1 Department of Cytology and Genetics, Novosibirsk State University, Novosibirsk
630090, Russia
2 Department of Cytology and Genetics, Novosibirsk 630090, Russia
* Author for correspondence (e-mail: zhimulev{at}bionet.nsc.ru)
Accepted 19 November 2002
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Summary |
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Key words: Heterochromatin, Polytenization, Chromosome 3, Drosophila melanogaster
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Introduction |
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At the cytological level, heterochromatin in mitotic chromosomes of
Drosophila melanogaster has been divided into 61 regions (designed
h1-h61) according to H- and N-staining
(Gatti et al., 1994), which
reflects heterogeneous contents of DNA. Sequences forming heterochromatin are
divided into highly repeated (satellites), moderately repeated (mainly
transposable elements) and occasional unique (genes). These sequences occur in
fixed positions within the heterochromatic regions
(Lohe et al., 1993
;
Pimpinelli et al., 1995
;
Carmena and Gonzalez, 1995
;
Makunin et al., 1999
). Large
blocks of simple satellite DNAs are interrupted by `islands' of complex
sequences (Le et al., 1995
).
The few heterochromatic genes are clustered within dull or moderately
fluorescent regions after H-staining
(Dimitri, 1991
;
Dimitri et al., 2003
;
Koryakov et al., 2002
).
Euchromatin and heterochromatin also differ in nucleosome and histone
organization. Transcriptionally active euchromatin has long tracts without
nucleosomes, which are hypersensitive for nucleases, whereas inactive
heterochromatin has long stretches with regular nucleosomal pattern
(Sun et al., 2001).
Heterochromatic histones carry epigenetically heritable modifications
including hypoacetylation of H3 and H4 histones and methylation of lysine-9 of
H3 histone (reviewed by Dillon and
Festenstein, 2002
; Richards
and Elgin, 2002
).
In addition to modified histones, Drosophila heterochromatin
contains the non-histone proteins HP1, Su(var)3-7, Su(var)3-9 and SuUR. The
HP1 protein is found mainly in pericentric heterochromatin, chromosome 4 and
telomeres (James and Elgin,
1986), where it binds to methylated lysine-9 of H3 histone
(Jacobs et al., 2001
). This
methylation is probably catalysed by Su(var)3-9 protein, as mammalian homologs
of Drosophila Su(var)3-9 have been shown to have this activity
(Aagaard et al., 1999
;
Rea et al., 2000
). The
Su(var)3-7 protein also interacts with HP1
(Cleard et al., 1997
). The
SuUR protein is present in pericentric and intercalary heterochromatin, but
its function remains unclear (Makunin et
al., 2002
).
The phenomenon of position effect variegation is caused by inactivation of
a euchromatic gene relocated to heterochromatin. Genetic inactivation results
from chromatin changes in the relocated region, namely under-replication and
compaction of DNA, histone modifications and the appearance of HP1
(Belyaeva et al., 1993)
(reviewed by Elgin, 1996
;
Wallrath, 1998
;
Richards and Elgin, 2002
).
There appear to be specific centers of inactivation within heterochromatin as
not all heterochromatic fragments can evoke position effect variegation
[(Pokholkova et al., 1993
) and
references therein].
The specific content of DNA and proteins causes late replication of
heterochromatin in the cell cycle in comparison with euchromatin. This
attribute is common for chromosomes of many organisms
(Lima-de-Faria and Jaworska,
1968), including Drosophila melanogaster
(Ananiev et al., 1977
;
Steinemann, 1980
).
Heterochromatin replicates late only when it forms dense heavily stained
blocks. In early embryogenesis, heterochromatin is indistinguishable in
appearance from euchromatin and replicates simultaneously
(Lima-de-Faria and Jaworska,
1968
). However, centromeric regions in deep heterochromatin
replicate at the same time as euchromatin despite the dense appearance
(Ahmad and Henikoff, 2001
).
