The interphase chromatin special state zones
The possibility is demonstrated of RecA protein of E. coli introduction into eukaryotic cell with preservation of its biologicall activity. The character of this protein affinity to chromatin of different degree of condensation is shown on cytological level (undirect immunofluorescent method combine...
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| Zitieren: | The interphase chromatin special state zones / S. P. Shpilevaya, I. E. Kostetsky, K. M. Bilitch, L. I. Lihatcheva, T. V. Stolar, D. M. Irodov, E. V. Kostetskaya, I. M. Spivak, V. D. Zhestyanikov, V. A. Kordyum // Биополимеры и клетка. — 1993. — Т. 9, № 5. — С. 89-100. — Бібліогр.: 24 назв. — англ. |
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irk-123456789-1562552019-07-04T16:04:05Z The interphase chromatin special state zones Shpilevaya, S. P. Kostetsky, I. E. Bilitch, K. M. Lihatcheva, L. I. Stolar, T. V. Irodov, D. M. Kostetskaya, E. V. Spivak, I. M. Zhestyanikov, V. D. Kordyum, V. A. Генно-инженерная биотехнология The possibility is demonstrated of RecA protein of E. coli introduction into eukaryotic cell with preservation of its biologicall activity. The character of this protein affinity to chromatin of different degree of condensation is shown on cytological level (undirect immunofluorescent method combined with cytophotometry) for different stages of cellular cycle (protein is not tested in meta- and anaphase chromosomes). The presence of bacterial RecA protein in the nuclei and cytoplasm of cells both in vitro and in vivo is confirmed with the help of immunoelectron microscopy. Studies were carried out on cultures of HeLa and Ltk⁻ cells and also in vivo on hepatocytes after direct injection on RecA protein and plasmid pKCR2 enclosed in liposomes into the liver of adult mice line BALB/c. Proceeding from the experimental data obtained and also considering the fact that RecA protein mainly connects with single-stranded DNA, the assumption is done about existence of special state chromatin zones (SSCZ). For these zones the active affinity to RecA protein serum is character with intensive fluorescence, thus they may correspond to some actively expressing genes, gathered in clusters. Показано можливість введення до еукаріотичної клітини RecA-білка Е. coli із збереженням його біологічної активності. На цитологічному рівні (непрямий імунофлюоресцентний метод з цитофотометрією) визначено характер зв'язування RecA- білка з хроматином різного ступеню спіралізації на окремих стадіях клітинного циклу (у мета- і анафазних хромосомах білок не тестується). Присутність бактеріального білка у цитоплазмі і ядрах клітин in vivo та in vitro підтверджена також імуноелектронною мікроскопією. Виходячи з отриманих експериментальних даних та враховуючи переважне зв'язування RecA-білка з онДНК, зроблено припущення про існування зон особливого стану хроматина (ЗOCX), які характеризуються активним зв'язуванням з сироваткою до бактеріального RecA-білка та інтенсивною флюоресценцією і можуть відповідати зібраним у кластери активно експресуючим генам. Експерименти виконано на культурах клітин HeLa і Ltk⁻, а також in vivo на гепатоцитах після прямого введення білка та плазміди pKCR2 у складі ліпосом до печінки дорослих мишей лінії BALB/c. Показана возможность введения в эукариотическую клетку RecA - белка Е. coli с сохранением его биологической активности . На цитологическом уровне (непрямой иммунофлюоресцентный метод с цитофотометрии ) определен характер связывания RecA - белка с хроматином разной степени спирализации на отдельных стадиях клеточного цикла (в мета- и анафазного хромосомах белок не тестируется ) . Присутствие бактериального белка в цитоплазме и ядрах клеток in vivo и in vitro подтверждено также имуноэлектронной микроскопией . Исходя из полученных экспериментальных данных и учитывая преимущественное связывание RecA - белка с онДНК , высказано предположение о существовании зон особого состояния хроматина ( ЗOCX ) , которые характеризуются активным связыванием с сывороткой к бактериальному RecA - белка и интенсивной флюоресценцией и могут соответствовать собранным в кластеры активно экспрессирующим генам . Эксперименты выполнены на культурах клеток HeLa и Ltk⁻ , ??а также in vivo на гепатоцитах после прямого введения белка и плазмиды pKCR2 в составе липосом в печени взрослых мышей линии BALB/c. 1993 Article The interphase chromatin special state zones / S. P. Shpilevaya, I. E. Kostetsky, K. M. Bilitch, L. I. Lihatcheva, T. V. Stolar, D. M. Irodov, E. V. Kostetskaya, I. M. Spivak, V. D. Zhestyanikov, V. A. Kordyum // Биополимеры и клетка. — 1993. — Т. 9, № 5. — С. 89-100. — Бібліогр.: 24 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.000378 http://dspace.nbuv.gov.ua/handle/123456789/156255 ru Биополимеры и клетка Інститут молекулярної біології і генетики НАН України |
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Генно-инженерная биотехнология Генно-инженерная биотехнология |
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Генно-инженерная биотехнология Генно-инженерная биотехнология Shpilevaya, S. P. Kostetsky, I. E. Bilitch, K. M. Lihatcheva, L. I. Stolar, T. V. Irodov, D. M. Kostetskaya, E. V. Spivak, I. M. Zhestyanikov, V. D. Kordyum, V. A. The interphase chromatin special state zones Биополимеры и клетка |
| description |
