Fluorescence intensity profiles of in situ hybridization signals depict genome architecture within human interphase nuclei
An approach towards construction of two-dimensional (2D) and three-dimensional (3D) profiles of interphase chromatin architecture by quantification of fluorescence in situ hybridization (FISH) signal intensity is proposed. The technique was applied for analysis of signal intensity and distribution w...
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| Дата: | 2008 |
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Інститут клітинної біології та генетичної інженерії НАН України
2008
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| Назва журналу: | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| Цитувати: | Fluorescence intensity profiles of in situ hybridization signals depict genome architecture within human interphase nuclei / I.Y. Iourov, S.G. Vorsanova, Y.B. Yurov // Цитология и генетика. — 2008. — Т. 42, № 5. — С. 3-8. — Бібліогр.: 20 назв. — англ. |
Репозитарії
Digital Library of Periodicals of National Academy of Sciences of Ukraine| _version_ | 1859545993576448000 |
|---|---|
| author | Iourov, I.Y. Vorsanova, S.G. Yurov, Y.B. |
| author_facet | Iourov, I.Y. Vorsanova, S.G. Yurov, Y.B. |
| citation_txt | Fluorescence intensity profiles of in situ hybridization signals depict genome architecture within human interphase nuclei / I.Y. Iourov, S.G. Vorsanova, Y.B. Yurov // Цитология и генетика. — 2008. — Т. 42, № 5. — С. 3-8. — Бібліогр.: 20 назв. — англ. |
| collection | DSpace DC |
| description | An approach towards construction of two-dimensional (2D) and three-dimensional (3D) profiles of interphase chromatin architecture by quantification of fluorescence in situ hybridization (FISH) signal intensity is proposed. The technique was applied for analysis of signal intensity and distribution within interphase nuclei of somatic cells in different human tissues. Whole genomic DNA, fraction of repeated DNA sequences (Cot1) and cloned satellite DNA were used as probes for FISH. The 2D and 3D fluorescence intensity profiles were able to depict FISH signal associations and somatic chromosome pairing. Furthermore, it allowed the detection of replicating signal patterns, the assessment of hybridization efficiency, and comparative analysis of DNA content variation of specific heterochromatic chromosomal regions. The 3D fluorescence intensity profiles allowed the analysis of intensity gradient within the signal volume. An approach was found applicable for determination of assembly of different types of DNA sequences, including classical satellite and alphoid DNA, gene-rich (G-negative bands) and gene-poor (G-positive bands) chromosomal regions as well as for assessment of chromatin architecture and targeted DNA sequence distribution within interphase nuclei. We conclude the approach to be a powerful additional tool for analysis of interphase genome architecture and chromosome behavior in the nucleus of human somatic cells.
Представлено метод побудови двомірних (2D) та тримірних (3D) профілів інтенсивності сигналів флуоресцентної гібридизації in situ (FISH), що заснований на кількісній FISH. Наведена методика була використана для аналізу розташування та розподілу сигналів в інтерфазних ядрах клітин різних соматичних тканин людини. Використання 2D профілів інтенсивності продемонструвало можливість визначення локалізації FISH-сигналів. Більш того, даний підхід дозволив ідентифікувати репліковані сигнали, дати оцінку ефективності гібридизації та провести порівняльний аналіз варіації вмісту ДНК специфічних ділянок хромосом. Побудова 3D профілів показала розподіл інтенсивності у межах площі сигналу. Використання цієї методики дозволило визначити зосередження різних типів послідовностей ДНК: класична сателітна та альфоїдна ДНК; генонасичені (G-позитивні полоси) і геноненасичені (G-негативні полоси) ділянки хромосом. Крім цього, методика надала можливість оцінити розташування хроматина в інтерфазних ядрах як культивованих, так і некультивованих клітин. Зроблено висновок, що наведений підхід є ефективною додатковою методикою для вивчення ядерної організації, специфіки варіації та розташування послідовностей ДНК в інтерфазних ядрах, а також поведінки ядер при приготуванні хромосомних препаратів соматичних клітин людини.