One of the main attributes of heterochromatin in polytene chromosomes is
its under-replication. For instance, euchromatin passes up to 10 replication
cycles in salivary glands (SGs), whereas heterochromatin passes only 1-2
cycles (Rudkin, 1965;
Berendes and Keyl, 1967
;
Mulder et al., 1968
). However,
the number of cycles can vary significantly in different heterochromatic
regions. For example, the light gene, situated in the most distal
region h35 of the 2L arm, polytenizes to the level of euchromatin
(Devlin et al., 1990
). The
rolled gene, located in the most proximal part of 2R heterochromatin,
also polytenizes almost to the level of euchromatin, whereas repeated
sequences flanking this gene (Bari-1 and AAGAC) are extremely under-replicated
(Berghella and Dimitri, 1996
).
Insertions of P-transposons in different blocks of heterochromatin also
display highly polytenized regions (Zhang
and Spradling, 1995
).
Different factors can influence the level of polytenization. In follicle
and nurse cells (NCs), the quantity of DNA was greater in strains containing
attached X and Y chromosomes compared with that in strains containing free and
Y chromosomes (Freed and Schultz,
1956). Relocating of a fragment of the X chromosome right arm,
containing the AAGAG satellite, to euchromatin by the inversion
In(1LR)pn2b increases the level of the satellite polytenization
considerably (Koryakov et al.,
1999
). Some genes also influence the polytenization level of
heterochromatic regions. The SuUR mutation suppresses
under-replication of pericentric and intercalary heterochromatin in SG
chromosomes, which results in full polytenization of intercalary
heterochromatin sites and the appearance of a new structure with reproducible
banding pattern in the pericentric heterochromatin of chromosome 3, called
Plato Atlantis (Belyaeva et al.,
1998
; Moshkin et al.,
2001
; Semeshin et al.,
2001
). Mutation of the other gene, cycE1672,
increases the polytenization level of satellite DNAs in ovaries
(Leach et al., 2000
).
The level of heterochromatin polytenization is higher in chromosomes of
germline cells than in somatic cells. These differences probably reflect the
fact that, in SG chromosomes, under-replication begins from the first
endocycle whereas the genome of NCs fully replicates during the first four
cycles (Dej and Spradling,
1999).
In D. virilis, satellite sequences are more highly replicated in
ovaries than in SGs and Malpighian tubes
(Endow and Gall, 1975). In
D. melanogaster, mutation otu causes formation of polytene
chromosomes in NCs (King et al.,
1981
), which show greater polytenization of heterochromatic
sequences than in SGs (Mal'ceva and
Zhimulev, 1993
; Koryakov et
al., 1996
).
The banded structures in chromosome 3 heterochromatin in both SuUR SGs and otu NCs raise several questions. Do the same DNA sequences polytenize in chromosomes of different cell types? Is this DNA heterochromatic? In this paper, we characterize the polytenized heterochromatic regions in chromosome 3 and correlate these novel heterochromatic features with heterochromatic blocks in mitotic chromosomes revealed by differential staining.
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Materials and Methods |
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Cytology and fluorecsent in situ hybridization (FISH)
NC and SG chromosomes were prepared as described previously
(Koryakov et al., 1996). DNA
clones were labeled with biotin-16-dUTP (Boehringer Mannheim, Mannheim,
Germany) either in PCR or in random-primed polymerase reaction with Klenow
fragment.