The possibility is demonstrated of RecA protein of E. coli introduction into eukaryotic cell with preservation of its biologicall activity. The character of this protein affinity to chromatin of different degree of condensation is shown on cytological level (undirect immunofluorescent method combined with cytophotometry) for different stages of cellular cycle (protein is not tested in meta- and anaphase chromosomes). The presence of bacterial RecA protein in the nuclei and cytoplasm of cells both in vitro and in vivo is confirmed with the help of immunoelectron microscopy. Studies were carried out on cultures of HeLa and Ltk⁻ cells and also in vivo on hepatocytes after direct injection on RecA protein and plasmid pKCR2 enclosed in liposomes into the liver of adult mice line BALB/c. Proceeding from the experimental data obtained and also considering the fact that RecA protein mainly connects with single-stranded DNA, the assumption is done about existence of special state chromatin zones (SSCZ). For these zones the active affinity to RecA protein serum is character with intensive fluorescence, thus they may correspond to some actively expressing genes, gathered in clusters. |
| format |
Article |
| author |
Shpilevaya, S. P. Kostetsky, I. E. Bilitch, K. M. Lihatcheva, L. I. Stolar, T. V. Irodov, D. M. Kostetskaya, E. V. Spivak, I. M. Zhestyanikov, V. D. Kordyum, V. A. |
| author_facet |
Shpilevaya, S. P. Kostetsky, I. E. Bilitch, K. M. Lihatcheva, L. I. Stolar, T. V. Irodov, D. M. Kostetskaya, E. V. Spivak, I. M. Zhestyanikov, V. D. Kordyum, V. A. |
| author_sort |
Shpilevaya, S. P. |
| title |
The interphase chromatin special state zones |
| title_short |
The interphase chromatin special state zones |
| title_full |
The interphase chromatin special state zones |
| title_fullStr |
The interphase chromatin special state zones |
| title_full_unstemmed |
The interphase chromatin special state zones |
| title_sort |
interphase chromatin special state zones |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| publishDate |
1993 |
| topic_facet |
Генно-инженерная биотехнология |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/156255 |
| citation_txt |
The interphase chromatin special state zones / S. P. Shpilevaya, I. E. Kostetsky, K. M. Bilitch, L. I. Lihatcheva, T. V. Stolar, D. M. Irodov, E. V. Kostetskaya, I. M. Spivak, V. D. Zhestyanikov, V. A. Kordyum // Биополимеры и клетка. — 1993. — Т. 9, № 5. — С. 89-100. — Бібліогр.: 24 назв. — англ. |
| series |
Биополимеры и клетка |
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Генноинженерная
биотехнология
S. P. Shpilevaya, I. Е. Kostetsky, К. М. Bilitch, L. I. Lihatcheva,
Т. V. Stolar, D. М. Irodov, Ε. V. Kostetskaya, I. М. Spivak,
V. D. Zhestyanikov, V. A. Kordyum
THE INTERPHASE CHROMATIN SPECIAL STATE ZONES
The possibility is demonstrated of RecA protein of E. coli introduction into eukaryotic
cell with preservation of its biologicall activity. The character of this protein affinity to
chromatin of different degree of condensation is shown on cytological level (undirect
immunofluorescent method combined with cytophotometry) for different stages of cellu-
lar cycle (protein is not tested in meta- and anaphase chromosomes).
The presence of bacterial RecA protein in the nuclei and cytoplasm of cells both
in vitro and in vivo is confirmed with the help of immunoelectron microscopy.
Studies were carried out on cultures of HeLa and Ltk~ cells and also in vivo on he-
patocytes after direct injection on RecA protein and plasmid pKCR2 enclosed in liposo/nes
into the liver of adult mice line BALB/c.
Proceeding from the experimental data obtained and also considering the fact that
RecA protein mainly connects with single-stranded DNA, the assumption is done about
existence of special state chromatin zones (SSCZ). For these zones the active affinity to
RecA protein serum is character with intensive fluorescence, thus they may correspond
to some actively expressing genes, gathered in clusters.
Introduction. The product of the gene recA of Escherichia coli due to its
great significance for normal functioning of cellular genome occupies
particular place among all products of the bacterial genes, revealed du-
ring two last decades. Polyfunctioning of this protein with comparative-
ly small weight (37 800 D), taking participation in the processes of re-
combination, replication, reparation, mutagenesis, cell division, synthesis
of new proteins, induction of prophages [1, 2] is suprising.
There may exist some SOS-functions both in eukaryotic and proka-
ryotic cells. It was shown even in the early studies, that mammalian cells
might response the inhibition of replication by induction of new proteins
(during the inhibition of proliferation in the E. coli cells the sygnal ca-
using induction of RecA protein is generated). Proliferation of the lympho-
id cells, peripheral lymphocytes, primary fibroblasts of the skin has been
inhibited by the delay of the replication. It has been found that under
these conditions the synthesis of two new proteins is induced, one of
them is localised in nucleus (nuclear localization of products supposes
their fermentive nature and interaction with DNA) [3]. May the pro-
ducts of genes recA (or their analoges) with different origin substitute
functionally one another? The presence of protein factors in eukaryots
functional analogous to recA protein of E. coli is shoun in a number
of studies.
Thus the protein RecA, isolated from Ustilago maydis, realises
homological coupling of overspyralised DNA with ss-DNA, requiring
ATP for the reaction [4]. Specifical to ss-DNA and ss-RNA endonuclease
was isolated from mouse cells plasmocytome mitochondria [5]. Activity
of endonuclease specifical to ss-DNA in a large extend is similar to the
enzymes from fungus mitochondrias, whose absence results in disturban-
ces in the processes of DNA reparation and recombination.