Представлен метод построения двухмерных (2D) и трехмерных (3D) профилей интенсивности сигналов флюоресцентной гибридизации in situ (FISH), основанный на количественной FISH. Настоящая методика была использована для анализа расположения и распределения сигналов в интерфазных ядрах клеток различных соматических тканей человека. Использование 2D профилей интенсивности продемонстрировало возможность определения колокализации FISH-сигналов. Более того, предложенный подход позволил идентифицировать реплицированные сигналы, дать оценку эффективности гибридизации и сравнительный анализ вариации содержания ДНК специфических участков хромосом. Построение 3D профилей показало распределение интенсивности в пределах площади сигнала. Применение этой методики позволило определить сосредоточение различных типов последовательностей ДНК: классическая сателлитная и альфоидная ДНК; геннонасыщенные (G-положительные полосы) и генноненасыщенные (G-отрицательные полосы) участки хромосом. Кроме того, методика дала возможность оценить расположение хроматина в интерфазных ядрах как культивированных, так и некультивированных клеток. В результате исследования был сделан вывод о том, что предлагаемый подход является эффективной дополнительной методикой для изучения ядерной организации, специфики вариации и расположения последовательностей ДНК в интерфазных ядрах, а также поведе- ния ядер при приготовлении хромосомных препаратов соматических клеток человека.
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I.Y. IOUROV 1, 2, S.G. VORSANOVA 1, 2, Y.B. YUROV 1, 2
1 National Research Center of Mental Health,
Russian Academy of Medical Sciences, Moscow, Russia
2 Institute of Pediatrics and Children Surgery, Roszdrav,
Moscow, Russia
FLUORESCENCE INTENSITY PROFILES
OF IN SITU HYBRIDIZATION SIGNALS
DEPICT GENOME ARCHITECTURE
WITHIN HUMAN INTERPHASE NUCLEI
An approach towards construction of two�dimensional
(2D) and three�dimensional (3D) profiles of interphase chro�
matin architecture by quantification of fluorescence in situ
hybridization (FISH) signal intensity is proposed. The tech�
nique was applied for analysis of signal intensity and distribu�
tion within interphase nuclei of somatic cells in different
human tissues. Whole genomic DNA, fraction of repeated
DNA sequences (Cot1) and cloned satellite DNA were used as
probes for FISH. The 2D and 3D fluorescence intensity pro�
files were able to depict FISH signal associations and somatic
chromosome pairing. Furthermore, it allowed the detection of
replicating signal patterns, the assessment of hybridization
efficiency, and comparative analysis of DNA content variation
of specific heterochromatic chromosomal regions. The 3D flu�
orescence intensity profiles allowed the analysis of intensity
gradient within the signal volume. An approach was found
applicable for determination of assembly of different types of
DNA sequences, including classical satellite and alphoid
DNA, gene�rich (G�negative bands) and gene�poor (G�posi�
tive bands) chromosomal regions as well as for assessment of
chromatin architecture and targeted DNA sequence distribu�
tion within interphase nuclei. We conclude the approach to be
a powerful additional tool for analysis of interphase genome
architecture and chromosome behavior in the nucleus of
human somatic cells.
Introduction. The investigation of high�order
chromatin arrangement is a promising direction in
cell biology and genetics due to the immense impact
of nuclear organization on functional genome
activity in somatic cells. Substantial progress made
throughout last decades has given us the look at how
positioning of chromosomes in the nucleus is linked
to genome functions. We know now that chromo�
somes are compartmentalized into specific vol�
umes, arrangement of chromatin is likely to be con�
served throughout mitotic division, transcriptional
activity is determined by positioning of a gene with�
in the chromosomal volume, chromatin arrange�
ment differs between cell types [1–3]. Despite of
these achievements, a number of gaps in our incom�
plete knowledge concerning nuclear organization in
interphase nuclei of different human somatic cells
evidence for the need of technology improvement
in chromatin arrangement studies. An approach to
quantify fluorescence in situ hybridization (FISH)
signals proposed earlier was shown to be useful for
applied molecular cytogenetics [4, 5]. This commu�
nication extends the potential of quantitative FISH
(QFISH) technique for chromatin arrangement
studies. Additionally, we propose for the first time
the construction of three�dimensional (3D) intensi�
ty profiles allowing the analysis of intensity distribu�
tion within signal area supposed to be useful for
determination of DNA sequence positioning and
variation in interphase.
