Spatial arrangement of constitutive heterochromatin and euchromatin in somatic cell

cycle of Rattus rattus, from Manipur, India.

Dhananjoy CH 1*, J.M. Laishram1, Bhagirath Th. 3,Ibemhal A1., Brajendro N.1,C B Singh2, L.
Surendrajit3 A., L. Surbala 3 , S. Monteswori 3
1
Central Agricultural University, PBG Deptt., Manipur-7985004, India.
2
Institute of Bioresources & Sustainable Development, Manipur, India.

3
Deparment of Life Sciences, Manipur University, Imphal-795003, India

* author for correspondence (dhananjoych@gmail.com)

Abstract
We demonstrate the importance of heterochromatin in studying the chromosome

territories in rat. The Rattus rattus species from Manipur, India has eight heterochromatic

blocks on p-arms of autosomes besides centromeric heterochromatins in bone marrow cells.

With these blocks as reference points, the chromosome territories in interphase nuclei and

different stages of cell divisions are studied. The heterochromatic blocks are always located

at the periphery as deeply stained spherical body in Giemsa staining. The euchromatic region

attached to heterochromatic as lightly stained region in interphase nuclei just like Pappas.

The increase in euchromatic regions after S-phase is towards the nuclear centre and lightly

stained. The size of heterochromatic block is almost constant throughout the interphase, and

prophases or pro-metaphases. However, the heterochromatic blocks decreases as segregation

of centromeres and p-arms of respective chromosomes take place in metaphase. In prophases

and pro-metaphases clear and distinct Rabl arrangement can be observed. In metaphase

stages chromosome territories are disturbed. This study gives an opportunity to study

chromosome territories in unavailability of much sophisticated techniques in cytogenetics

using heterochromatin as markers.

Key words: heterochromatin, Chromosome territories, rat, nuclei, euchromatin, interphase.

Introduction

The cytogenetics in recent years focus on the chromosome architecture than the

number, structure and abnormalities both in plants and animals. Since the late 19 th century,

an uncounted number of microscopic studies have appeared on numerous aspects of nuclear

structure and on the observation of mitotic chromosomes (Creamer and Creamer 2010). A

territorial organization of interphase chromosomes was first suggested for animal cell nuclei

by Carl Rabl (Rabl 1885),but it was Theodor Boveri who introduced the term chromosome

territory (CT) in his seminal studies of blastomere stages of the horse roundworm Parascaris

or Ascaris megalocephala (Boveri 1909). Recent experiments concerning the radial

positioning of chromosomes in the nuclear volume of human and primate lymphocyte cell

suggest a relationship between the gene density of a chromosome territory (CT) and its

distance to the nuclear center (Kreth et. al. 2004). The simplest reason to organize

chromosomes into domains in the nucleus is to prevent them from becoming entangled,

(Essers et. al. 2005). To decide about the random or non-random distribution of a given

target, such as a CT, chromosomal sub region, or gene, it is important to define a simple or

sometimes multiple 3D(three dimensional) reference points, which represent the target in

question in the nuclear space. For a painted CT, its intensity gravity center can be chosen as a

single reference point or the CT surface may be used to define multiple reference points.

Next, proper reference structures must be defined to decide whether the chosen target is

distributed randomly or non-randomly with respect to them. For reference structures, one can

choose other chromatin targets or distinct nuclear structures, e.g., the nuclear lamina,

nucleoli, or splicing speckles (Ronneberger et. al. 2008).

 In the present study, we emphasized the heterochromatin present in the genome as

reference point and investigate the interphase and pro-metaphase in the wild rat having much

heterochromatic arms compare to the other rat species in the genes Rattus. Besides the non-

random arrangement of euchromatin and heterochromatin inside nuclei can be ascertained.

The present study will give an alternative and simpler method to study the CT in the absence

of much sophisticated techniques.

Materials and methods

Five live specimens (2 males and 3 females) of Rattus rattus were collected from

Kharam Waiphei, Singda of Manipur in 2010 and processed the cytological works from the

bone marrow cells of femur. After tentative identification of the specimens with the help of

reference book one representative specimen was sent to Zoological Survey of India for

confirmation of the identity and confirmed the same. Approval of the Institutional Ethics

Committee (IEC) was obtained for using live animals and protocols of the IEC were followed

throughout the study. Metaphase chromosomes were prepared from five males and five

females using standard colchicines- hypotonic spreading technique. The chromosomes were

stained with Giemsa or for C-bands (Sumner 1972) or for G-bands (Sumner et. al. 1971).