Squashes of polytene chromosomes were incubated in 2xSSC for 1 hour at 65°C, denaturated in 2xSSC, 0.07 N NaOH for 1.5 minutes, dehydrated in steps of increasing concentrations of cold ethanol (70%, 80%, 100%) for 3-5 minutes each, and air dried. Labeled probe was added to the hybridization mix (50% formamide, 2xSSC, 10% dextran sulphate) to a final volume of 30 µl. Hybridization was performed overnight at 37°C. Unbound probe was removed with three 15 minute washes in 0.2xSSC at 42°C, slides were incubated in blocking solution (2% Blocking reagent (Boehringer Mannheim) for 30 minutes, 4xSSC, 0.1% Triton X-100). Slides were stained with avidin-FITC for 30 minutes at 37°C and washed three times for 5 minutes with 4xSSC, 0.1% Triton X-100. Chromosomes were counterstained with propidium iodide (2 µg/ml in 0.2xSSC) at room temperature for 1-5 minutes, then were treated with Hoechst33542 (2 µg/ml in 0.2xSSC) at room temperature for the same time and rinsed in 0.2xSSC for a few seconds. Finally, 10 µl of antifade solution (2.5 mg/ml, 1,4-diazobicyclo-[2.2.2]-octane in 2xSSC; Sigma) was added before examination by fluorescence microscope. If necessary, the signal was enhanced by treatment with biotinilated anti-avidin antibodies and subsequent staining with avidin-FITC.
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Results |
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In SuUR mutants, a series of distinct bands appear in 80A-C, small
bands in 81F become a large compact block and new, banded material is visible
between chromosome 3 arms. This novel material was called Plato Atlantis, and
its banding pattern was divided into six sections: PAA, PAB, PAC, PAD, PAE and
PAF. There are constrictions in the PAA and between PAE and PAF regions
(Belyaeva et al., 1998;
Semeshin et al., 2001
)
(Fig. 2B).
In otu mutant NCs, the proximal 3L euchromatin comprises diffuse
bands in section 80, whereas the proximal 3R contains a distinct band in 81F.
Novel chromosomal material, visible between sections 80 and 81, was divided
into four zones, separated by constrictions, named phD, phE, phF and phG
(polytene heterochromatin) by analogy with chromosome 2
(Koryakov et al., 1996)
(Fig. 2C). Constrictions
indicate that DNA is under-replicated, resulting in frequent chromosomal
breakage. Breakage was observed between phE and phF, phF and phG, but not
between phD and phE; thus, the latter two regions are always present together
in non-rearranged chromosomes.
The pericentric region of chromosome 3 in NCs of flies homozygous for both otu and SuUR mutations undergoes minimal changes in comparison with otu mutants, unlike SG chromosomes. All the structures become larger: if in strain otu, the phG section looks like a narrow, unstructured region (Fig. 2C), whereas in strain otu; SuUR, the phG section is larger and also may contain fine bands (Fig. 2D).
Morphology of heterochromatic zones may vary from chromosome to chromosome.
For example, the region phD may have a clear banding pattern with at least
three bands (Fig. 2C), or may
be very compact, without dividing into bands
(Fig. 2E). The region phE
consists of puff-like material without a banding pattern
(Fig. 2D,G), but occasionally
fine friable bands can be seen in this region
(Fig. 2F). Usually, the phF
block resembles net-like ß-heterochromatin
(Fig. 2C), but sometimes phF is
represented by a block of -heterochromatin
(Fig. 2E).
Localization of chromosomal rearrangements
To find a correlation between definite heterochromatic blocks in mitotic
chromosomes and newly polytenized regions, we analyzed several chromosomal
rearrangements with known break points within mitotic heterochromatin.
Heterozygous deficiencies Df(3L)1-16, Df(3L)2-66, Df(3L)6B-29 +
Df(3R)6B-29 and Df(3R)10-65, which remove regions h47-h51,
h49-h50d, h50-h58p and h58, respectively, were used with the homozygous
mutation SuUR as a background (see map of the deficiencies in
Fig. 1A). In addition to these
deficiencies, we studied the rearrangement Df(3R)4-75, which contains
a long paracentric inversion (Koryakov et
al., 2002).
