Download H1 and HMG17 extracted from calf thymus nuclei are

Gene Therapy (1997) 4, 586–592
 1997 Stockton Press All rights reserved 0969-7128/97 $12.00
H1 and HMG17 extracted from calf thymus nuclei are
efficient DNA carriers in gene transfer
SV Zaitsev1,2, A Haberland1, A Otto3, VI Vorob’ev2, H Haller1 and M Böttger1,4
1
Franz Volhard Clinic at the Max Delbrück Center for Molecular Medicine, 3Max Delbrück Center for Molecular Medicine, BerlinBuch; 4Medical Clinic, Department of Cardiology, Charité, Humboldt University, Berlin, Germany; and 2Institute of Cytology of the
Russian Academy of Sciences, St Petersburg, Russia
In this article we describe the chromatographic separation
of acid nuclear protein fractions which have previously
been shown to be active in DNA transfection experiments.
By combining anionic and cationic ion exchangers, we
were able to separate and identify some of the active proteins. In addition to HMG1, already known for its transfection activity, we have identified histone H1 and HMG17 as
further transfection-active proteins. The highest transfection activity was associated with H1 and another nonidentified protein showing a somewhat higher electrophoretic
mobility than H1. We have also found that the presence of
CaCl2 in a low concentration in the cell culture medium is
an important requirement for transfection.
Keywords: nuclear extract; histone H1; HMG1; HMG17; reporter gene; transfection
Introduction
Effective gene transfer is an important prerequisite for
gene therapy. Because nonviral delivery systems are
expected to have little or no harmful side-effects, the
development of such systems with high efficiency and
avoiding toxic and immunogenic problems is an urgent
task.1,2 One approach in this direction is the use of protein–DNA complexes as carriers of exogeneous genes into
the host cell. While polylysine-ligand conjugates for targeting surface receptors of the host cell in complex with
DNA via the endocytotic pathway3 or viral capsid proteins 4 have been used successfully in gene transfer,
nuclear proteins have received little attention. The nonhistone protein HMG1 is the first example of a transfectionactive nuclear protein described.5–7
In a separate article, we showed that a series of nuclear
protein fractions can be isolated from acid nuclear
extracts by fractionated precipitation with acetone. These
fractions proved to be transfection-active in complex with
DNA in the presence of a low concentration of Ca ions
in the cell culture medium. We demonstrated that these
fractions contain histone H1, the nonhistone proteins
HMG1, HMG2, HMG17 and other not yet identified proteins in different ratios.8 The aim of this study is to
resolve further the constituents of these fractions by chromatographic methods, to identify the purified proteins by
sequencing and to test their activity as DNA carriers for
gene transfer. We envisage that with continuous purification and functional testing for transfection efficiency of
the identified proteins at each purification step, we will
be able to improve our delivery system based on such
Correspondence: M Böttger, Franz Volhard Clinic, Wiltberg Strasse 50,
13122 Berlin, Germany
Received 19 August 1996; accepted 13 February 1997
proteins. Our final goal is to establish an efficient transfection method by using physiological agents and physiological conditions which do not perturb or damage cells
and which can be used in vivo.
Results
In a separate article8 we demonstrated that a series of
nuclear protein fractions from calf thymus termed 2V–
5.5V exhibit the ability to package DNA into transfectionactive protein–DNA complexes. These protein fractions
were obtained by extraction of nuclei with 5% perchloric
acid and subsequent gradual acetone precipitation of
these extracts. In this article, we describe the purification
of the protein fractions 2V–3.5V by chromatographic
methods (FPLC) which results in a considerable increase
in the transfection efficiency. Each step of the purification
process was checked by transfection experiments using
the protein samples as DNA packaging agents. Only
positive samples were investigated further.