Materials and methods. Peripheral blood lym�
phocytes were obtained from individuals with nor�
mal male and female karyotype. Chromosomal
preparations of blood lymphocytes were made
according to standard protocols with fewer modi�
fications [6, 7]. Interphase nuclei of chorionic villi
were obtained from specimens of spontaneous
abortions, which were identified to possess diploid
chromosome complement [8]. The fresh�frozen
brain tissue samples were acquired from the Brain
Bank of the National Research Center of Mental
Health of Russian Academy of Medical Sciences
(NRCMH RAMS). The processing of the adult
brain tissue for FISH was performed according to
a step�by�step protocol described earlier [9]. To
prepare the suspension of volumetric interphase
nuclei an acetic acid free processing procedure was
applied. In total, 20 samples of blood lympho�
cytes, 20 samples of chorionic villi, and 3 samples
of the adult brain were studied. FISH was per�
formed according to the previously described pro�
tocols [6, 10–12]. DNA probes used were those
ІSSN 0564–3783. Цитология и генетика. 2008. № 5 3
Оригинальные работы
© I.Y. IOUROV, S.G. VORSANOVA, Y.B. YUROV, 2008
ISSN 0564–3783. Цитология и генетика. 2008. № 54
I.Y. Iourov, S.G. Vorsanova, Y.B. Yurov
painting classical satellite DNA of chromosomes 1,
9, and 16; alphoid satellite DNA of chromosomes
17, and X; Cot1�DNA probe, cloned classical satel�
lite DNA sequences, all the centromeric DNA,
short interspersed repetitive elements, produces flu�
orescent R�banding or paints gene�rich euchromat�
ic regions of the genome depending on temporal
hybridization conditions; total�human�DNA�probe
suppressed by Cot1 DNA during hybridization
(paint euchromatic regions of the genome). All the
probes, taking part of the original collection of labo�
ratory of cytogenetics of NRCMH RAMS, were
described previously [6, 7, 13, 14]. QFISH was per�
formed as described previously [4, 5, 15]. The con�
struction of intensity profiles was made by digital
capturing of microscopic image by the monochrome
CCD camera, LG�3 grayscale scientific PCI frame
grabber, and Scion Image Beta 4.0.2 software
acquired from www. scioncorp.com. Both 2D (two
dimensional) and 3D profiles of FISH signal inten�
sities were obtained via the use of corresponding
software options and the macros supplied by the
manufacturer. Volumetric brain derived interphase
nuclei were analyzed through the corresponding
stacking options of the software. For 2D profile
analyses, 50 nuclei were evaluated per sample per
probe (DNA probes for chromosomes 1, 9, 16, 17, X
and Y) and 30 nuclei were evaluated per sample per
probe in case of 3D profile analyses.
Results and discussion. To test the versatility of
the approach dividing and postmitotic cells were
analyzed. The technique was elaborated through
studying interphase nuclei of chorionic villi, cul�
tured peripheral blood lymphocytes, and cells of
the adult brain. First, 2D profiles have demon�
strated interphase paired signals for autosomes,
which have been present in all the tissues analyzed.
This appeared as single signal with doubled relative
intensity in a nucleus. The occurrence of paired
signals significantly differed between tissues being,
however, slightly different from sample to sample
of the same tissue (Fig. 1). The higher incidence
was in human brain cell suspensions (average
48 %), while chorionic villi and blood lympho�
cytes demonstrated significantly lower incidences
of paired signals, average– 6.4 and 7.3 %, respec�
tively. As to the X chromosome, paired signals were
occasionally detected (1–5 nucleus per sample).
Nuclei exhibiting single signal with relative inten�
sity similar to each single signal of disomic nuclei
Fig. 1. QFISH with construction of 2D intensity profiles on
human interphase nuclei: I – QFISH on interphase nuclei
of chorionic villi showing that left nucleus have a single sig�
nal (relative intensity 3364 pixels) in contast to right one
with two signals (relative intensities 1540 and 1842 pixels).