Investigation of the rearrangements in heterochromatin is a very complex problem for several reasons. First, both in SGs and in NCs, banded structures do not arise in every chromosome, and often instead of these structures one can see a mass of unstructured heterochromatin. Second, even the presence of a banding pattern does not mean full polytenization; by contrast, it remains partial, which results in multiple ectopic contacts. Third, the structures under investigation are small in size, and frequently it is very difficult to map precisely a deficiency, removing only 2-3 small heterochromatic blocks. Taking into account all of these complexities, we made the conclusions about rearrangements based on analysis of many chromosomes of every stock.
Mapping the deficiency Df(3L)1-16 in SG chromosomes shows that it removes material from the proximal part of Plato Atlantis from the region PAA to PAF. Results (Fig. 3A) demonstrate that all the bands of Plato Atlantis are present in the normal homolog, whereas the region of compact bands of the section PAA joins PAF in the deleted homolog. Analysis of the same rearrangement in NC chromosomes demonstrates that it removes the most part of phD, except the proximal portion, as indicated by an arrow in Fig. 3B.
|
Chromosome 3 in the SGs of larvae with the deficiency Df(3L)2-66 lacks material from the proximal part of PAC to PAF (Fig. 3C), whereas both phD and phE regions are visible in the normal homolog of NC chromosomes. At the same time in the rearranged homolog, only the most distal part of phE is present along with phD (Fig. 3D). The blocks phF and phG are not affected by the rearrangement (not shown).
The deficiency Df(3L)6B-29 + Df(3R)6B-29 removes the longest parts of chromosome 3 heterochromatin. In the SG chromosomes, one of the break points is in the distal part of PAA, whereas the proximal part of this region, indicated by an arrow, is visible only in one homolog. The second break point was mapped in the euchromatic region 82B of chromosome 3R. In the rearranged homolog, region 82B ectopically connects to the region 101E of chromosome 4, which is followed by diffused material marked by a white arrow and the region PAA (Fig. 3E). In NCs, chromosome 3L with the deficiency lacks phD and phE (Fig. 3F), and chromosome 3R lacks material up to the region 82B (Fig. 3G). As found in SGs, the rearranged homolog in NCs has a small amount of friable material between break points (marked by an arrow in Fig. 3F). This material probably corresponds to h53 or at least a part of this region, containing the centromere, which could not be deleted.
Mapping of the rearrangements Df(3R)10-65 and Df(3R)4-75
showed that they are more complex than it was known before. According to
genetic (Marchant and Holm,
1988a; Marchant and Holm,
1988b
) and cytological data in mitotic chromosomes
(Koryakov et al., 2002
), the
rearrangement Df(3R)10-65 is a simple deficiency; however, analysis
of NC polytene chromosomes reveals that this stock contains a long pericentric
inversion. Break points of the inversion are located in constrictions 80C-phD
and phG-81F. We could not determine what part of the chromosome is deleted,
probably due to its small size (data not shown).
It was shown genetically that Df(3R)4-75 is a deficiency of part
of the 3R arm (Marchant and Holm,
1988a; Marchant and Holm,
1988b
), whereas analysis of the mitotic chromosome demonstrated
that this stock contains a heterochromatic inversion
(Koryakov et al., 2002
).
Mapping of the rearrangement in SG chromosomes with SuUR mutation
showed that, in addition to heterochromatic, there is at least one euchromatic
inversion with break points in 79E and 83F (data not shown). The presence in
so short a chromosome length of at least two inversions and numerous ectopic
contacts does not allow us to perform more-detailed mapping.
Summarizing all the data, we can conclude that newly appeared heterochromatic structures are formed by pericentric heterochromatin rather than by proximal euchromatin. Also, left and right arms make an unequal contribution to formation of heterochromatic structures. We determined that, in SG chromosomes of the SuUR mutants, heterochromatic structures are formed by the blocks h47-h50d; by contrast, in NC chromosomes, in addition to these blocks, h50p, h53 and h57 are also present.