The fractions 2V-3.5V were separated on a Mono Q column by elution with a linear salt gradient of 3–73% of
buffer B. Only the breakthrough material (2VA–3.5VA),
not bound to the column, proved to be transfection-active
and was used for additional separations using the Mono
S column. Typical electrophoretograms of the Mono Q
purified 2.5V fraction are shown in Figure 1a. With the
exception of the transfection-active fraction 2.5VA (lane
2), all other fractions (2.5VB etc) were rejected. A second
step of purification of the 2VA–3.5VA fractions was performed by means of the Mono S column under the same
conditions as with the Mono Q column but using buffer
C instead of buffer B. The result of this purification step
for 2.5VA is shown in Figure 1b. Figure 2 shows the elution profiles of the 3VA and 3.5VA fractions after Mono
S separation as examples for the resolution of the FPLC.
There are large differences in the number of bands
Transfection-active nuclear proteins
SV Zaitsev et al
587
Figure 2 Elution profiles after Mono S chromatography of protein fractions 3.5VA (a) and 3VA (b). The fraction numbers given in the order of
elution are identical with the numbers of the lanes of the electrophoresis
experiment shown in Figure 1c and provide the denotation of the protein fractions.
Figure 1 Acetic acid/urea polyacrylamide gel electrophoresis of protein
fractions obtained from acid nuclear extracts by acetone precipitation and
ion exchange chromatography (FPLC). (a) Fraction 2.5V after chromatography on Mono Q. Lane 2 contained transfection-active material indicated by an arrow (2.5VA) and was identified as histone H1. Lanes 1,
12: protein markers; lanes 3, 4: 2.5VB; lanes 5, 6: 2.5VC; lanes 9–11:
2.5VD. (b) Fraction 2.5VA after chromatography on Mono S. Lane 1:
2.5VA; lane 2: 2.5VASB (breakthrough); lanes 3–10: 2.5VAS1, lanes 11,
12: 2.5VAS2. (c) Fractions 3V and 3.5V after chromatography on Mono
S. Lanes 1, 9, 12: protein marker (histone H1); lane 2: 3.5VAS3; lane 3:
3.5VAS4; lane 4: 3.5VAS5; lane 5: 3.5VAS6; lane 6: 3.5 VAS7; lane 7:
3.5VAS8; lane 8: 3.5VAS9; lane 10: 3VAS1, lane 11: 3VAS2. Bands
designated by arrows in lanes 2 and 10: HMG17; in lane 4: HMG1; in
lane 5: HMG2; in lane 11: H1.
between both fractions. All bands were collected and
studied by electrophoresis. The results are shown in Figure 1c. Fraction 2.5VA contains a single band, 3VA two
bands and 3.5VA several bands. The fractions 4.5V-5.5V
containing HMG1 and 2 proteins are omitted here as the
transfection activity of HMG1 was previously demonstrated (Böttger et al5,6). The transfection activity of
HMG1 is smaller than that of the 2V–3.5V fractions.8
Before summarizing the results of the chromatographic
purification, it is necessary to describe the transfection
experiments in more detail. We used the luciferase gene
(pCMV Luc) as a reporter gene. pCMV Luc DNA (2 mg)
were mixed with protein at a weight input ratio r i of 10–
20 and added to 2 × 105 NIH 3T3 cells, containing 8 mm
CaCl2 in the culture medium. In the range of 1 , r i , 30
no significant differences in the transfection efficiency
have been observed previously, although the variation in
the transfection rates of several experiments of the same
fraction was high. 8 Ca ions were compulsory for high
transfection activity in this system. However, as shown
later, the Ca concentration could be further decreased.
The transfection efficiency is given in terms of relative
light units. Figure 3 shows the results of transfection
experiments performed with the total nonfractionated
protein extract, S, the nuclear protein fraction 3V and following protein fractions 3VA, 3VAS1 and 3VAS2
Transfection-active nuclear proteins
SV Zaitsev et al
588
Figure 3 A comparison of transfection efficiencies in terms of relative
light units (RLU) obtained with free DNA, different protein–DNA complexes and Lipofectamin. The protein fractions S, 3V, 3VA, 3VAS1,
3VAS2 representing different purification levels by ionic exchange chromatography are used for complex formation with pCMV Luc DNA (2 mg).
