Vol. 56, no. 3: 289-294, 2003
CARYOLOGIA
Evidences for continuous genome in Impatiens
balsamina (Balsaminaceae)
S. GHOSH
Department of Botany, M.U.C. Women’s College, Burdwan, 713 104, West Bengal, India.
Abstract - The connections between chromosomes through telomeres by chromatin-containing connectives are on record. In the present investigation these
connectives are studied using acetic-orcein and acetic-carmine as well as DNAspecific Giemsa and fluorochrome staining methods in the various stages of
mitosis in cells derived from both haploid and diploid phases of Impatiens balsamina L. (2n=14). Results of this study suggest that the fully disentangled, taut
interchromosomal chromatin-positive connectives between discrete chromosomes are inherent structures that reflect a continuous genome in this plant.
During divisional stages, especially at metaphase, when chromosomes have
become maximally coiled and folded, interchromosomal chromatin connectives
are no longer resolvable. These results suggest that, in this plant, end-to-end
chromosome connections may provide the physical basis for maintenance of the
positional stability of the chromosomes throughout the cell cycle. The role of
ordered chromosome topology is discussed in the light of the developing concepts.
Key words: Chromosome connection; Continuous genome; Interchromosomal
chromatin connectives.
INTRODUCTION
Chromosomes are the coiled and folded state
of interphase chromatin found in the metaphase
stage of cell division. Each chromosome in
eukaryotic cells is thought to be composed of
single linear DNA molecule that replicates to
form the two chromatids that are held together
at the centromere. The number of chromosomes
is species-specific and, at interphase, each chromosome appears to occupy its own nuclear territory, with little or no overlap with adjacent
chromosome territories (LODISH et al. 2000). A
single chromosome has its own individuality and
is capped at each end by telomere. In order to
* Corresponding author: e-mail: [email protected]
duplicate and segregate correctly each chromosome must possess intact telomeres since excessive telomere shortening has been linked to chromosome “end sticky” and improper segregation
at mitosis. End-to-end attachments of chromosomes in cells from a variety of plants and animals have been reported (W AGENAAR 1969;
COSTELLO 1970; TAKAYAMA 1975; BAHR 1977;
A SHLEY 1979; L AVANIA and S HARMA 1984;
GAVRILA et al. 1995). Tandemly linked “haploid
sets” containing 23 chromosomes each was
demonstrated by NAGELE et al. (1998) in human
cells. NAGELE et al. (1999) also reported the existence of an ordered interphase chromosome
topology in quiescent human cell. Topological
separation of parental genomes in preimplantation mouse embryos was reported by MAYER et
al. (2000). However, information is currently
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GHOSH
being accumulated regarding the topology of the
chromosomes in the nucleus and its implications.
The results of the present investigation suggest
that the positional stability of the chromosomes
in mitosis is maintained by the aboriginal end-toend attachment of the chromosomes by interchromosomal chromatin connectives during the
mitotic divisional stages of both haploid and
diploid phases in Impatiens balsamina (2n=14).
This study raises a question with regard to the
individuality of each of the several discrete chromosomes in a haploid set and favours the continuity of the haploid set of chromosomes in this
plant material.
MATERIAL AND METHODS
Pink “Camellia flower” cultivar of Impatiens balsamina L. (2n=14) from Sutton and Sons (India)
Pvt. Ltd., Kolkata, was used for this investigation.
Root-tip cells were studied by staining with the conventional acetic-orcein method. In some cases, the
C-banding method was also employed (GHOSH and
B HANJA 1982). In addition, the fluorochrome
Hoechst 33258 (0.02%) was used according to VOSA
and MARCHI (1972) during the study of root tip cells
and observed under the fluorescence microscope.
Divisional stages in the pollen grain were studied
using conventional acetic carmine stain and
observed under phase contrast microscope. Division of the generative cell was studied using supravital staining with Hoechst 33258 (GHOSH and BHANJA 2000).
RESULTS
Prometaphase cells stained using the C-banded method in the root tip of I. balsamina revealed
thin and faintly stained chromatin material interconnecting chromosomes end-to-end (Fig. 1).
Some of the metaphase chromosomes were
found to be connected by interchromosomal
chromatin connectives (Figs. 2 and 3). Connections between the chromosomes of metaphase
stage were frequently observed in preparations
subject to chromosome banding with the DNAspecific fluorochrome Hoechst 33258 (Fig. 3).