Comparing the intensities of these signals, we come to the
conclusion that left nucleus have a paired signal; II – the fre�
quency of paired signals revealed by chromosome enumer�
ation probes for chromosomes 1, 9, 16, and 17 in the adult
brain (a), chorionic villi (b) and blood lymphocytes (c)
were considered as monosomic and were excluded.
Both chorionic villus and blood lymphocyte were
characterized by occurrence of replicated signals.
The rate of their incidence exceedingly varied from
sample to sample being observed in all samples
analyzed. Thus, the range of occurrence of repli�
cated signals was between 4 and 46 % in blood
lymphocytes and was 10–51 % in chorionic villi.
The human brain occasionally demonstrated
replicated signals. Replicated signals appeared as a
doubled signal with two peaks of identical relative
intensity having a connecting fluorescent track
(data not shown). Replicated signals were usually
coupled with a single signal in the case of chromo�
some X or occurred simultaneously in the case of
autosomes. However, deviated patterns of signal
appearance were observed in a small proportion of
cells (less than 2 %).
Additional interesting observation made during
the testing of the approach was referred to the pos�
sibility of assessment of both hybridization efficien�
cy and DNA sequence size variation in a chromo�
somal region. To monitor hybridization efficiency,
dependence between signal relative intensity and
time was obtained. The 100 % of hybridization effi�
ciency was considered the time point when relative
intensity of hybridization signals stopped to
change. Using different DNA probes, we succeed�
ed to define this point as nearly 960 minutes or
16 hours (Fig. 2). This feature of the approach was
not only useful for selecting optimal hybridization
conditions, but also provided for selecting condi�
tions for the analysis of DNA seuquences distribu�
tions (using Cot1�DNA� and total�human�DNA�
probe) in 3D intensity profiles assay. Through
comparing the ratio of relative signal intensities of
homologous chromosomes in metaphase spreads
and interphase nuclei, we found almost complete
equality between these values in all the samples
analyzed (data not shown). Therefore, the assay
proposed can define relative size of DNA sequence
in a specified chromosomal region in interphase.
Comparing the intensity of profiles obtained by
sequential quantification of FISH signals through
the stacks of volumetric nuclei, we found that the
area of intensity profiles (the value of relative inten�
sity) changed insignificantly, whereas the shape of
the curve tended to change. This means that analy�
sis of intensity profiles of FISH signals allows to
analyze DNA size and behavior independently
from processes happening with nucleus during pre�
paration of cell suspensions.
During analyses with DNA probes painting dif�
ferent repetetive DNAs of cellular genome, we
have noticed that the distribution of intensity with�
in area is non�random and tends to vary within
nuclear area. To address this type of DNA behav�
ior, QFISH constructing 3D intensity profiles was
applied. The latter has shown that satellite DNAs
tend to gather forming large assemblies, which
corresponded to single, double and multiple 3D
fluorescent intensity peakes (Fig. 3, a). Using this
tentative classification, we were able to show tis�
ІSSN 0564–3783. Цитология и генетика. 2008. № 5 5
Fluorescence intensity profiles of in situ hybridization signals depict genome
Fig. 2. QFISH identification of optimal hybridization condi�
tions. Relative intensities were measured at different time
points. The 100 %�efficiency was considered the point when
relative intensities ceased to change: 960 minutes or 16 hours
Fig. 3. QFISH with construction of 3D intensity profiles on
interphase nuclei of the adult human brain with Cot1�DNA
probe at temporal conditions when all large satellite DNAs
are painted: a– assembly of satellite DNA appearing as single
peak; b– assemblies of satellite DNA appearing as multiple
peaks (mainly 3 peaks)
sue�specific patterns of repetetive DNA assem�
blies. Thus, large assemblies with single 3D fluo�
rescent intensity peak per nucleus were more fre�
quent in adult brain cells (mean 34 %), whereas in
chorionic villi and blood cells satellite DNA assem�
blies were relatively uncommon (mean 5 and 3 %,
respectively). Large assemblies appearing as dou�
ble 3D fluorescent intensity peak per nucleus were
less common as to single�peak�assemblies in the
adult brain being, however, more frequent as to
other tissues; mean frequencies of such assemblies
were 17 % (the adult brain), 7 % (chorionic villi),
and 4 % (blood lymphocytes). Satellite DNA assem�
blies characterized by multiple 3D fluorescent
intensity peaks per nucleus (more than 2 relatively
large 3D intensity profiles) were rare in the adult
brain (mean frequency 3 %), but were more com�
mon in chorionic villi (mean frequency 9 %), and
even more common in blood lymphocytes (mean
frequency 33 %). The assemblies were almost exclu�
sively referred to satellite DNA (both classical and
alpha satellite), whereas gene�rich euchromatic
regions and gene�poor euchromatic regions tended
to be distributed regularly within nuclear area.