Complexes of ri ratios between 10 and 20 were added to 2 × 105 NIH 3T3
cells. The values are means ± s.e. from at least six different experiments.
obtained after two FPLC purification steps as described
above. Negative and positive controls obtained with free
DNA and Lipofectamin respectively, were added. Compared with the unpurified total protein extract, S, a
strong increase in the expression of the luciferase gene in
terms of RLU could be seen after acetone fractionation
(3V) and Mono Q separation (3VA). The increase in RLU
was more than 30-fold. The purification on Mono S
resulting in 3VAS1/S2 did not result in a further increase
in RLU. Free DNA and cells without added DNA as a
negative control were inactive (approximately 2000 RLU
both). The transfection efficiencies of the fractions 3VA
and 3VAS2 were comparable with that of Lipofectamin.
Analogous results were obtained with 3.5V and the
samples derived from it by FPLC (Figure 4). Chromatographic purification resulted in a considerable increase in
the transfection efficiency. The protein fractions 3.5VAS7
and 3.5VAS8 reached the highest RLU values.
In connection with the transfection studies, the results
of the purification procedure can be summarized as follows. After purifying the 2V and 2.5 V fractions in two
steps according to the scheme 2V→2VA→2VAS1 and
2VAS2 (2.5V in the same way), a single protein component was found in 2VAS1/S2 and 2.5VAS1/S2 which was
identified as histone H1 (see Figure 1b). However, the
results of purification of the 3V and 3.5V fractions were
more complicated (Figure 1c). Purification was performed as before. Histone H1 was still only present in a
small concentration (3VAS2, 3.5VAS9). A small but
significant increase in transfection activity with
(2.6 ± 0.9) × 105 RLU was found in the fractions 3VAS1
and 3.5VAS3 which was identified as bovine HMG17 by
its N-terminal sequence (not shown). Furthermore, it was
found that other samples such as 3.5VAS5 and in parti-
Figure 4 Transfection efficiencies in terms of RLU obtained from different
protein–DNA complexes. The protein fractions 3.5V, 3.5VA and
3.5VAS3–3.5VAS9 are used, 2 mg pCMV Luc, 2 × 105 NIH 3T3 cells.
The values are means ± s.e. (six experiments).
cular 3.5VAS7 and 3.5VAS8 were highly active in transfection (Figure 4). These results together with electrophoresis data suggest that another transfection-active
protein with a somewhat higher electrophoretic mobility
in the acetic acid/urea system other than H1 (Figure 1c)
could be present. According to these results, we believe
that the increase in transfection activity is due to both the
loss of inhibiting protein components and the presence
of new, nonidentified proteins.
Because the presence of Ca ions is compulsory for
obtaining high transfection efficiencies, it was necessary
to investigate the role of Ca ions in more detail. In particular we wanted to know if Ca ions are a necessary part
of the protein–DNA complex itself, ie whether they are
required for the complex formation with DNA and/or as
stabilizers against dilution dissociation. This latter question arose from the need to dilute the complexes during
addition to the cell culture medium. However, H1–DNA
complexes should be stable (see Ref. 9). We performed
transfection experiments with Ca in the complex mixture
using both Ca-free medium and Ca-containing cell culture medium. Only low expression could be detected if
Ca was not present in the cell culture medium, independent of the presence or absence of Ca in the complex
mixture (Figure 5). Thus, the presence of Ca ions is not
necessary for the complex formation, but seems to be
needed as a stabilizing agent against dilution dissociation. On the other hand, as discussed later, Ca ions
could also play a protective role for the complexes inside
the cell. In another experiment, we optimized the Ca concentration in the medium. Since Ca is known to be toxic
for cells in high concentration, we also varied the time of
exposure of the transfecting complexes in the Ca-containing cell culture medium to the cells. The results of such
experiments using protein fraction 2.5VAS1 (H1 histone)
are shown in Figure 5. After the recorded times of cell
contact, the transfection mixture was replaced with fresh
culture medium. It is noteworthy that the presence of
10% calf serum in the medium had no negative effect on
Transfection-active nuclear proteins
SV Zaitsev et al
589
Figure 5 Dependency of the transfection efficiency of the protein fraction
2.5VAS2 in the complex with DNA on the concentration of CaCl2 . Transfection efficiency in terms of RLU (2 mg pCMV Luc, 2 × 105 NIH 3T3
cells). (a) Four hours transfection time; (b) 12 h transfection time. The
values are means ± s.e. (at least three experiments).