Sometimes chains of tandemly connected
metaphase chromosomes were also found in
preparations stained with conventional aceticorcein staining (Fig. 4). Although present, but
more difficult to resolve, connections were
observed between the orderly arranged chromosomes in the prometaphase stage of pollen
mitosis using negative phase contrast microscopy
(Fig. 5). During metaphase of pollen mitosis broken chains of haploid sets of chromosomes were
also visible (Fig. 6). The prophase stage (Fig. 7)
of the generative nucleus of the pollen grain and
the prometaphase stage of the same (Fig. 8) from
an in vitro germinating pollen grain display chromosomes arranged into a chain that are connected to each other by interchromosomal chromatin connectives. Occasionally, these chains
appear to be closed (Fig. 8). Interchromosomal
chromatin connections in prometaphase stage
of generative nucleus were found in about 70%
cases. The linear chain like array of metaphase
chromosomes of the generative nucleus from the
in vitro grown pollen tube might be the result of
the telomeric connections (Fig. 9). The continuity of the chromatin in between several discrete
chromosomes was discernible in each of the two
daughter chains of chromosomes at anaphase
stage of the generative nucleus (Fig. 10) from
the in vitro grown pollen tube. Figure 11 shows
a chain of the chromosomal blocks in a sperm
nucleus, in a stretched condition, again supporting the continuity of chromosomes in the
haploid set of this material and thereby ensuring
the positional stability of the chromosomes in
the sperm nucleus.
DISCUSSION
Interchromosomal chromatin connections in
the prometaphase root tip cell (Fig. 1) partially
support the idea of CHIARELLI (1976) in which it
was claimed that the whole haploid set of chromosomes are connected one after another. But in
this figure the presence of two chains are not distinguishable. Specific end-to-end attachments of
chromosomes in Ornithogalum virens (n=3) were
demonstrated by ASHLEY (1979) and it was suggested that the proximity of homologous ends
and consequently homologous alignment might
facilitate initiation of pairing at meiosis. In Impatiens balsamina the interconnected genome containing 7 chromosomes represents a nonhomologous alignment and thus end-to-end attachment
might not facilitate initiation of pairing at meiosis.
The interchromosomal chromatin connectives in
Impatiens are mostly of euchromatin nature.
However, LAVANIA and SHARMA (1984) reported
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CONTINUOUS GENOME IN IMPATIENS BALSAMINA
1
1 µm
3
4
1 µm
2
1 µm
5
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1 µm
1 µm
6
Fig. 1 – A prometaphase cell showing 14 interconnecting somatic chromosomes in Impatiens root tip squash stained with 1
percent Giemsa solution at pH 7.0.
Fig. 2 – Metaphase from the root tip cell showing interchromosomal connections mostly between telomeres of some chromosomes stained with 1 percent Giemsa solution at pH 7.0.
Fig. 3 – Metaphase chromosomes fluorochromed with Hoechst 33258 showing connections in between some chromosomes
in the complement.
Fig. 4 – Acetic-orcein stained metaphase plate with 11 chromosomes most of them are connected through interchromosomal
chromatin connectives.
Fig. 5 – Prometaphase stage of pollen mitosis observed under negative phase contrast microscope. This plate indicates the
continuation of the genome with orderly arrangement of chromosomes.
Fig. 6 – Metaphase stage of pollen mitosis within pollen grain stained with acetic carmine. Four out of 7 chromosomes show
connection.
that, in the majority of cases, connections between
metaphase chromosomes in root tip cells of various plant species in Leguminosae are made up of
constitutive heterochromatin. In this material the
chromatin connections delineated are of the endto-end type, and other types such as interstitial,
centromere to telomeres etc. as noted by BAHR
(1977), LAVANIA and SHARMA (1984) and the present study (Figs. 2, 3 and 6) may be due to some
problem in spreading of metaphase chromosome
adequately. However, it was found that end-toend connections are most frequently observed in
this material as observed by others in Allium cepa,
Crepis capillaries, Callitriche hermaphroditica,
Nigella arvensis and Secale cereale (WAGENAAR
1969), in Rumex acetosa (MOUTSCHEN et al. 1970),
and in various species of Leguminosae (LAVANIA
and SHARMA 1984). In the present study the possibility that connectives are fixation-artifacts can
safely be ruled out by study of the generative and
sperm nuclei since these were observed in a
supravital condition using aqueous fluorochromes
and immediately after preparation. CHALY and
BROWN (1988) examined early mitotic stages in
HeLa cells and proposed a model considering the
prometaphase configuration as an important linking step to maintain intragenomic order. The
results of the present experiment suggest that
interchromosomal chromatin connectives, clearly
visible at prometaphase may conserve the positional stability of the chromosomes in genome
throughout the cell cycle. Nonrandom spatial
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GHOSH
7
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10
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Fig. 7 – Hoechst 33258 stained prophase stage of the generative nucleus squashed out from pollen grain showing chain of haploid set (n=7) of interconnected chromosomes with positional stability.