© S. P. Shpilevaya, I. E. Kostetskvf K. M. Bilitch, L. I. Lihatcheva, Т. V. Stolar,
D. M. Irodov, Ε. V. Kostetskaya, I. M. Spivak fV. D. Zhestyanikov, V. A. Kordyum, 1993
89 ISSN 0233-7657 БИОПОЛИМЕРЫ II КЛЕТІ\Л. 1993. Т. 9. № 5
Partially purified factor specifical to ss-DNA dependent ATP acti-
vity and promoting DNA homologous pairing with heteroduplex forma-
tion and DNA strands transferation between corresponding DNA-sub-
strates has been isolated from human skin fibroblasts nuclear extract by
chromatography on biogel [6], Activity of this factor was tested in ex-
periments with phages circular ss-DNA in linear ss-DNA. Factor-catali-
zed reactions were found similar to those, in which protein recA from
E. coli participates.
So extensive spectrum of eukaryotic proteins which are able in vitro
to fulfil RecA protein functions such as homologous pairing of superhe-
lical DNA with ss-DNA, transfer of DNA strands between corresponding
substrates has been found. For some of them, for example, for RRAl [7]
nuclear localization has been shown. The presence of the same elements
in the system of general recombination of pro- and eukaryots lets us as-
sume under certain conditions the possibility of the participation of RecA
protein of E. coli in the processes of homologous recombination and re-
paration of the eukaryotic genome. However the possibility of penetra-
tion of ReeA protein through the nuclear membrane and its ability to
interact effectively with eukaryotic DNA being in part of chromatin has
been studied to our day in a few works [8—10]. For solving of this prob-
lem at first one had to make sure of the possibility of ReeA protein of E.
coli penetration inside eukaryotic cell and, in part, into nucleus. Choice
of method of test penetration of ReeA protein in cytoplasm and nucleus
was of principal importance. As it was known that in vivo ReeA protein
is related with DNA while carrying out this work for its test indirect
immunofluorescent (IF) method was chosen to define visually penetra-
tion and localization of ReeA protein in nuclei of eukaryotic cells. It was
suspected that use on the one side of the method permiting to make vi-
sual the connection of ReeA protein with chromatin and, on the other
side, of suitable model system (rapidly deviding cells of HeLa culture)
would allow to determine on the morphological level the character of the
connection of ReeA protein with chromatin of different degree of spirali-
zation of the isolated stages of cellular cycle.
In addition to method described above the method of immunochemi-
cal electron microscopy was applied to clear up some questions concer-
ning the localization of RecA protein in different cellular compartments.
Thus an attempt of the spatial distinguishing of the expressing si-
tes of chromatin (in which there must be firm connection of ReeA prote-
in with single-stranded DNA) from the rest of unreplicating chromatin
mass was made.
Beside this it was supposed that in differentiated cells, such as, for
example, hepatocytes of adult mice, in which mitotic cell-divisions took
place rarely, evidence of connection of ReeA protein with expressing ge-
nes might be useful for determination of their morphological and structu-
ral localization of these expressing genes in nucleus. Initially the presence
of the so called special state chromatin zones (SSCZ) in nuclei, in which
there were actively expressing genes gathered in clusters was postulated.
Materials and methods. O b j e c t s of s t u d y . Studies in vitro we-
re carried out on culture of HeLa cells from the collection of cells of
Sankt-Petersburg Institute of cytology of AS of Russia and Ltk- cells
from the collection of cells of 1MB & G of Ukraine. Culture were culti-
vated on the Eagle medium containing 10 % bovine serum and antibio-
t ics—penici l l in (200 v/ml) and streptomycin (1 mg/ml ) . In system in
vivo experiments were made on hepatocytes of mouses of BALB/c line
(2-month age, mascular) for one varient the group of 2-3 animals was
picked out.
E x t r a c t i o n of RecA p r o t e i n . Purified ReeA protein — poly-
peptide with molecular weight 37 800 D — was extracted from E. coli cells
superproducing this protein (stamp DH 1, defective on the system of re-
combination) under the condition of SOS-function by nalidixic acid [11].
Cells of E. coli DH 1 transformated by plasmid pxl3 had high content
90 ISSN 0233-7657 БИОПОЛИМЕРЫ II КЛЕТІ\Л. 1993. Т. 9. № 5
of protein owing to the presence of spr mutation and persuation by na-
lidixic acid. Cultivation of E. coli for accumulation of its biomass was
carried out on the medium LB (1 % of pepton, 0.5 % yeast extract, 1 %
NaCl). The protein was purified on a Sepracryl S-200 column [12]. Pu-
rity of- RecA protein on the SDS electroporation in 14 % polyacrylamid
gel was 96-98 %. Functional activity of RecA protein was controlled by
its ability to hydrolise ATP in the presence of single-stranded DNA. Con-
trolled by such way activity was 1.84=0.4 pM A T P /mg of protein in
hour [13].
P I a s m id . BamHI-SalGI fragment from plasmid pxl3, containing
full-size recA gene of E. Coli1 was cloned on BamHI-site (with filling in
by DNA-polymerase of uncomplementary ends) on plasmid pKCR [14].
After restriction analyse thf clones, in which recA gene was in the direct
orientation to the promoter SV40, were selected. Resulting plasmid pKCR2
was induced in mammalian cells in vitro and in vivo.