the transfection process. Maximum expression was seen
at 4–8 mm CaCl2 after 4 h (Figure 5a) and at 1–2 mm
CaCl2 after 12 h (Figure 5b) of incubating the cells with
the transfection mixture. An inverse relationship between
incubation time and Ca concentration with respect to
transfection efficiency seems to exist. On the other hand,
at extended transfection times the morphological appearence of the cells deteriorates. Thus, we prefer to work at
a transfection time of 4 h.
The question whether the transfection effects observed
here could be due, at least in part, to the usual calcium
phosphate-mediated DNA coprecipitation was answered
by our experiments. This possibility cannot be excluded
because of the presence of phosphate in RPMI. Figure 6
shows the results of the DNA-Ca phosphate coprecipitation technique after preforming 2.5VAS1–DNA complexes and performing the Ca phospate precipitation in
the usual way. The conditions of DNA and protein concentration were the same as in the control experiments
using the 2.5VAS1 fraction as the transfection-active protein. We observed only a small positive effect of 2.5VAS1
on the transfection efficiency of the Ca phosphate method
as compared with the control. It is further noteworthy
with regard to this question that the control with free
plasmid DNA in RPMI and in the presence of Ca++ was
negative in transfection (the RLU value corresponds to
that of the untreated cells of about 2000 RLU).
A further point which provokes interest with respect
to the practical application of these protein fractions is
their cytotoxicity. Many of the nonviral vectors such as
Figure 6 A comparison of the transfection efficiencies in terms of RLU
obtained from 2.5VAS1–DNA complexes (open columns) and of the Ca
phosphate coprecipitation method of preformed 2.5VAS1–DNA complexes
(hatched columns), pCMV Luc (2 mg), 2 × 105 NIH 3T3 cells; the values
are means ± s.e. (at least 10 experiments). Free DNA in the presence of
CaCl2 is negative in transfection.
lipofectin and polylysine are cytotoxic. Using the XTT
test, we checked the cytotoxicity of 2.5VAS1 alone, in the
presence of CaCl 2 and as a protein–DNA complex with
and without Ca++ (Figure 7). There was no evidence for
cytotoxicity under incubation conditions as used for the
transfection experiments. After 16 h of incubation, the
viability slowly decreased (not shown here).
Finally, in order also to demonstrate the validity of our
conclusions for other cell lines including primary cells,
we transfected a number of different eukaryotic cell types
with 2.5VAS1–pCMV Luc complexes (Figure 8). The
results confirm our findings on the efficacy in transfection
of these nuclear proteins. HUVEC cells are transfectable,
however, with less reproducibility. There were experiments in which no transfection was observed. The permanent endothelial cell line ECV304 was constantly able to
be transfected.
Discussion
These results confirm and extend our observations that
histone H1 and other nuclear proteins like HMG1 (HMG1
however to a lower extent) are efficient mediators of transfection in complex with DNA (Böttger et al5,8). An
important requirement for this activity is the presence of
Ca ions in the cell culture medium. In the absence of Ca++
the transfection activity is essentially lower (Figure 5).