Chromosome number and morphology were recorded from 500 Giemsa – stained metaphases

from each specimen directly under 100X bright field optics of microscope- Olympus BX-41

as well as from photographs of selected cells. C-band and G-bands expression pattern were

examined in 500 metaphases each. The types of chromosome and karyotype were done

according to Yosida (1983). A total number of 5000 each of interphase nuclei, prophases,

pro- metaphases and metaphases were studied under 100X oil immersion and most cleared

observations were taken photographs in Olympus BX-41 phase contrast light microscope.
The snaps were taken under oil immersion with zoom(not digital) at 200 ASA of Olympus

digital camera attached to the microscope.

Results and discussion

In Giemsa stained the diploid number of the rat species, Rattus rattus was 42 with

#1,6,7,8,9,10 homomorphic subtelocentrics; #2,3,4,5,11,12,13 acrocentrics; #14-20 small

metacentrics and telocentric sex chromosomes(Figure 1). The longest chromosome had six

bands and least of two in sex chromosomes interphase nuclei(Figure 2). The C-banded

metaphase had eight distinct heterochromatin blocks as p arms of #,6,7,8, and 9 and most of

chromosomes had centromeric heterochromatin except #1 that had least centromeric

heterochromatin(Figure 3). The Rabl arrangement can be seen mostly in pro-metaphases

where heterochromatin blocks and centromeric heterochromatins at one pole and telomeres at

opposite pole(Figure 4). The un spreaded G-banded metaphase plate show clustering of the

heterochromatins of centromeres and p-arms and few chromosomes with less

heterochromatins(Figure 5). The clustering of heterochromatin was also visible in G-banded

early metaphase(Figure 6). When early metaphases were seen through C-banding, the

congregated chromatin region was C-band positive and most of the chromosomes were

detaching from that region(Figure 7). The interphase-G2 phase cells when examined through

normal Giemsa, without any pretreatment just after S-phase the deeply stained spherical body

corresponded to heterochromatic blocks and centromeres and lightly stained chromatin

corresponded to the non heterochromatic chromosomes(Figure 8) and always located at

periphery. G-banded cell of the same stage show dark spherical body and light stained

chromatin bodies(Figure 9). The darkly stained chromatin materials had connection with the

lightly stained chromatin materials(Figure10 lined). The connection between the

heterochromatin and euchromatin seem to be through the ring like structure of

heterochromatic regions as evidenced from Figure11(long arrowed).The early S-phase the

light stained seem to came out from the cage of darkly stained congregation of

heterochromatins in side view in normal Giemsa stained cells(Figure12).When viewed from

above of the same stage of the cell, the fine chromatin threads were physically connected to

the heterochromatin block(Figure 13). The C-banded in early S-phase cell show physical

connection between hetero- and euchromatin materials(Figure 14). Before the formation of

chromatin threads, euchromatin and heterochromatin were closely associated(Figure 15). In

G1-phase the presumed euchromatin was attached to the heterochromatin materials(Figure

16).