Fig. 8 – Hoechst 33258 stained prometaphase stage of the generative nucleus crushed out from the in vitro germinating pollen
grain showing the continuation of the genome forming a ring of 7 chromosomes.
Fig. 9 – The haploid set of 7 late metaphase chromosomes, squashed out from in vitro grown pollen tube and stained by acetic
carmine, remains in the form of a distinct chain. Here the interchromosomal chromatin connectives can barely be seen between
the highly compacted chromosomes.
Fig. 10 – Anaphase stage of generative nucleus, stained with Hoechst 33258, in which the mother chin splits into two distinct
daughter chains maintaining the topology of the chromosomes due to continuation of the genome.
Fig. 11 – Two fluorochromed sperm nuclei show a chain of chromosomes in each. Stretching of one chain shows obvious connections resulting the specific position of the chromosomes in the sperm nucleus.
positioning of the 46 human chromosomes during
division was reported by MOSGOLLER et al. (1991).
The relative location of several chromosomes
within the wheel-shaped prometaphase chromosome rosettes of human fibroblast and HeLa cells
were determined by NAGELE et al. (1995), and
they suggested that chromosomes are separated
into two haploid sets, each derived from one parent. From this study it appears that in Impatiens
the topology of the chromosomes in the paternal
set remains intact due to the end-to-end connections between chromosomes. NAGELE et al. (1998)
also suggested that human chromosomes within
rosettes are segregated into tandemly linked “haploid sets” containing 23 chromosomes each. Analyzing the division of the generative nucleus in
Impatiens, it may be argued that all chromosomes
in the paternal set pass from one cell division to
the next with intact topology due to their attachments to one another by way of interchromosomal
chromatin connectives. The sperm nuclei in this
material showed the existence of an ordered linear
arrangement of interphase chromosomes (Fig. 11)
and this finding corroborates the observations by
N AGELE et al. (1999) in nuclei of quiescent
human-cells. Specific locations for chromosomes
in diploid lymphoblasts and primary fibroblasts
were also reported by BOYELE et al. (2001).
MAYER et al. (2000) argued that the genome
separation during early mammalian development
was functionally important, and further speculated that disturbances in genome separation are
not compatible with normal embryogenesis. If
this is true in this plant material, it may be proposed that continuation of the genome through
interchromosomal chromatin connectives is the
physical basis of genome separation. LAVANIA
and SHARMA (1984) suggested that interchromosomal connections play a role in maintaining
the coordinate functioning of the whole genetic
apparatus in the eukaryotic system. GAVRILA et
al. (1995) reported that the huge ring of non-
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CONTINUOUS GENOME IN IMPATIENS BALSAMINA
homologous chromosome is the proper arrangement of interphase chromosomes that is a “ sine
qua non” for an ordered function of genes and
for a correct replication of the genetic material in
eukaryotes. A linear array of 7 chromatin blocks
(n=7) in interphase (GHOSH and BHANJA 2000)
and prophase (Fig. 7) in generative and sperm
nuclei (Fig. 11) of Impatiens balsamina suggest
that the positional stability of chromosomes is
maintained throughout the cell cycle due to continuation of the genome through chromatin connectives. This topology of the chromosomes may
be associated with the coordinate functioning
of the genome. NAGELE et al. (2001) reported
that the telomeres associations in interphase
nuclei of human cells played a role in maintenance of interphase chromosome topology, and
that disruptions of this topology, such as in aneuploid cancer cells, may be somehow related to
their abnormal gene expression pattern. During
mitosis in chaffinch intercentromeric connectives between nonhomologous chromosomes
maintain the order of chromosomes (SAIFITDINOVA et al. 2001). From the present study it
appears that function of the end-to-end interchromosomal chromatin connectives is to maintain the order of chromosomes throughout the
cell cycle.
Thus, from the above discussion, more than
one linkage group in the form of several discrete
chromosomes is apparent in each cell and the
inherent continuity between the chromosomes
through the interchromosomal chromatin connectives is barely visible due to the fully disentangled taut fibril nature of the connectives. In
some cases especially at prometaphase stages of
the mitotic division in both haploid phase and
diploid phase the connections becomes discernible. However, from this study as well as
from the corroborative reports by others, it may
be concluded that the genome of Impatiens balsamina is continuous, and the interchromosomal chromatin connectives are the physical basis
of this continuation that, in turn, maintains the
positional stability of the chromosomes throughout the cell cycle.
Acknowledgements – The author is grateful to
Dr. Robert Nagele (UMDNJ-SOM, Department of
Molecular Biology, Two Medical Center Drive,
Stratford, New Jersy 08084) for his valuable suggestions and editorial comments during the preparation of this manuscript.
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Received November 27, 2002; accepted February 7, 2003