Recently, Fritz et al10 demonstrated an effective in vitro gene
transfer using histones including H1 and a modified H1
molecule with a SV 40 nucleophilic signal as the DNA carrier. However, for obtaining high expression results, the
presence of chloroquine was necessary. CaCl2 was not
present in the transfection mixture. Using galactosylated
histones complexed with DNA and targeting the asialo-
Transfection-active nuclear proteins
SV Zaitsev et al
590
Figure 7 Cell viability of NIH 3T3 cells as the percentage of controls in
the presence of 2.5VAS1 alone (open columns), additionally in the presence of 4 mM CaCl2 (upward hatched columns), in the presence of the
2.5VAS1–DNA complex (downward hatched columns) and, additionally,
in the presence of 4 mm CaCl2 (crossed-hatched columns) as a function
of the protein concentration determined by the XTT assay. The test was
performed in 96-well plates with 4 × 104 NIH 3T3 cells, protein concentrations as given and 2 mg/ml pCMV Luc DNA, mean ± s.d., three
experiments.
Figure 8 Comparison of 2.5VAS1-mediated transfection of different cell
lines and HUVEC cells. All cell types (2 × 105 cells each) were transfected
with 2 mg DNA, ri ratios between 2 and 20, means ± s.e. from at least
four experiments.
glycoprotein receptor, Chen et al11 were able to transfect
human liver cells. The best result was obtained with galactosylated H1. Without galactosylation there was, however,
no detectable transfection activity. These authors used
CaCl2 in the culture medium. In contrast to the results cited,
we found in the presence of Ca a high transfection activity
for NIH 3T3 cells and other cell lines, including human
liver cells, using H1 containing protein fractions in the complex with DNA. A commercial H1 sample was also active.8
Therefore, we need to discuss the ability of certain
nuclear proteins to mediate DNA transfection and the
role of Ca ions in this process. We suggest that there are
common features in H1 histone and other nuclear proteins such as HMG1/2 and HMG17 which can mediate
DNA transfection. We suggest that the ability to bind and
condense DNA9,12 together in the presence of nuclear
location sequences13 are such common features. The
structures of H1–DNA and HMG1–DNA complexes
under similar ionic conditions are well documented by
means of physicochemical and electron microscopic
methods. Whereas HMG1–DNA complexes resulted in
nearly spherical condensed particles of about 40 nm
diameter involving one or a few DNA molecules,5,12 H1–
DNA complexes often have a cable-like condensed
appearance involving many DNA molecules.14,15 Furthermore, refolded DNP double fibers and large aggregated
condensates (@100 nm) were found. The small size of
HMG1–DNA complexes should be well suited for in vivo
gene transfer studies. It is worth mentioning in this context that core histones were also shown to be transfectionactive but with lower efficiency10 (and our unpublished
data). The effect that H1 inhibits or blocks transcription
in cell-free systems can obviously be overcome in intact
cells.16 This is not astonishing given the large number of
DNA molecules involved and of the dynamic character
of the electrostatic binding of these proteins to DNA.
An aspect which needs further elucidation is the pathway by which the protein–DNA complexes are taken up
by the cells and reach the nucleus. Possibly, Ca ions in
the culture medium play an important role in this process. They could for instance transform the cytoplasmic
membrane into a state more suitable for penetration of
the condensed and aggregated protein–DNA complexes
(see Epstein et al17). On the other hand, endocytosis cannot be excluded. In this context it is worth mentioning
that we also, similar to Fritz et al,10 were able to transfect
HepG2 cells by means of the fraction 2.5VAS1 representing H1 in the presence of chloroquine (data not
shown). Chloroquine neutralizes the acid pH of endocytotic vesicles, inhibiting hydrolases in endosomes and
lysosomes which results in decreasing degradation of
macromolecules. This results in enhanced transfection
rates. Hence, chloroquine could indicate that endocytosis
is involved in the H1-mediated transfection. Also, the
size of the complexes far exceeding 100 nm which are
taken up by the cells could indicate an endocytotic mechanism. However, in the presence of Ca ions (6 mm) chloroquine is inactive in enhancing transfection (data not
shown). This could mean that Ca competes with chloroquine in the function of releasing intact protein–DNA
complexes into the cytoplasm. It is noteworthy that,
according to our experiments, Ca does not enhance the
transfection efficiency of Lipofectamin (unpublished)
which supports this hypothesis.