L i p o s o m e s . To transfer RecA protein and plasmid DNA negati-
vely-charged unilamellar liposomes were used 10 to 100 nm in diameter
with next compounds: lecitin : cholesterol : phosphat ide acid : phosphati-
dylethanolamin in proportion 7 : 2 : 1 : 0.2, received by ultrasonic method
with phase conversation [15, 16].
RecA protein incorporation in liposomes was carried out by adding
of protein in lipid mixture on the «gel» stage in the process of liposome
preparation. DNA plasmid's pKCR2 were included into liposomes by Ca-
fusion method and amount of liposome's included DNA was 7-10 % from
that was introduced. Sizes, lamellarity and safety of liposomes were con-
trolled in electrone microscope on negatively stained grids [17].
E x p e r i m e n t s i n v i t r o . HeLa cells were sown on the cover
glasses and were cultured under standard conditions in Eagle's medium
with additions for 24 h.
Cells were transfected with plasmid pKCR2 by the Ca-phosphate and
DEAE-dextran technique [21]. Besides, introduction of plasmid pKCR2
into cells in part of liposomes was applied.
RecA protein when it was introduced into mammalian cells in vitro
was added directly to cultural medium in account of 0.5 mg/ml with next
cells exposition during 30 min. Then medium was withdrown and cells
were subjected to glycerol shock (15 % glycerol) for 3 s.
Besides, in other varients of experiments HeLa cells were incubated
with pure RecA protein without liposomes and with unloaded liposomes.
Fixation of preparations in several changes of aceton cooled up to 4 °С
for 20 min was conducted after 24 h after treatment.
E x p e r i m e n t s i n v i v o . Volume of material, which was intro-
duced directly in the mouse liver was 60 μΐ/ individual. In variations of
injections of plasmid pKCR2 being in part of liposomes mixture contained
30 μg of lipids and 10 μg of plasmid DNA. Animals were sacrified and
liver was extracted 24 h and 48 h after injection of material. Hepatocytes
was prepared as early described [15]: from the same lobe of liver in which
the material was injected.
R e c e i v i n g a n d p u r i f i c a t i o n of s e r u m s . Shinshila race
rabbits were immunized by electrophoratively purified RecA protein on
the next scheme. During 3 weeks RecA protein was induced in acount
100 μg/individual in the region of two cervical and two popliteal lympha-
dens of rabbits with seven-day intervals. The first immunization was car-
ried out with complete Freind adjuvant and the next two — with uncom-
plete one.
In a week after third injection it was done buster-injection —100 μg
of RecA protein was injected through rabbit's ear vein and blood collec-
tion was made in 7-8 days after last injection. Antibody titer was deter-
mined by double gel-diffusion method ( 1 : 1 6 - 1 : 3 2 ) . Gamma-globulin
fraction was isolated by double precipitation of serums by ammonium
sulphate with 33 % of saturation with following dialysis to 0.0175 M of
sodium phosphated buffer with pH 6.3. In emergency gamma-globulins
91 ISSN 0233-7657 БИОПОЛИМЕРЫ II КЛЕТІ\Л. 1993. Т. 9. № 5
were additionally purified on ion exchange column with cellulose (1.5X
X25 sm) using the same buffer. IgS fraction removed from ion exchange
column was concentrated by PEG-3500-4000 and was kept under 4 °С
with addition of NaN3.
Before staining of cytological preparations serum to ReeA protein
was purified additionally 1-2 times by liver powder [19]. Labelled by
fluoresceinisothiocyanit (FITC) donkey's antirabbit globulin was puri-
fied from unconnected fluorochrom on the 1 % agarose.
Working dilution of serum to RecA protein for preparations staining
was 1 : 5-1 : 10; luminescent — 1 :16 .
S t u d y i n g of c y t o l o g i c a l p r e p a r a t i o n s . ReeA protein
was tested by undirect IF method [23]. Preparations were looked through
on the luminescent microscope ML-2 with photometrical addition on the
520 nm and with the probe 0.5 μΐη. The objective X70 for water immersion
was used. Stained ReeA protein fluoresced in green area of spectrum.
Measurement on the each varient of experiments were done on 50 cells
with measuring by probe fluorescene of nucleus and cytoplasm separately.
E l e c t r o n i m m u n o c y t o c h e m i s t r y . 24 h after injections of
material the cubes of liver tissue were fixed in 2.5 % glutaraldehyde pre-
pared on the 0.1 M sodium-cacodylate buffer (pH 7.4) at 4 °С for 2.5 h.
After r insing in the same buffer the tissue was postfixed in 2 % osmium
tetroxid. Then after dehydration in a graded series of alcohols the samp-
les were embedded in Epon-Araldite.
Monolayers of HeLa cells were cultured on pieces of lavsan film
and after 24 h of transfection processed according classic scheme with
the addition of 2 % sucrose at the first steps. The film with cells was
embedded in special metal rings by «sandwich» method. Under the cont-
rol of light microscope separate groups of cells were separated from film,
fastened to standard epoxy blocks and then sectioned as usually.
Ultrathin sections for immunochemical assay were cut on ultratome
LKB III (Sweden) and mounted on parlodian metal grids without any
film-coating.