FISH of DNA clones from heterochromatin
Another approach for heterochromatin mapping in polytene chromosomes is
fluorescent in situ hybridization (FISH). We hybridized clones of satellite
DNA, defining distal portion of left arm heterochromatin (block h48, satellite
AATAACATAG), centromeric area (block h53, dodecasatellite) and distal portion
of right arm heterochromatin (block h57, satellite AAGAG)
(Lohe et al., 1993;
Abad et al., 2000
)
(Fig. 1C), the third chromosome
1.688 satellite (Koryakov et al.,
1999
; Abad et al.,
2000
), and AACAC, which was not shown for chromosome 3
(Makunin et al., 1999
). All
these clones were hybridized to SG polytene chromosomes of OR-R strain as a
control.
In SGs of the wild-type strain, all these clones demonstrate a signal of hybridization as one or several spots in the chromocenter. In the case of the dodecasatellite, in addition to spots we revealed diffused labeling around the chromocenter (data not shown).
Satellites AAGAG (Fig. 4A) and AATAACATAG (Fig. 4B) in SG chromosomes of the SuUR mutants label region PAF. The dodecasatellite displays two types of signals: a diffuse weak signal distributed uniformly all around Plato Atlantis, 80A-C and 81F; and a bright site in PAF (Fig. 4C). Also, we noted that, if satellites AAGAG and AATAACATAG after first enhancement of FISH produce a bright signal of hybridization, then the dodecasatellite gives a very weak signal. Probably, this results from a higher polytenization level of AAGAG and AATAACATAG in comparison with the dodecasatellite.
|
FISH in SG chromosomes reveals the 1.688 satellite in the central part of Plato Atlantis, in region PAC (Fig. 4D). For the satellite AACAC in SG chromosomes after enhancement of brightness, we found a hybridization signal not only in pericentric heterochromatin of chromosome 3, but also in numerous euchromatic sites both in OR-R and in SuUR strains (data not shown).
Satellite AAGAG in NC chromosomes hybridizes with the region phG (Fig. 4E). We observed an intensive fluorescence signal even without brightness enhancement both in chromosomes of otu; SuUR and otu mutants. Satellite AATAACATAG labels the proximal portion of phE (Fig. 4F). In this case, the signal was visualized only after a second FISH enhancement. In addition, we noted significant differences in the rate of signals in chromosomes of otu and otu; SuUR strains: a very weak signal in otu, and a bright signal in otu; SuUR. The label of dodecasatellite is situated in the distal part of phF (Fig. 4G), and signal was clearly seen even after one enhancement. Sequence AACAC in NC chromosomes, like in SG chromosomes, hybridizes diffusely all around heterochromatin (Fig. 4H).
Results of hybridizations described above demonstrate that Plato Atlantis and structures in NC chromosomes are not identical. If, in Plato Atlantis, the satellites AAGAG, AATAACATAG and dodecasatellite are found in one narrow zone (PAF) then, in NC chromosomes, they mapped in three clearly distinguished regions (phE, phF and phG).
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Discussion |
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The possibility that the novel banded structures are not heterochromatic,
but originate from proximal euchromatin, was investigated by deletion mapping.
The location of satellites from different parts of heterochromatin to the same
region in SGs (Fig. 5A) or
close regions in NCs (Fig. 5C)
supports an origin of these structures from the most proximal euchromatin of
the 3L arm. However, deletion mapping conflicts with this conclusion
(Fig. 5D-F). Satellite
AATAACATAG, found in the region h48 (Lohe
et al., 1993), should be located in regions PAA-PAD of SG
chromosomes and in phD of NC chromosomes. This contradiction can be resolved
if there is a minor site of this satellite in addition to the major one.
According to this hypothesis, the minor site, situated somewhat proximally to
h48, polytenizes in SG and NC chromosomes, and this site is seen in FISH
experiments, while the major site remains under-replicated.
|
Availability of several sites, both major and minor, was already shown for
the 1.688 satellite. At first, this sequence was found in the X chromosome as
a unique large block in heterochromatin
(Lohe et al., 1993), and as
very short repeats (2-3 copies) in euchromatin
(DiBartolomeis et al., 1992
).