Materials and methods
Protein extraction and fractionation
The proteins were extracted from calf thymus nuclei with
5% perchloric acid at 0°C as suggested by Sanders and
Transfection-active nuclear proteins
SV Zaitsev et al
Johns18 with minor modifications. Total proteins contained in the acid extract were fractionated by gradual
addition of increasing volumes of cold acetone/0.1 m
HCl. Fractions termed 1V, 2V,...6.5V (V, abbreviation for
volumes of acetone), were obtained in this way. Each
fraction was collected by centrifugation, dried and stored
at −20°C (for more details, see Böttger et al8).
Chromatographic separation, purification and sample
labeling
Chromatography was performed using an automatic
FPLC gradient system with UV monitor and fraction collector from Pharmacia Biotech, Uppsala, Sweden using
both the anionic and cationic exchange columns Mono Q
HR 5/5 and Mono S HR 5/5 (Pharmacia Biotech, Freiburg, supplier). The transfection efficiency of the separated protein samples was taken as a criterion for a successful purification step. Chromatographic separations
were performed after loading of 2 ml of fractions 2V–
3.5V in a concentration of 3 mg/ml in buffer A on the
Mono Q column and elution by a linear salt gradient of
3–73% of buffer B. Then the material not bound to the
column (breakthrough), exhibiting a high transfection
activity, was further purified on Mono S under the same
elution conditions using buffer C. All fractions obtained
were precipitated by acetone, dried and stored at −20°C.
The following buffers were used: A: 0.02 m NaCl, 0.05 m
Tris-HCl, pH 7.4, 0.02 m b-mercaptoethanol; B: 0.82 m
NaCl, 0.05 m Tris-HCl, pH 7.4, 0.02 m b-mercaptoethanol;
C: 1.82 m NaCl, 0.05 m Tris-HCl, pH 7.4, 0.02 m b-mercaptoethanol.
The following system was used for labeling and
characterizing the samples obtained by chromatography.
2V–5.5V: fractions obtained by PCA extraction and subsequent precipitation with 2 volumes (V) to 5.5V acetone.
2VA–3.5VA: fractions 2V–3.5V, but chromatographed on
Mono Q (breakthrough fractions only). 2VAS1, 2VAS2
etc: samples obtained from fraction 2VA, but additionally
separated on Mono S, where numbers 1,2 etc give the
order of elution.
Polyacrylamide gel electrophoresis
Electrophoresis was performed according to Paniym and
Chalkley19 using 15% polyacrylamide slab gels and
Coomassie blue staining.
Protein identification
Proteins were sequenced by N-terminal amino acid
sequencing using a 477A sequencer (Applied Biosystems,
division of Perkin Elmer, Foster City, CA, USA) and
identified after a search in the SwissProt database using
the Fasta programme (HUSAR).
Cells
NIH 3T3 cells (mouse fibroblasts) were grown in RPMI
1640 medium with l-glutamine (Serva, Heidelberg,
Boehringer Ingelheim, Heidelberg, Germany, supplier)
supplemented with 10% heat-inactivated fetal calf
serum (FCS).
The human hepatoma cell line HepG2 20 and the human
hepatocellular carcinoma line Huh721 were maintained in
Dulbecco’s modified Eagle’s medium (DMEM) from
Gibco BRL (supplied by Life Technologies, Eggenheim,
Germany) containing 10% heat-inactivated FCS.
The mouse colon tumor cell line CT2622 was main-
tained in RPMI 1640 medium with l-glutamine supplemented with 10% heat-inactivated FCS.
Endothelial cells (HUVEC cells) were prepared from
human umbilical cord veins according to Jaffe et al23 and
cultivated in a modified manner published by Griesmacher et al. 24
The permanent human endothelial cell line ECV304
originally established from the vein of an apparently normal umbilical cord25 was propagated in Medium 199
(Gibco BRL, Paisley, UK, Life Technologies, Eggenstein,
Germany, supplier) with 10% heat-inactivated FCS.