Although gene�rich euchromatic regions more
commonly were distrubted along nuclear periph�
ery in contrast to gene�poor euchromatic regions
that more commonly tended to position closely to
nuclear center, no definitive conclusion could be
drawn, inasmuch as consistent pattern of this
DNA behavior was not observed due to exreme
arrangement variation of these DNA types.
The behavior of DNA within nuclear volume is
highly variable and appears to be crucial for func�
tional genome activity [1–3]. However, an inte�
grated view of direct relationship between chro�
mosome or specific DNA type positioning in the
nucleus and critical intracellular processes (i.e.
transcription or translation) is far from being cre�
ated. In major part this is due to technical limita�
tions, which are inteneded to be overcomed by
enhancing and modifying molecular cytogenetic
techniques [4]. The approach proposed seems to
be an interesting additional tool for studying chro�
matin behavior in interphase. Moreover, it granted
to make important observations of DNA position�
ing in interphase nuclei.
We found that genome organization varies
between tissues, as the incidence of paired chro�
mosome regions and large type�specific DNA
assemblies was different in each tissue analyzed.
This agrees with a previous study of murine ge�
nome orgnization in different tissues that conclud�
ed the existence of tissue�specific patterns of chro�
matin arrangement in interphase [16]. Futher�
more, we have observed that non�transribed DNA
sequences (satellite DNAs) possess rather complex
behavior allowing to suggest a functional role of
their positioning in interphase. The organization
of different DNA types on chromosomes (chro�
mosome banding relationship to base composition
in a chromosomal region) is featured by specific
gene content: G�negative/R�positive bands are
gene�rich regions, G�positive/R�negative bands –
gene�poor regions, chromosomal region consist�
ing of constitutive heterochromatin (C�bands) –
non�transcribed sequences apparently lacking
genes (satellite DNA) [17]. This classification was
previously used for analyses of genome organiza�
tion in lymphocytes and cancer cell lines. As a
result, there was found that chromatin arrange�
ment closely correlates with banding patterns of
human chromosomes: gene�poor regions and C�
band�regions are preferentially localized at
nuclear periphery and around the nucleolus,
whereas gene�rich regions are more frequently
positions inside the nuclear volume rarely tether�
ing nuclear membrane and the nucleolus [18]. The
present findings confirm these observation in some
extent as well as adding a new type of chromatin
behavior referred to as association of chromosome
regions or large�scale type�specific DNA assemblies
in interphase. Together, this suggests the present
approach to expand current possibilities of intr�
erphase genome organization studies and allows to
identify new types of chromatin behavior.
Another technical key point for all the studies of
chromosomes refers to their behavior during prepa�
rations of metaphase spreads or interphase nuclei
including tissue processing, fixation and delivery
on slides. Current concepts suggest these proce�
dures to influence rather chromosome length and
spreading of G�positive bands than chromatin
behaviour as the positioning of chromosomes
appears to be relatively conserved during the deliv�
ery of nuclei/metaphase suspension on slides [19].
Therefore, one can modulate chromatin behavior
in living cells skipping sophisticated volumetric
preparations. Considering this suggestion, it ap�
pears to be useful to monitor different conjunctions,
ISSN 0564–3783. Цитология и генетика. 2008. № 56
I.Y. Iourov, S.G. Vorsanova, Y.B. Yurov
associations and assemblies of different DNA types
and define the differences between volumetric and
routine nucleus preparations. The approach pro�
posed could be also applied for these aims.