To identify the molecules of ReeA protein the method of indirect im-
munolabelling was used that is based on the formation of special linking:
antigene — antibody — protein A — colloidal gold. Biospecific probe «pro-
tein A — colloidal gold» (PAG) was obtained from scientific-industrial
centre «BIOFROMEKOLOGIA» (Saratov, Russia). The main characte-
ristics of the probe were the following: the mean diameter of the gold
particle 20.6 nm; concentration 3.5· IO12 pt /ml. The final concentration of
gold probe was defined cytophotometrically upon the absorption O D 5 2 O
and settled for electron microscopy in ranges 0.1-0.2 by diluting the ini-
tial reagent 20-40 times. To avoid the formation of gold depositions on
thin sections the probe after diluting was centrifugated (up to 5 min
with 10 000 rot/min) with the subsequent control in electron microscope.
The labelling of thin sections was settled according to method [21] with
special attention on blocking procedure, i. e. preliminary incubation of
sections on the drops of bull serum albumin to diminish the unspecific
labelling.
After incubation in drops of PAG the grids with sections were rinsed
thoroughly in phosphate buffer saline and distilled water and then, avoi-
ding them from final drying, stained with uranyl acetat followed by lead
citrate. The preparations were examined in «TESLA» electrone micro-
scope at 90 kV.
E x p e r i m e n t s w i t h HeLa c u l t u r e . Controls: 1) cells per-
suated by unloaded «empty» liposomes during 30 min with subsequent
glycerine shock of 30 s of duration; 2) cells without any treatment (intact
cells) and stained with serum to ReeA protein; 3) intact cells, stained
with serum to bacterial β-galactosidase.
Fluorescence of cytoplasm under staining of control intact cells with
serum to ReeA protein was not considerable and on quantitative indices
approached to values of autoluminescence (luminescence of unstained fi-
92 ISSN 0233-7657 БИОПОЛИМЕРЫ II КЛЕТІ\Л. 1993. Т. 9. № 5
xed cells) (fig. Ι , α ) . The chromatin net was slightly revealed in nuclei
(fig. 1, c). After preparations staining with serum to bacterial β-galacto^
sidase no fluorescence of cytoplasm was noted and no morphological
structures were revealed (fig. 1, rf) in nuclei; the luminescence of ceils
corresponded to autolurninescence level.
Fig. 1. Immunofluorescence of HeLa cells (controls: a—d): a — autolurninescence; b —
treatment with unloaded «empty» liposomes, serum to RecA protein; с — cell without any
treatment stained with antiserum to RecA protein; d — cell without any treatment stained
with antiserum to β-galactosidase; e—after 30 min exposition with pure ReeA protein;
f , g, h — after 30 min exposition with ReeA protein enclosed in liposomes; і — after con-
tact with plasmid pKCR2 enclosed in liposomes; g, і — groups of metaphase chromosomes
that do not reveal any fluorescence
After staining of preparations previously treated by «empty» lipo-
somes with antibodies to RecA protein of Et coli dim luminescence of cy-
toplasm was noted and slightly fluorescent chromatin and nucleolus co-
uld be identified (fig. 1, b).
Obviously that after staining of preparations with serum to bacterial
RecA protein chromatin structures of nucleus revealed themselves, while
after using of other serum — to bacterial β-galactosidase — they did not.
93 ISSN 0233-7657 БИОПОЛИМЕРЫ II КЛЕТІ\Л. 1993. Т. 9. № 5
On these grounds it may be suppossed that there are specifical to anti-
serum to RecA protein determinants in the structure of eukaryotic chro-
matin, and it may testify that there are functional analogues of ReeA
protein of E. coli in eukaryotic nucleus. But low intensity of luminescence
of stained chromatin structure shows that either the quality of such pro-
teins is not considerable, or their affinity with antibodies received by im-
munization of animals by RecA protein of E. coli is not large.
I n c u b a t i o n of HeLa c e l l s w i t h p u r e RecA p r o t e -
i n a n d RecA p r o t e i n , e n c l o s e d i n l i p o s o m e s . At is was
demonstrated in seria of experiments, described in detail in previous pub-
lication [8], the bright fluorescence was observed in cytoplasm and nuc-
lei on cell preparations 24 h after their incubation for 0.5 h with pure
RecA protein or RecA protein enclosed in liposomes. Increasing of lumi-
nescence intensity of this cellular structures (in comparison with control
varients) after treatment of preparations with serum to bacterial RecA
protein of E. coli was explained as a consequence of E. coli protein pene-
tration in eukaryotic cell and its nucleus. In such cases the localization
of fluorescence on nuclear chromatin structures was observed visualy.
Karyoplasm of nuclei did not luminescence and went on to be dark, un-
fluorescent (fig. l,e, f , g, h). Fluorescence distribution on length of chro-
matin thread is ununiform and next to intensive luminescent sites there
are sections of chromosomes without luminescence.
Observation of chromatin luminescence peculiarities on the different
stages of cellular cycle allowed to note reccurence in its fluorescence de-
pending on degree of chromatin condensation. Nuclei with greatly decon-
densated chromatin, according to .stages of G and S periods of cellular
cycle by visual observations differed also by intensive fluorescence. Lu-
minescence of nuclei decreased according to chromatin condensation in
the late prophase and was completely absent in metaphasic chromosomes
(fig. 1, g", i). For this photometrical measurements demonstrated increa-
sing of quantity (in relative units) of tested RecA protein in cytoplasm,
which exceeded one in next undivided cells, thus, bacterial RecA orotein
during metaphase— anaphase could be tested only in cytoplasm. Nuclei
luminescence was renewed after the formation of nucleus covers around
separated groups of chromosomes and decondensation of chromatin, for-
mation of nucleolus in telophasic nuclei.