Later, homologous sequences were discovered in chromosomes 2 and 3
(Koryakov et al., 1999
;
Abad et al., 2000
). For this
reason, it is possible that sites of dodecasatellite and AAGAG, detected in
polytene chromosomes, also correspond to minor sites, which are invisible in
mitotics, rather than to major sites in regions h53 and h57. However, data of
FISH for satellites AAGAG and dodeca at least do not conflict to rearrangement
mapping data.
The situation with the satellite AACAC is somewhat different. In mitotic
chromosomes, this sequence was located in chromosomes 2 and Y. In SG polytenes
of OR-R and SuUR strains, it was found in two bright sites and across
the chromocenter as a fine net (Makunin et
al., 1999). In our study, this satellite was identified in
chromosome 2 after brightness enhancement (not shown) and in numerous sites in
hetero- and euchromatin. Probably there are several minor sites of AACAC all
around heterochromatin and in euchromatin as well. Also, unspecific
hybridization to the sequence AC/TG is possible. This dinucleotide repeat is
found in the Drosophila genome approximately once per every 30-100 kb
(Cirera et al., 1994
).
Unspecific hybridization may result from errors of Taq polymerase during PCR
labeling.
The data obtained mean that, in both types of heterochromatic regions with banding pattern (NCs of otu mutants and SGs of SuUR mutants), there are no long tracts of satellite DNAs. There are only small sites of 1.688 satellite and, probably, short fragments of AACAC or at least AC/TG. Large blocks of satellites locate either in the narrowest region of Plato Atlantis (PAF), or in friable ß-heterochromatic portions of NC chromosomes (Fig. 5A-C).
There is an interesting correlation between the polytenized heterochromatic
regions and the location of essential `heterochromatic' genes. There are 16
complementation groups in chromosome 2 heterochromatin
(Hilliker and Holm, 1975;
Hilliker, 1976
;
Schupbach and Wieschaus, 1989
;
Eberl et al., 1993
), which can
be divided into three groups in the right arm (in distal, proximal and middle
parts) and one group in the distal part of the left arm
(Dimitri, 1991
;
Dimitri et al., 2003
). In NC
chromosomes, namely gene-containing regions form large diffuse heterochromatic
blocks. For example, the major part of the left arm does not contain known
genes, and no noticeable structures are found in NC chromosomes, but right arm
heterochromatin consists of at least two large blocks
(Koryakov et al., 1996
). A
similar situation was found in chromosome 3. It was shown that all the genes
of the left arm are grouped in regions h47-h51, and of the right arm in region
h58 (Koryakov et al., 2002
).
Mapping of deletions indicates that almost all Plato Atlantis are formed by
the sections of chromosome 3 removed by the deficiencies Df(3L)1-16
and Df(3L)2-66, which contain all known genes for 3L arm
heterochromatin (Marchant and Holm,
1988a
; Marchant and Holm,
1988b
) (Fig. 5D,E).
The part of chromosome 3 heterochromatin in NCs, which can produce the banding
pattern (phD and phE), is formed by these regions as well
(Fig. 5E,F). Right arm
heterochromatin contains only two known genes locating in the most distal part
(Marchant and Holm, 1988a
;
Marchant and Holm, 1988b
;
Koryakov et al., 2002
). In
Plato Atlantis, 3R heterochromatin is not presented almost completely and, in
NCs, it consists of a pair of typical ß-heterochromatic diffuse blocks
(phF and phG) (Fig. 5D-F).