In summary, a QFISH protocol may be not
only applied for analysis of chromosome abnor�
malities and chromosomal mosaicism or identifi�
cation of chromosome parental origin [4, 5, 15],
but also for studying nuclear organization. Futher�
more, the construction of 3D intensity profiles pro�
vides for additional information concerning DNA
behavior in interphase being, therefore, a new
technical milestone in chromatin behavior studies
achieved. It is noteworthy, that recent molecular
cytogenetic studies of the brain have proposed a
new biomedical direction termed molecular neu�
rocytogenetics (encompassing studies of chromo�
some numbers and behavior in the central nervous
system), which requires additional technical tools
to take the well�deserved place in current bio�
medecine [20]. Positive results, that were obtained
here studying the adult human brain, present the
approach described as an additional powerful
technique for genetic and cell biology investiga�
tions of the human brain.
We express our gratitude to Dr. Ilia V. Soloviev
for original ideas that form our current research
directions. Technical assistance of Alexei D. Kolotii,
Victor V. Monakhov, and Oxana S. Kurinnaya is
acknowledged. Support of this study is partially pro�
vided by INTAS 03–51–4060.
И.Ю. Юров, С.Г. Ворсанова, Ю.Б. Юров
АНАЛИЗ ПРОФИЛЯ ИНТЕНСИВНОСТИ
СИГНАЛОВ ФЛЮОРЕСЦЕНТНОЙ
ГИБРИДИЗАЦИИ IN SITU
ДЛЯ ИЗУЧЕНИЯ ОРГАНИЗАЦИИ ГЕНОМА
В ИНТЕРФАЗНЫХ ЯДРАХ КЛЕТОК ЧЕЛОВЕКА
Представлен метод построения двухмерных (2D)
и трехмерных (3D) профилей интенсивности сигна�
лов флюоресцентной гибридизации in situ (FISH),
основанный на количественной FISH. Настоящая ме�
тодика была использована для анализа расположения
и распределения сигналов в интерфазных ядрах кле�
ток различных соматических тканей человека.
Использование 2D профилей интенсивности проде�
монстрировало возможность определения колокали�
зации FISH�сигналов. Более того, предложенный
подход позволил идентифицировать реплицирован�
ные сигналы, дать оценку эффективности гибридиза�
ции и сравнительный анализ вариации содержания
ДНК специфических участков хромосом. Построение
3D профилей показало распределение интенсивности
в пределах площади сигнала. Применение этой мето�
дики позволило определить сосредоточение различ�
ных типов последовательностей ДНК: классическая
сателлитная и альфоидная ДНК; геннонасыщенные
(G�положительные полосы) и генноненасыщенные
(G�отрицательные полосы) участки хромосом. Кроме
того, методика дала возможность оценить расположе�
ние хроматина в интерфазных ядрах как культивиро�
ванных, так и некультивированных клеток. В резуль�
тате исследования был сделан вывод о том, что
предлагаемый подход является эффективной допол�
нительной методикой для изучения ядерной организа�
ции, специфики вариации и расположения последова�
тельностей ДНК в интерфазных ядрах, а также поведе�
ния ядер при приготовлении хромосомных препаратов
соматических клеток человека.