So from visual observations or contrasting of chromatin after treat-
ment of HeLa cells with pure RecA protein or RecA protein in compound
of liposomes the next conclusion may be done:
1) in described experiments it was shown that RecA protein of E.
coli penetrated in eukaryotic cell and nucleus that is proved by appearan-
ce in them of specifical fluorescence;
2) in nucleus protein is associated with chromatin and thus its vi-
sualization on chromosomes after staining of preparations by antibodies
to RecA protein becomes possible;
3) RecA protein independent on the way of introduction into euka-
ryotic cell (in compound of liposomes or without them) preserves its bio-
logical activity and, obviously, functions in eukaryotic cells with revea-
ling of some (for example, DNA-connecting) biochemical activities;
4) RecA protein of E. coli association with chromatin unmasked by
undirect IF method depends on the degree of chromosomes condensation
and charges during cellular cycle. In compactly condensated metaanapha-
sic chromosomes protein is not revealed, diffusely dispersing in cytoplasm.
Simultaneously with karyokinesis protein begins to be revealed associated
with chromatin;
5) connection of RecA protein with chromatin is observed not along
whole chromatin strand and brightly fluorescent sites alternate with sec-
tions of chromatin, uncontrasted by protein.
G e n e recA i n t r o d u c t i o n i n t o HeLa c e l l s . Fullsized gene
recA of E. coli was cloned in plasmid pKCR [14] on BamHI site. Plas-
mid was introduced in HeLa culture by several ways (Ca-phosphatic and
94 ISSN 0233-7657 БИОПОЛИМЕРЫ II КЛЕТІ\Л. 1993. Т. 9. № 5
DEAE-dextran and in compound of liposomes), in experiments we have
chosen two last ones as giving stable results. Beside, this the possibility
of introduction of plasmid DNA enclosed in liposomes into cultivated
cells was studied. Presence of protein-product in nuclei and cytoplasm
was determined 24 h after transfection.
RecA protein according to the presence of fluorescence was revealed
both in nuclei and in cytoplasm of transformated cells. Cells fluorescence
(fig. l , i ) did not differ from one under influence of pure ReeA protein on
HeLa preparations or of protein being contained in liposomes. The same
regularities in character of luminescence of chromatin in dependence of
degree of its spiralization were observed. In cells transformed by plasmid
pKCR2 enclosed in liposomes (approximately 36 %) , different quantities
(in relative units) of protein-product were revealed [8]. ,For this in nuc-
lei of some cells the content of ReeA protein greatly exceeded one in nuc-
lei of cells from other variations of experiments — after influence by pure
ReeA protein or by protein enclosed in liposomes. Untransformated cells
(about 64 %) showed fluorescence within the limits of luminescence of
cells treated by unloaded «empty» liposomes, that is they were on the
level of control.
So this study has shown:
1) the possibility of introduction of recA gene in part of plasmid
pKCR2 in HeLa cells by X)EAE-dextran method or using liposomes with
the effect of transfection;
2) product of recA gene expression in nucleus is associated with
chromatin.
E x p e r i m e n t s i n v i v o . Controls: 1) hepatocytes of animals
without any treatment; 2) hepatocytes of animals after injection in liver
of unloaded liposomes; preparation in both cases were stained with se-
rum to RecA protein; 3) besides preparation from liver of intact animals
for comparison were stained with serum to β-galactosidase.
Morphological revealings of chromatin in nuclei of intact animals
(fig. 2, a) and after introduction of unloaded «empty» liposomes (fig. 2, b)
under staining of preparations of liver by antibodies to bacterial ReeA
protein were the same with one in analogous experiments in HeLa cul-
ture. Chromatin was contrasted slightly though after injection of «empty»
liposomes the structure of nucleus revealed more clear. Controlled stai-
ning of hepatocytes preparations with antiserum to bacterial β-galactosi-
dase did not reveal any structures in nuclei (fig. 2, e).
In variants of experiments — after injection into liver of ReeA pro-
tein (fig. 2, d) or plasmid pKCR2 (fig. 2, f ) enclosed contained in lipo-
somes fluorescence in hepatocyte nuclei was observed after two days.
Vizualized chromatin was seen as thin luminescent net (fig. 2 ,d ) , or in
other nuclei on the more compactly condensated chromatin treads inten-
sive luminescent regions was marked (fig. 2 , / ) . Photometrical measure-
ments fixed considerable amounts of ReeA protein in nuclei and cyto-
plasm of hepatocytes.
So it was shown that:
1) after injection into liver of ReeA protein being contained in lipo-
somes or plasmid coding expression of bacterial RecA protein the pene-
tration of injected material into separate hepatocytes takes place;
2) ReeA protein in hepatocytes is tested in nuclei and cytoplasm af-
ter both variants of introduction;
3) in nuclei ReeA protein is associated with chromatin and after
s taining by antibodies to bacterial ReeA protein is vizualized on the nuc-
leus structures;
4) staining of hepatocyte preparations of intact animals allows to
observe slight luminescence of chromatin, that is the evidence of some
determinants in eukaryotic chromatin connected with antibodies to bac-
terial ReeA protein.
T h e i m m u n o e l e c t r o n r e v e a l i n g of RecA p r o t e i n
i n v i t r o a n d i n v i v o . On fig. 3 (a — i) one can observe the frag-
95 ISSN 0233-7657 БИОПОЛИМЕРЫ II КЛЕТІ\Л. 1993. Т. 9. № 5
ments of ultrathin sections of mouse liver and HeLa culture cells after
incubation with antibodies for bacterial RecA protein followed by PAG
complexes. Judging by distribution of colloidal gold particles ReeA pro-
tein is revealed both in nuclei and in cytoplasm of those two test-systems.