The results of our cytogenetic investigation provide critical and much important information
concerning the location of heterochromatins inside nucleus particularly in wild rats of species
Rattus rattus. Since Heitz (1928) introduced the term heterochromatin for a specific portion
the chromosome, it has been a mysterious in cytogenetics( Brown 1966; Yunis and Yasmineh
1971; Comings 1972). Traditional cytology classifies chromatin into less condensed
euchromatin and non-condensed heterochromatin. Heterochromatin has been further
subdivided into permanently condensed constitutive heterochromatin and facultative
heterochromatin, which becomes condensed/decondensed at some point during
development (Wegel and Shaw 2005). Constitutive heterochromatin is considered to have
discernible genetic function, although it interacts with euchromatic genes causing position
effect variegation in Drosophila(Hannah 1951; Baker 1968). Furthermore, centromeric
heterochromatin appears to be involved in changes of morphology through breakage and re-
fusion. Constitutive heterochromatin has been regarded to have a distinct role in
evolution(Sharma 1985) and also it is believed to play a significant role in evolution of the
karyotype in plants and animals(Swanson 1957; White 1973; Pathak et. al. 1973). Groups of
organisms possessing relatively more heterochromatin may be more successful in speciation
providing a variety of different karyotypes. Such an increased number of viable
chromosomal rearrangement favours reproductive isolation as a preliminary for speciation.
Constitutive heterochromatin arise through saltatary replication, they may induce an old
species, which has reached a dead end of evolution to rise to new forms(Sharma 1985). In
mammals , two principal components of mitotic chromosomes can be distinguished: G-light
bands(also called R-bands) replicate early during S-phase and contain most of the house
keeping but relatively few tissue-specific genes. G-dark bands replicate later, are gene poor,
and contain tissue-specific genes (Craig and Bickmore 1994). Recently it has been
demonstrated that these chromosome bands are maintained in interphase nuclei as focal
chromatin aggregations (Sadoni et. al. 1999) built up by a number of chromatin domains in
the order of ~ 1Mb. These domains apparently persist through all interphase stages, show
distinct nuclear localization patterns, and may provide an important component of the higher
order nuclear architecture(Cremer and Cremer 2001). Since during interphase the C-
heterochromatin is manifested as chromocenters, it may be easily visualized using either
fluorescent(Vosa 1970), or Giemsa(Sumner 1972). In our observations not a single
interphase nucleus with chromocenters is observed, so fusions of these structures might be
the reason for observation. The dark spherical body is the evidence for fusion of
chromocenters in the interphase stage in the absence of chromocenters(Figure 11). The size
of the heterochromatic regions throughout interphase stages remain constant(Figures 9 to 16)
despite little structural changes(Figures 8 and 10). The chromatin arrangement after the end
of telophase in cell cycle is not appreciated so far. Our observation in G 1 stage of interphase
suggest that euchromatin are attached to non replicative constitutive heterochromatin(Figure
15 and16). Heterochromatin is typically highly condensed, gene-poor, and transcriptionally
silent, whereas euchromatin is less condensed, gene-rich, and more accessible to
transcription. Besides acting as a graveyard for selfish mobile DNA repeats, heterochromatin
contributes to important biological functions, such as chromosome segregation during cell
division. Multiple features of heterochromatin-including the presence or absence of specific
histone modifications, DNA methylation, and small RNAs-have been implicated in
distinguishing heterochromatin from euchromatin in various organisms (Tamaru 2010 ). Our
observations point that the heterochromatin present on p-arms are non-replicative constitutive
and remain congregated with centromeric heterochromatins. They occupied specific domain
inside nuclear boundary most probably at the periphery. Probably the main determinant of
nuclear organization is the spatial distribution of chromosomes that occupy confined and
mostly non- overlapping volumes inside the boundaries set by the nuclear envelope(Cremer
and Cremer 2001). The spatial arrangement of chromosomes is non-random and follows
certain rules(Parada and Misteli 2002). First, during chromatin condensation and
decondensation, no major relative positional changes occur(Manders et. al. 1999 and 2003).
Second, during congregation of chromosomes to the metaphase plate, relative
neighbourhoods are still maintained(Gerlich et. al. 2003; Chally and Brown 1988). Third,
symmetrical chromosome positions were observed in post mitotic sister cells, suggesting a
dependence on the metaphase configuration, the “mitotic preset”(Sun and Yokota 1999). Our
observations are in accordance with above rules. In the C-banded metaphase plate
heterochromatic blocks on p-arms are more or less at the proximity one another(Figure 3)
and in prophases, prometaphases, the heterochromatic blocks are congregated(Figures
4,5,6,7) as in S-stage (Figures 8,9,10,11). The heterochromatin proteins that have been
shown to interact with nuclear membrane proteins(Ye and Worman 1996; Furukawa 1999)
might represent the molecular link that tethers gene-poor chromosomes rich in
heterochromatin to peripheral positions when they contact the nuclear envelope by random
movement during early G1 (Garlich and Ellenberg 2003). The euchromatin and
heterochromatin are seems to be connected at certain points(Figure 10,11,12,13) and almost
remain connected till prophases and early metaphases as reported by Visser and Aten (1999).
Our observation is against the occurrences of interchromatin compartments (Visser et. al.
2000) at least during interphase between eu- and heterochromatin. The deeply stained
heterchromatin are formed by the fusions of chromocenters of respective chromosome
( Sumner 1972) and euchromatic regions would be whole length of arms chromosomes.