І.Ю. Юров, С.Г. Ворсанова, Ю.Б. Юров
АНАЛІЗ ПРОФІЛЮ ІНТЕНСИВНОСТІ
СИГНАЛІВ ФЛУОРЕСЦЕНТНОЇ
ГІБРИДИЗАЦІЇ IN SITU
ДЛЯ ВИВЧЕННЯ ОРГАНІЗАЦІЇ ГЕНОМУ
В ІНТЕРФАЗНИХ ЯДРАХ КЛІТИН ЛЮДИНИ
Представлено метод побудови двомірних (2D) та
тримірних (3D) профілів інтенсивності сигналів флу�
оресцентної гібридизації in situ (FISH), що заснова�
ний на кількісній FISH. Наведена методика була ви�
користана для аналізу розташування та розподілу
сигналів в інтерфазних ядрах клітин різних соматич�
них тканин людини. Використання 2D профілів ін�
тенсивності продемонструвало можливість визначен�
ня локалізації FISH�сигналів. Більш того, даний
підхід дозволив ідентифікувати репліковані сигнали,
дати оцінку ефективності гібридизації та провести по�
рівняльний аналіз варіації вмісту ДНК специфічних
ділянок хромосом. Побудова 3D профілів показала
розподіл інтенсивності у межах площі сигналу. Вико�
ристання цієї методики дозволило визначити зосеред�
ження різних типів послідовностей ДНК: класична
сателітна та альфоїдна ДНК; генонасичені (G�пози�
тивні полоси) і геноненасичені (G�негативні полоси)
ділянки хромосом. Крім цього, методика надала
можливість оцінити розташування хроматина в інтер�
фазних ядрах як культивованих, так і некультивова�
них клітин. Зроблено висновок, що наведений підхід є
ефективною додатковою методикою для вивчення
ядерної організації, специфіки варіації та розташуван�
ня послідовностей ДНК в інтерфазних ядрах, а також
поведінки ядер при приготуванні хромосомних препа�
ратів соматичних клітин людини.
ІSSN 0564–3783. Цитология и генетика. 2008. № 5 7
Fluorescence intensity profiles of in situ hybridization signals depict genome
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Received 26.05.07
ISSN 0564–3783. Цитология и генетика. 2008. № 58
I.Y. Iourov, S.G. Vorsanova, Y.B. Yurov
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| id | nasplib_isofts_kiev_ua-123456789-8218 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 0564-3783 |
| language | English |
| last_indexed | 2025-11-26T01:42:32Z |
| publishDate | 2008 |
| publisher | Інститут клітинної біології та генетичної інженерії НАН України |
| record_format | dspace |
| spelling | Iourov, I.Y. Vorsanova, S.G. Yurov, Y.B. 2010-05-14T14:15:19Z 2010-05-14T14:15:19Z 2008 Fluorescence intensity profiles of in situ hybridization signals depict genome architecture within human interphase nuclei / I.Y. Iourov, S.G. Vorsanova, Y.B. Yurov // Цитология и генетика. — 2008. — Т. 42, № 5. — С. 3-8. — Бібліогр.: 20 назв. — англ. 0564-3783 https://nasplib.isofts.kiev.ua/handle/123456789/8218 An approach towards construction of two-dimensional (2D) and three-dimensional (3D) profiles of interphase chromatin architecture by quantification of fluorescence in situ hybridization (FISH) signal intensity is proposed. The technique was applied for analysis of signal intensity and distribution within interphase nuclei of somatic cells in different human tissues. Whole genomic DNA, fraction of repeated DNA sequences (Cot1) and cloned satellite DNA were used as probes for FISH. The 2D and 3D fluorescence intensity profiles were able to depict FISH signal associations and somatic chromosome pairing. Furthermore, it allowed the detection of replicating signal patterns, the assessment of hybridization efficiency, and comparative analysis of DNA content variation of specific heterochromatic chromosomal regions. The 3D fluorescence intensity profiles allowed the analysis of intensity gradient within the signal volume. An approach was found applicable for determination of assembly of different types of DNA sequences, including classical satellite and alphoid DNA, gene-rich (G-negative bands) and gene-poor (G-positive bands) chromosomal regions as well as for assessment of chromatin architecture and targeted DNA sequence distribution within interphase nuclei. We conclude the approach to be a powerful additional tool for analysis of interphase genome architecture and chromosome behavior in the nucleus of human somatic cells. Представлено метод побудови двомірних (2D) та тримірних (3D) профілів інтенсивності сигналів флуоресцентної гібридизації in situ (FISH), що заснований на кількісній FISH. Наведена методика була використана для аналізу розташування та розподілу сигналів в інтерфазних ядрах клітин різних соматичних тканин людини. Використання 2D профілів інтенсивності продемонструвало можливість визначення локалізації FISH-сигналів. Більш того, даний підхід дозволив ідентифікувати репліковані сигнали, дати оцінку ефективності гібридизації та провести