X chromosome heterochromatin does not contain unique genes (reviewed by
Gatti and Pimpinelli, 1992),
no new blocks appear in polytene chromosomes, and new bands in division 20 in
SuUR mutants are not related with heterochromatin
(Koryakov et al., 1999
;
Kolesnikova et al., 2001
). It
seems evident that predominantly gene-containing regions of heterochromatin
undergo polytenization or banding pattern formation in polytene chromosomes of
SGs of SuUR mutants and NCs of otu mutants. This conclusion
is in good correlation with data about polytenization of heterochromatic genes
(Devlin et al., 1990
;
Berghella and Dimitri,
1996
).
Differences in heterochromatin polytenization in somatic and germline
cells
In spite of many common features, there is a very important difference
between chromosomes of SGs and NCs. Polytene chromosomes of normal morphology
do not develop in NCs and appear only in some mutants such as otu
(King et al., 1981). This gene
influences egg chamber formation, the system of actin microfilaments and the
transfer of substances from NCs to the growing oocyte
(Storto and King, 1988
). In
some way, the absence of normal product of this gene causes polytene
chromosome formation, but it does not increase the polytenization level of
heterochromatin. In SGs, the situation is opposite, polytene chromosomes
develop normally, but heterochromatin is not polytenized, and partial
polytenization can be caused only by the mutation SuUR. In addition
to this, in SG chromosomes this mutation makes global changes in pericentric
heterochromatin, but has little effect in NCs.
Plato Atlantis and blocks in NC chromosomes are different with respect to several other traits. First, a single banded structure was found between the arms of chromosome 3 in SGs whereas, in NC chromosomes, four structures appear, which have both euchromatic and heterochromatic morphology. Second, three satellite DNAs locate at the same site in SG chromosomes, whereas they are in three clearly distinguishable blocks in NCs. Third, regions affected by deficiencies Df(3L)1-16 and Df(3L)2-66 in NC and SG chromosomes are different.
According to genetic data, these deficiencies do not overlap
(Marchant and Holm, 1988a;
Marchant and Holm, 1988b
), but
they partially overlap by cytological data
(Koryakov et al., 2002
)
(Fig. 5E). Based on
rearrangement mapping, we can determine where heterochromatic genes are
located in Plato Atlantis. Deficiencies Df(3L)1-16 and
Df(3L)2-66 do not overlap in sections PAA-PAC, which means that seven
distal genes (l(3)80Fa to l(3)80Fg) are in this interval. In
PAD-PAE, these deficiencies overlap, and proximal break points coincide with
each other. It is followed by the conclusion that three proximal genes
(l(3)80Fh to l(3)80Fj) locate somewhere in the narrowest
portion of PAE, which could not be distinguished by cytological methods in
SGs. The position of satellite AAGAG and mapping of Df(3L)6B-29 +
Df(3R)6B-29 allows us to conclude that the gene l(3)81Fa is
situated in 81F-82B, and the gene l(3)81Fb is situated distally to
82B (Fig. 5D).
In NC chromosomes, deficiencies Df(3L)1-16 and Df(3L)2-66 are clearly discerned, and the overlapping zone seems to be very small and locates between blocks phD and phE. This makes it possible to put genes l(3)80Fa to l(3)80Fg into phD, and genes l(3)80Fh to l(3)80Fj into phE. Like in SGs, one of the right arm genes (l(3)81Fa) is situated in 81F-82B, and another one (l(3)81Fb) locates distally to 82B (Fig. 5F).
Summarizing data of the morphological analysis and detailed comparison of rearrangements, we construct a scheme comparing two types of polytene and mitotic chromosomes (Fig. 5G). We conditionally marked out three levels of polytenization: absence, middle level with diffuse ß-heterochromatin formation and high level with banding pattern formation. It is seen that the same regions of mitotic heterochromatin behave differently in NC and SG chromosomes. In both cases, the regions PAA-PAE and phD are homologous and formed by the blocks h47-h50d, whereas other blocks (h50p, h53 and h57) are partially polytenized in NCs, but absent in SGs. It is clear that the genetic systems controlling replication of DNA and especially heterochromatin in somatic and germline cells may be different.
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Acknowledgments |
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References |
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