In cytoplasm the greatest amount of gold particles is located over
the channels of granular endoplasmic reticulum (GER) and clusters of
free ribosomes and polysomes (fig. 3, a ) . In some cases the label is dis-
posed directly in cytosol. The high level of PAG binding is shown for
Fig. 2. Immunofluorescence of mice hepatocytes 2 days after injection of «empty» liposo-
mes (b), RecA protein of E. coli (d, g) and plasmid pKCR2 (e, f ) enclosed in liposomes.
Controls: hepatocytes of intact animals stained with antibodies to ReeA protein of E. co-
li (a) and to bacterial β-galactosidase (c)
some autophagic vacuoles (fig. 3, b), which can be explained by proteo-
lysis of deposited ReeA protein in these structures. Less intensively the
mitochondrion and microbodies are labelled Very scanty labelling of the
background level is observed over Golgi elements and in the vesicles of
agranular endoplasmatic reticulum.
In the nuclei both of hepatocytes and of HeLa cells the label is dis-
posed unevenly. The tendency is seen for its predominance over peri-
membrane chromatin, perinuclear space and nuclear membrane comple-
xes (fig. 3, a, e — g). In some cases after the injection of liposomes con-
taining plasmid pKCR2 the peculiar chains of gold particles can be ob-
served, located perpendicularly to the nuclear membrane and crossing it
(fig. 3 , g ) . On the control grids no deposition of colloidal gold is found,
thus, such «chains» probably correspond to the true distribution of pro-
96 ISSN 0233-7657 БИОПОЛИМЕРЫ II КЛЕТІ\Л. 1993. Т. 9. № 5
Fig. 3. Electron immunocytochemistry of hepatocytes (a, b, d, e, g) and HeLa culture cells
(c, f, h) 24 h after injection/applying of liposomes containing RecA protein (a—e) or
plasmid pKCR2 { f , g, h). All sections are stained with rabbit antiserum to ,RecA protein
and protein A—col lo ida l gold complexes according to [21]. Enlargements: a — 64 000;
b — 96 000; c —34 000; d— 12 000; e — 5 5 000; f — 67 000; g — 48 000; h — 42 000. Abbre-
viations: N— nucleus; Nu— nucleolus; P — nuclear pore complex; Av — autophagic vacuo-
le; CcH — condense chromatin; Deh — disperse chromatin; G and F — granular and fib-
rillar components of nucleoli; GER — granular endoplasmic reticulum; C — cytoplasm.
Arrows indicate the clusters of colloidal gold grains
tein molecules and depict to some- extent their transport from cytoplasm
to the nucleus.
The attempt to identify the localization of gold particles with certa-
in structural type of ,chromatin had met some methodological difficulties.
According to method [21], the preliminary etching of sections should be
done (with 10 % H2O2) to facilitate the access of reagents to antigenic
determinants. The binding of the latter with antibodies after these pro-
ISSN 0233-7657 БИОПОЛИМЕРЫ И КЛЕТКА. 1993. Т. 9. № 5 7 - 3 . 7 0 5 O7
cedure raised to some extent but at the same time the contrast of ultra-
structures beneath the label reduced. Nevertheless it can be affirmed that
label is mainely localized not in the mass of perimembrane chromatin but
on the periphery of its condensed clusters, in the border between the con-
dense chromatin and the disperse one. It is here, in the bordering zone7
that the perichromatin fibrils are situated — the sites of initial despira-
lization of expressing chromatin [22].
Both after injection of bacterial protein and of plasmid with its gene
the nucleoli are labelled intensively in two test-systems. There is howe-
ver certain difference between the distribution of probe over hepatocyte
nucleoli in comparison with those of the HeLa cells.
The nucleoli of hepatocytes (often having nucleolonemal and some-
times compact structure) are labelled more intensively and gold particles
are distributed more evenly (fig. 3, h). There is no preference of label
to fibrillar or granular zones and only on the periphery of nucleolonemal
loops there is sometimes the label ,of more than 2 particles.
In cells of HeLa culture, where nucleoli often have one large vacuole
and well-defined borders between granular and fibrillar components, the
label is distributed unevenly forming clusters of 5-10 particles (fig. 3 , / ) .
These latter are placed either between two zones or over granular com-
ponent, mostly on the periphery of organelle. It is here, in granular com-
ponent of nucleolus, that most mature sorts of RNA are placed, the ribo-
somes precursors [23].
Both in HeLa cells and in hepatocytes the intensity of labelling irs
different nucleoli even on the same section varies g rea t ly—from abun-
dant to practically zero.
In control immunochemical variants the ultrathin sections were in-
cubated with PAG omitting the preliminary incubation with antibodies.
On such sections there were practically no label at all, which is the evi-
dence for comparatively high specificity of method. Nevertheless some
methodological aspects of this investigation should be discussed. The
authors of method [21], having applied it to the variety of tissues con-
cluded that in principle one can obtain the high specific label of protein
for tissue embedded in routine conditions, without usage of low tempera-
ture embedding media. However in all these papers the proper proteins
of the cells were investigated whereas in our work the protein was fo-
reign. May be in such case the antigene determinants are particularly
tender and can be distroyed in the process of outine embedding. On the
other hand, taking in mind that the serum used was polyclonal, it is
possible that some portion of protein molecules with high specificity co-
uld be washed out from sections of the step of blocking. Both these rea-
sons explain to some extent the fact that the label obtained, although spe-
cific enough, is less intensive in whole comparing with IF method, where
the preparatory procedure is shorter and less harmful for antigene de-
terminants.
