. 9, 2431–2438. P. O. Box 6162, Havana 10600, Cuba
5. Bourious, M., Morre, P., Ruel, L., Grau, Y., Heitzler, P., and
Simpson, P. (1990) EMBO J. 9, 2877–2884.
Received May 20, 1998
6. Plyte, S. E., Hughes, K., Nikolakaki, E., Pulverer, B. J., and
Woodgett, J. R. (1992) Biochem. Biophys. Acta 1114, 147–162.
7. Siegfried, E., Chou, T.-B., and Perrimon, N. (1992) Cell 71, UV shadowing is the most commonly used method
1167–1179. for the micropreparative detection of polyacrylamide
8. Puziss, J. W., Hardy, T. A., Johnson, R. B., Roach, P. J., and gel electrophoresis (PAGE)-separated oligodeoxynucle-
Heiter, P. (1994) Mol. Cell Biol. 14, 831– 839. otides; it is simple, rapid, and appropriate for a variety
9. Plyte, S. E., Feoktistova, A., Burke, J. D., Woodgett, J. R., and
Gould, K. L. (1996) Mol. Cell. Biol. 16, 179 –191.
1
10. Harwood, A. J., Plyte, S. E., Woodgett, J., Strutt, H., and Kay, To whom correspondence should be addressed. Fax: 0537 21 80
R. R. (1995) Cell 80, 139 –148. 70. E-mail: protchem@cigb.edu.cu.

ANALYTICAL BIOCHEMISTRY 264, 127–129 (1998)

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128 NOTES & TIPS

FIG. 1. Recovery of oligodeoxynucleotides from zinc-imidazole-stained gels by rapid passive elution. Identical samples of a 32P-labeled oligode-
oxynucleotide (45-mer, 522 ng, about 20,000 cpm) with the sequence 59-AGTCGTCCGGCAGCGAGGGGGGAGGAGGTGGTGGTGGTGGTG-
GTC-39 were electrophoresed in parallel lanes of a 15% polyacrylamide gel (1). Triplicate lanes were stained with zinc-imidazole (2). The visualized
bands were excised and processed for oligodeoxynucleotide mobilization as described in (6) (h), which includes an incubation (2 3 5 min) in 100
mM EDTA to chelate zinc ions (destaining) and three changes in distilled water for gel washing (wash). Alternatively, distilled water was replaced
with 70% ethanol (v/v) in water for gel washing (■). After oligodeoxynucleotide mobilization, bands were crushed through a 1-mL polypropylene
syringe containing two metal sieves at its bottom to yield gel microparticles of 32 mm. The resulting gel slurry was incubated in elution buffer (20
mM Tris-acetate, 0.5 M NH4Ac, 1 mM EDTA, pH 7.4) under vortexing (2 3 10 min) and the eluted oligodeoxynucleotides were collected in the
supernatant after centrifugation (elution). On-gel corresponds to total radioactive counts remaining in the gel after elution. Results are the average
of three independent experiments, followed by the standard deviation in parentheses.

of uses (1). Unfortunately, the short-wavelength UV slice after zinc chelation; losses of oligodeoxynucleoti-
light may damage the integrity of nucleic acids (2–5) des were significantly lowered to 1.4% and the overall
and has the added disadvantage of being hazardous to elution yields increased to 89% (Fig. 1, ■).
eyes and skin (1). To avoid these problems, we have We have eluted a variety of oligodeoxynucleotides
recently developed a new zinc-imidazole-based method from zinc-imidazole-stained gels by using this modified
for the visualization of polyacrylamide-separated DNA procedure, ranging from 8 to 50 mers, with recoveries
under UV light-free conditions (2). similar to those described in the legend to Fig. 1. The
As zinc-imidazole is a reversible fixative method, recovered oligodeoxynucleotides migrated as single,
double-stranded DNA can be eluted with a high recov- highly purified bands upon reelectrophoresis, without
ery, 85% or higher (6); it is supposed that oligode- discernible degradation products. The quality of our
oxynucleotides can also be recovered from the gel if oligodeoxynucleotide preparations was further evalu-
required. To verify this, we used our recently developed ated by standard sensitive techniques.
purification method (6) for the rapid elution of a 45-mer
oligodeoxynucleotide from gel microparticles (Fig. 1, 59-End radioactive labeling. The synthetic oligode-
h). However, we found that an appreciable (approx. oxynucleotide 59-ATCCCGATCCAAAACAGC-39 was
20%) amount of DNA diffuses to the solution during labeled with [g-32P]ATP using T4 polynucleotide ki-
the originally established mobilization step for zinc- nase (1). The percentage of radioactivity incorporated,
imidazole-stained dsDNA, which includes zinc chela- as measured by trichloroacetic acid precipitation (1),
tion with EDTA followed by water washes to remove averaged 88% in three independent experiments with a
excess EDTA. Recently, it has been demonstrated that specific activity of 109 cpm/mg by using primers pre-
diffusion of ethidium bromide-stained DNA on agarose pared by the zinc-imidazole passive diffusion technique
gels can be prevented by inclusion of 70% ethanol in or 78% after the conventional method [detection by UV
the storage buffer (7). Taking advantage of this knowl- shadowing after PAGE followed by elution with the
edge, we used 70% ethanol in water to wash the gel crush and soak method (1)].
 NOTES & TIPS 129