порівняльний аналіз варіації вмісту ДНК специфічних ділянок хромосом. Побудова 3D профілів показала розподіл інтенсивності у межах площі сигналу. Використання цієї методики дозволило визначити зосередження різних типів послідовностей ДНК: класична сателітна та альфоїдна ДНК; генонасичені (G-позитивні полоси) і геноненасичені (G-негативні полоси) ділянки хромосом. Крім цього, методика надала можливість оцінити розташування хроматина в інтерфазних ядрах як культивованих, так і некультивованих клітин. Зроблено висновок, що наведений підхід є ефективною додатковою методикою для вивчення ядерної організації, специфіки варіації та розташування послідовностей ДНК в інтерфазних ядрах, а також поведінки ядер при приготуванні хромосомних препаратів соматичних клітин людини. Представлен метод построения двухмерных (2D) и трехмерных (3D) профилей интенсивности сигналов флюоресцентной гибридизации in situ (FISH), основанный на количественной FISH. Настоящая методика была использована для анализа расположения и распределения сигналов в интерфазных ядрах клеток различных соматических тканей человека. Использование 2D профилей интенсивности продемонстрировало возможность определения колокализации FISH-сигналов. Более того, предложенный подход позволил идентифицировать реплицированные сигналы, дать оценку эффективности гибридизации и сравнительный анализ вариации содержания ДНК специфических участков хромосом. Построение 3D профилей показало распределение интенсивности в пределах площади сигнала. Применение этой методики позволило определить сосредоточение различных типов последовательностей ДНК: классическая сателлитная и альфоидная ДНК; геннонасыщенные (G-положительные полосы) и генноненасыщенные (G-отрицательные полосы) участки хромосом. Кроме того, методика дала возможность оценить расположение хроматина в интерфазных ядрах как культивированных, так и некультивированных клеток. В результате исследования был сделан вывод о том, что предлагаемый подход является эффективной дополнительной методикой для изучения ядерной организации, специфики вариации и расположения последовательностей ДНК в интерфазных ядрах, а также поведе- ния ядер при приготовлении хромосомных препаратов соматических клеток человека. We express our gratitude to Dr. Ilia V. Soloviev for original ideas that form our current research directions. Technical assistance of Alexei D. Kolotii, Victor V. Monakhov, and Oxana S. Kurinnaya is acknowledged. Support of this study is partially provided by INTAS 03–51–4060. en Інститут клітинної біології та генетичної інженерії НАН України Оригинальные работы Fluorescence intensity profiles of in situ hybridization signals depict genome architecture within human interphase nuclei Аналіз профілю інтенсивності сигналів флуоресцентної гібридизації in situдля вивчення організації геному в інтерфазних ядрах клітин людини Анализ профиля интенсивности сигналов флюоресцентной гибридизации in situ для изучения организации генома в интерфазных ядрах клеток человека Article published earlier |
| spellingShingle | Fluorescence intensity profiles of in situ hybridization signals depict genome architecture within human interphase nuclei Iourov, I.Y. Vorsanova, S.G. Yurov, Y.B. Оригинальные работы |
| title | Fluorescence intensity profiles of in situ hybridization signals depict genome architecture within human interphase nuclei |
| title_alt | Аналіз профілю інтенсивності сигналів флуоресцентної гібридизації in situдля вивчення організації геному в інтерфазних ядрах клітин людини Анализ профиля интенсивности сигналов флюоресцентной гибридизации in situ для изучения организации генома в интерфазных ядрах клеток человека |
| title_full | Fluorescence intensity profiles of in situ hybridization signals depict genome architecture within human interphase nuclei |
| title_fullStr | Fluorescence intensity profiles of in situ hybridization signals depict genome architecture within human interphase nuclei |
| title_full_unstemmed | Fluorescence intensity profiles of in situ hybridization signals depict genome architecture within human interphase nuclei |
| title_short | Fluorescence intensity profiles of in situ hybridization signals depict genome architecture within human interphase nuclei |
| title_sort | fluorescence intensity profiles of in situ hybridization signals depict genome architecture within human interphase nuclei |
| topic | Оригинальные работы |
| topic_facet | Оригинальные работы |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/8218 |
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