These observations are substantiate by diminutions of heterochromatin block and appearance

of centromeres or p-arms in metaphases and pro-metaphases(Figure 3). So, it can be

concluded that constitutive heterochromatin and euchromatin regions of chromosomes are

non-randomly arranged in the interphase nuclei that persist till the early metaphases.

Furthermore, there is always an overlapping between two types of chromatin that remained

connected up to early metaphases particularly in rat nuclei.

 Acknowledgement

The authors are grateful to Dean, of Central Agricultural University, Imphal for allowing us

to carry on the work and all the persons working in the Plant Breeding & Genetics,

Department namely K. Romesh Singh and I. Dinachandra Singh. We are thanking to Subash,

ISOR for critical evaluation of the manuscript and Department of Biotechnology, India (dt.7th

2009) for supporting the work. The paper is dedicated to respected departed Prof. Ch.

Dhanachand, Parasitoloy, Manipur University, who taught how to go on researched and TH

Yosida for his contribution in study of rat cytology.

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 “Fig”

Fig1 Normal Giemsa stained metaphase plate, 2n=42 with # 1, 6, 7, 8, 9, 10,

homomorphic subteloccentric chromosomes ; # 2, 3, 4, 5, 11, 12, 13 telocentric
chromosome pairs ; # 14-20 small metacentric chromosomes ; telocentric XY.
X4500
Fig2 G-banded metaphase plate. 4500X
Fig3 C-banded metaphase plate, showing the 8 distinct heterochromatic blocks on p-arms
(arrow head) and centromeric heterochromatins. 4500X
Fig4 Rabl arrangement in early prophase in relation with heterochromatins (arrow) and
telomeres (arrow head). X 7500
Fig5 G-banded metaphase plate with the deeply stain heterochromatic blocks (arrow) and
euchromatic chromosomes (arrow head). X 7500
Fig6 G-banded early metaphase plate with distinct connection (long arrowed) between
the heterochromatic blocks and some of the chromosomes. X 7500
Fig7 C-banded prophase chromosomes with distinct centromeres (arrow head) and
heterochromatic block (arrow). X 7500

Fig8 The interphase cells stained with normal Giemsa show oval shaped dark
heterochromatin(arrow) and lightly stain chromatin materials(arrowhead). X 9000
Fig9 The interphase cell stained with G-banding shows clear connections among
condensed heterochromatin (arrow)connected with euchromatin(arrow head). X
9000
Fig10 G-banded interphase cell show irregular shaped heterochromatins (arrow) and
lightly stained euchromatin (arrow head) that were connected (line). X 9000

Fig11 C-banded early prophase stage of mitosis, showing deeply stained congregations of
Heterochromatins (short arrow) and elongated lightly stained euchromatins (arrow
head) with dark strips in between the two chromatins (long arrow). X 9000

Fig12 Normal Giemsa stained interphase cell with prophase chromosomes. The
interphase (S- phase ) show Pappas like structure in which the lightly stained
 chromatins(arrow head) seem to be coming out dark stained
heterochromatin(arrow) and line represent the connection of eu- and
heterochromatin.X 4500
Fig13 The S-phase cell stained with Giemsa in which small fibres (arrow head) were
connected with the deeply stained spherical body of heterochromatin (arrow).
7500X

Fig14 C-banded S-phase cell in which lightly stained euchromatic thin fibres (arrow head)
were connected with congregation of dark stained heterochromatic body(arrow).
7500X

Fig15 The early S-phase cell with stained with normal Giemsa stain the spherical shaped
heterochromatins (arrow) seem to caped over the lightly stained euchromatic mass
(arrow head). X 9000
Fig16 The interphase staged stained with normal Giemsa stain showing a short
euchromatic (arrow head) protrusion from a big rounded spherical heterochromatic
body(arrow). X 6000