PCR. The subtype P1.7,16 from the porA gen-con- Cytosines Adjacent to Methylated CpG Sites
taining chromosomal DNA was prepared from cell cul- Can Be Partially Resistant to Conversion
tures of N. meningitidis H44/76 strain as described in
(8). The PCR was performed in a DNA thermal cycler
in Genomic Bisulfite Sequencing Leading
(MiniCycler, U.S.A.). Approximately 1 mg of chromo- to Methylation Artifacts
somal DNA was added to the PCR mixture in a volume
of 100 mL. The mixture comprised 20 mM Tris-HCl (pH
Janet Harrison,* Clare Stirzaker,*
8.4), 50 mM KCl, 1.5 mM MgCl2, 0.5% (v/v) glycerol,
0.01 mM dithiothreitol, 0.2 mM each dNTP, 50 pmol and Susan J. Clark*,†,1
each primer (oligodeoxynucleotides 59-ATCCCGATC- *Kanematsu Laboratories, Royal Prince Alfred Hospital,
CAAAACAGC-39 and 59-ATCCGATCCTGGCTTGC-39), Missenden Road, Camperdown, New South Wales,
and 3 U Taq DNA polymerase. After denaturation at 2050, Australia; and †Molecular Science, CSIRO,
94°C for 4 min, we carried out 2 amplification cycles, P.O. Box 184, North Ryde, New South Wales,
comprising denaturation at 94°C for 1 min, annealing 1670, Australia
at 40°C, and extension at 72°C for 30 s. This PCR was
completed after another 30 amplification cycles, com-
Received June 8, 1998
prising denaturation at 94°C for 1 min, annealing at
55°C for 1 min, and extension at 72°C for 30 s. The Key Words: non-CpG methylation; genomic bisul-
PCR-amplified products were analyzed by 2% (w/v) fite sequencing; methylation artifacts.
agarose gel electrophoresis followed by ethidium bro-
mide staining as usual. The DNA amplification value
by using primers prepared by our zinc-imidazole pas-
Methylation of DNA in eukaryotes is known to
sive diffusion technique versus the conventional crush
play a role in the processes of gene regulation (1),
and soak method after UV shadowing (1) was 3.8 3 104
imprinting (2), and oncogenesis (3, 4) and may
vs 2.8 3 104. Values represent the average of two
also be involved in genome defence (5). In plant
independent experiments.
genomes 5-methylcytosine is present in CpG dinu-
In addition to the above experiments, good readable
cleotides and in CpNpG trinucleotides (6). In mam-
sequencing ladders with appropriate signal-to-noise
malian genomes, while the presence of 5-methyl-
ratio have been obtained (not shown) by using primers
cytosine at CpG dinucleotides is well established,
prepared by this technique. Together, these data sug-
the existence of non-CpG methylation in vivo re-
gest that PAGE–zinc-imidazole followed by enhanced
mains controversial. There is evidence of non-CpG
passive elution from gel microparticles can be used as
methylation in mammals using a number of different
a reliable oligodeoxynucleotide micropurification method.
methods, including restriction enzyme analysis (7,
Although not tested, this method may be useful for
8), hydrazine cleavage (9), and nearest neighbor se-
oligodeoxynucleotide structural analysis (e.g., by mass
quence analysis (10 –12). However, each of these
spectrometry), as it does not introduce any alteration
techniques has resulted in different estimates of
in the structure of nucleic acids (2, 6), something that
non-CpG methylation in the genome, possibly due
cannot be assured with the traditional transillumina-
to the individual inherent shortcomings of each
tion-based methods.
method (13).
The question of non-CpG methylation can now be
REFERENCES
addressed by genomic bisulfite sequencing, a tech-
1. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular nique that enables the methylation status of every
Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor cytosine residue within a sequence to be determined
Laboratory, Cold Spring Harbor, NY.
(14 –18), including methylated cytosines at sites
2. Hardy, E., Pupo, E., Casalvilla, R., Sosa, A. E., Trujillo, L. E.,
López, E., and Castellanos-Serra, L. (1996) Electrophoresis 17, other than CpG dinucleotides (19). In plants,
1537–1541. genomic bisulfite sequencing has demonstrated ex-
3. Hartman, P. S. (1991) BioTechniques 11, 747–748. tensive non-CpG methylation of a repeat-induced
4. Carielo, N. F., Keohavong, P., Sanderson, B. J. S., and Thilly, point mutation gene (20) and the foreign hph gene
W. G. (1988) Nucleic Acids Res. 16, 4157. in Neurospora crassa (21), a transgene in Petunia
5. Gründeman, D., and Schöming (1996) BioTechniques 21, 898 – hybrida (22), and the SUPERMAN gene in Arabidop-
903. sis (23). The results of the genomic bisulfite se-
6. Castellanos-Serra, L., Hardy, E., and Sánchez, J. C. (1998) Anal.
Biochem. 257, 227–228.
7. Jacobs, D., and Neilan, B. A. (1995) BioTechniques 19, 892– 894. 1
To whom correspondence should be addressed at Molecular Sci-
8. Guillén, G., Alvarez, A., Lemos, G., Paredes, T., Silva, R., and ence, CSIRO, P.O. Box 184, North Ryde, NSW 1670, Australia. Fax:
Martı́n, A. (1993) Biotecnol. Apl. 10, 108 –113. (612) 9490-5100. E-mail: susan.clark@molsci.csiro.au.

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