Thus the presence of bacterial RecA protein in the nuclei and cyto-
plasm of cells both in vitro and in vivo is confirmed with the help of im-
munoelectron microscopy. In cytoplasm the protein is revealed in the
channels of granular endoplasmatic reticulum, in clusters of free ribo-
somes and polysomes and also in some autophagic vacuoles. In the cell
nuclei of both systems the protein is distributed unevenly — with the dis-
placement to the periphery of organelles. In the nucleoplasm ReeA pro-
tein is bound presumably with perichromatin fibrils the sites of initial
despiralization of expressing chromatin. Nucleoli, that in both test-sys-
tems depose the protein quite intensively although very unequally, accu-
mulate it mainly in the granular component. There is no substantial dif-
ference in the intercellular distribution of ReeA protein after its own pe-
netration to the cell or as the plasmid containing recA gene.
C o n c l u s i o n . Proceeding from our investigations one can conclu-
de that ReeA protein is able to recognise not only «naked» DNA, but al-
so DNA in part of chromatin of yeast [13] and chromatin of higher euka-
98 ISSN 0233-7657 БИОПОЛИМЕРЫ II КЛЕТІ\Л. 1993. Т. 9. № 5
ryots. Available in literature data don't contradict to the possibility of
prokaryotic proteins participation in substitution of eukaryotic proteins
functions in vivo and in vitro [1, 2, 24]. Thus RecA protein of E. coli in
vitro interacts equally effective both with pro- and eukaryotic DNA. The-
re is a number of proteins with functional specialities of RecA protein of
E. coli in eukaryotic cells [4-7], so functioning of RecA protein in euka-
ryotic cells in vivo in spite of unsufficient studying of this question se-
ems to be interesting.
We have demonstrated possibility of RecA protein of E. coli intro-
duction into eukaryotic cell with preservation of its fermentative activity.
Both the product itself and recA gene expression products penetrated in
nuclei of HeLa cells and hepatocytes in vivo with equal success and af-
ter cytological preparations treatment by rabbit's serum to RecA protein
the protein was revealed in nuclei to be associated in whole with chro-
matin. It is suprising that chromatin and nucleoli in control preparations
also became available to microscopic observation, one could see it very
well in the intact HeLa cells treated by «empty» liposomes. Besides, it
is obviously that RecA protein affinity with chromatin definitely depends
on degree of chromatin condensation and, correspondingly, on cellular
cycle phases. It can be cytologically proved by metaphasic and anaphasic
chromosomes groups being revealed as dark structures on brightly fluo-
rescent cytoplasm background while interphase nuclei chromatin fluores-
cence brightly.
Proceeding from of these data and also based on those facts that
RecA protein mainly connects with single-stranded DNA and that genes
are localized on chromosome ununiformly the assumption was done about
existence of special state chromatin zones (SSCZ). We postulate unu-
niform distribution of interphasic chromosome. Structuraly they are lo-
calized in nucleus in such a way, that majority of expressing genes are
collected in separate clusters. Length of each chromosome amounts to
santimeters. That 's why each of them in limit of nucleus with the diame-
ter of thousands times smaller than interphase chromosome length may
form pluralcurves-repetitions. And under unocasional distribution secti-
ons with intensive expressing genes (or at all expressing) are situated
one next another. So functional compartments being special state chroma-
tin zones are formed in nucleus. In result expressing sites spationaly
standout for the rest mass of unexpressing chromatin.
One may suspect different amounts of SSCZ for different tissues, this
number would be depend also on stage of cellular cycle. In its turn si-
zes of SSCZ must depend on degree of functional loading on the cell,
and also on the age of the cell.
Photometrical measurements often demonstrated higher acount of
RecA protein in interphasic nuclei, than in cytoplasm. Apparently the
possibility of RecA protein to contrast chromatin that we have seen may
be used in cytological practice for carrying out experiments with this pro-
tein, studying of expression of this gene in vivo and for ,studying of func-
tional activity of chromatin.
Р е з ю м е . Показано можливість введення до еукаріотичної клітини RecA-б'ілк&
Е. coli із збереженням його біологічної активності. На цитологічному рівні (непрямий
імунофлюоресцентний метод з цитофотометрією) визначено характер зв'язування RecA-
білка з хроматином різного ступеню спіралізації на окремих стадіях клітинного циклу
(у мета- і анафазних хромосомах білок не тестується). Присутність бактеріального біл-
ка у цитоплазмі і ядрах клітин in vivo та in vitro підтверджена також імуноелектрон-
ною мікроскопією. Виходячи з отриманих експериментальних даних та враховуючи пе-
реважне зв'язування RecA-білка. з онДНК, зроблено припущення про існування зон особ-
ливого стану хроматина (ЗOCX), які характеризуються активним зв'язуванням з сиро-
ваткою до бактеріального RecA-білка та інтенсивною флюоресценцією і можуть відпові-
дати зібраним у кластери активно експресуючим генам. Експерименти виконано на куль-
турах клітин HeLa і L t k - , а також in vivo на гепатоцитах після прямого введення біл-
ка та плазміди pKCR2 у складі ліпосом до печінки дорослих мишей лінії BALB/c.
11 ISSN 0233-7657 БИОПОЛИМЕРЫ II КЛЕТІ\Л. 1993. Т. 9. № 5
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