Transfection experiments
Vector DNA and proteins as specified in the text were
mixed in buffer D in the presence of CaCl2 (final concentration 2 mm) at room temperature. The protein was
gradually added in two to three portions to the DNA
under agitation in order to avoid turbidity. Protein concentration was determined by dissolving weighed
samples of lyophilized material into known volumes of
buffer and, where possible (H1 and HMG1), by spectrophotometry.26 After mixing, the samples were shaken for
at least 15 min. The weight input ratio ri of protein to
DNA was adjusted to 10–20. The final complex volume
was 100 ml. About 2 × 105 cells (here NIH 3T3 cells) per
well of a 24-well plate (Falcon, Franklin Lakes, NJ, USA)
grown in RPMI 1640 medium with l-glutamine supplemented with 10% FCS, were used for transfection. The
plasmid pCMV Luc was used as a reporter gene. The culture medium was removed before transfection and the
cells were washed twice with RPMI. The protein–DNA
complexes were added directly to 0.9 ml of culture
medium containing 10% FCS and 8 mm CaCl2 . This transfection mixture was then added to the cells. After 4 h of
incubation under 4.5% CO2 at 37°C, the transfection mixture was removed and replaced by 1 ml RPMI 1640 containing 10% FCS. After incubation for about 30 h the cells
were harvested and the luciferase activity measured by
the Promega assay system (supplied by Boehringer
Ingelheim, Germany) using a Lumat LB 9501/16 luminometer (Berthold, Bad Wildbad, Germany).
Transfection with Lipofectamin (Gibco BRL) was performed according to the manufacturer’s recommendations. DNA (2 mg) and Lipofectamin (3 ml) were both
diluted separately into 100 ml Optimem I (Gibco BRL).
After gently mixing both solutions at room temperature
and incubation for 15 min, the mixture was diluted with
0.8 ml Optimem I and added to the washed cells. Transfection time was 4 h, then the transfection medium was
replaced with normal growth medium. Further procedures were as given previously and Buffer D: 0.15 m
NaCl, 10 mm Tris-HCl, pH 7.6.
Calcium phosphate coprecipitation was performed
using the Profection Kit from Promega (supplied by
Boehringer Ingelheim, Germany). According to the
manufacturer’s recommendations, the amount of solution
was adapted to 20 mm dishes (24-well plates). DNA (2
mg) were mixed with a 2 m CaCl2 solution and water.
Amounts of 2.5VAS1 (histone H1) according to the input
ratio ri protein–DNA (w/w) selected were added to this
mixture. Then, the same volume 2 × Hepes-buffered
saline was added to this mixture with vortexing, final
volume 150 ml. The transfecting mixture was added dropwise to 2 × 105 cells in 1 ml culture medium.
591
Transfection-active nuclear proteins
SV Zaitsev et al
592
Viability measurements
Viability measurements were performed in 96-well plates
by means of the cell proliferation kit II (XTT) from Boehringer Mannheim, Germany. NIH 3T3 cells (4 × 104 per
well) were washed after a 4 h exposure to the protein
fraction 2.5VAS1 in three different concentrations (20
mg/ml, 60 mg/ml, 100 mg/ml) in 150 ml each, additionally
in the presence of CaCl2 (4 mm) to the protein, and in the
presence of the protein–DNA complex (protein concentration as above and 2 mg/ml pCMV luc DNA) and of 4
mm CaCl2. Then the XTT procedure was performed as
recommended by the manufacturer. The test measures
the ability of cells to reduce the water soluble tetrazolium
dye, XTT (sodium 3′-[1-(phenyl-amino-carbonyl)-3,4tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic
acid hydrate), to its insoluble formazan salt. The insoluble salt is coloured and can be determined spectrophotometrically.
9
10
11
12
13
14
15
Acknowledgements
16
We thank Lieselotte Winkler for skillful technical assistance, Professor Charles Coutelle (London) for a critical
reading of the manuscript, and Professor Burkhard
Micheel (Berlin) for support. This work was supported
by the Volkswagen foundation and the BMBF.
17
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