Institute of Cytology and Genetics, Siberian Division of Russian Academy of Sciences, Novosibirsk, 630090, Russia
* Author for correspondence (e-mail: zhimulev{at}bionet.nsc.ru)
Accepted 27 September 2002
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Summary |
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Here we describe novel structural modifications of polytene chromosomes
(swellings) and show that SuUR controls chromatin organization in
polytene chromosomes. The swellings develop as the result of SuUR
ectopic expression in the transgene system Sgs3-GAL4; UAS-SuUR+.
They are reminiscent of chromosome puffs and appear in 190 regions of
intercalary, pericentric and telomeric heterochromatin; some of them attain
tremendous size. The swellings are temperature sensitive: they are maximal at
29°C and are barely visible at 18°C. Shifting from 29°C to
18°C results in the complete recovery of the normal structure of
chromosomes. The swellings are transcriptionally inactive, since they do not
incorporate [3H]uridine. The SuUR protein is not visualized in
regions of maximally developed swellings. Regular ecdysone-inducible puffs are
not induced in cells where these swellings are apparent.
Key words: SuUR gene, Heterochromatin, Silencing, Polytene chromosomes, Drosophila
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Introduction |
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No homology with full-length SuUR protein was found in databases when a
BLAST search was used (Altschul et al.,
1997). However, the first 250 amino acids from the N-terminus show
a moderate similarity to the N-terminal part of the ATPase/helicase domain
found in the SWI2/SNF2 family of proteins
(Makunin et al., 2002
).
In lines containing two to six additional transgenic doses of the
SuUR+ gene the degree of DNA underreplication and ectopic
pairing in regions of IH is sharply enhanced; that is, the SuUR gene
functions as an enhancer of underreplication causing many late replication
sites to become underreplicated. Overexpression of SuUR+
under an ubiquitously active promotor is lethal for the organism, whereas
overexpression of the gene under a promotor that is continuously active in the
salivary gland cells results in development of tiny salivary glands (E.I.V.
and I.V.M., unpublished). In this paper we describe visible modifications of
polytene chromosome structure and morphology resulting from ectopic expression
of UAS-SuUR under the control of the Sgs3-Gal4 driver. It contains the
promoter region from the tissue-specific gene Sgs3 of D.
melanogaster and a coding sequence for the yeast transcription activator
GAL4 (Do et al., 2002). The
Sgs-3 gene is active only in cells of larval salivary glands and only
during the second part of the third larval instar
(Biyasheva et al., 2001
). This
peculiarity of the driver permits us to analyze the overexpression of
SuUR in a single larval organ, which normally histolyses soon; this
overexpression presumably will not damage the normal development of the whole
organism. We expected that strong overexpression of the
SuUR+ gene under the Sgs3-Gal4 driver would
result in a further enhancement of underreplication and ectopic pairing.
However, by contrast, in the polytene chromosomes of the Sgs3-Gal4;
UAS-SuUR+ larvae and prepupae unexpected and unusual swellings
appeared in the regions of IH, PH and in some telomeric regions. The most
interesting characteristics of these swellings are described in this
paper.
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Materials and Methods |
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Transgenic larvae Sgs3-Gal4/+ UAS-SuUR+/+ obtained from mating of lines Sgs3-Gal4 and UAS-SuUR+ were raised on standard medium at 18, 25 or 29°C. Both backgrounds, SuUR and SuUR+, were used for transgene expression. Polytene chromosomes of larvae and prepupae obtained from mating of these strains were analyzed.
The Drosophila strain containing UAS-lacZ was given by F. Karch.
Cytology
Preparations of salivary gland polytene chromosomes stained with acetic
orcein were made by the standard method and analyzed under a phase-contrast
microscope. Polytene chromosome maps were taken from a previous paper
(Bridges, 1935).
For autoradiography, salivary glands were dissected in Ephrussi and Beadle
solution (Ephrussi and Beadle,
1936) and transferred to the same medium containing
[3H]uridine (25 mCi/ml, specific activity 38 Ci/mM, Amersham) for
30 minutes. They were then fixed in alcoholacetic acid (3:1) mixture, covered
with liquid emulsion Illford L4, exposed for two weeks and then developed (for
details, see Zhimulev,
1999
).
Squashes for EM purposes were prepared as described earlier
(Semeshin et al., 2001).
Sections 120-150 nm thick were cut with an LKB-IV ultratome and examined under
the JEM-100C electron microscope at 80 kV. Immunostaining of polytene
chromosome was performed according to a previous paper
(Elgin, 1996
) with minor
modifications.
Constructs for transformations
The clone f27 contains the full ORF and 3'UTR of the SuUR
transcript cloned into pBluescript SK+ between
PstI and XhoI sites (for details, see
Makunin et al., 2002). The
insert of the f27 clone was excised with NotI and Acc65.I
and subcloned into pUAST (Brand
and Perrimon, 1993
) that had been digested with NotI and
Acc65.I. The resulting U6 clone contains the SuUR ORF and
3'UTR under the control of the UAS-containing minimal Hsp70
promoter in the P-element vector. For transformation 8 µg of the
U6 DNA were mixed with 2 µg of DNA of helper plasmid pUChspi delta 2-3
turbo in a total volume 20 µl, and this mixture was injected in y
w embryos by standard procedures, and several independent transformed
lines were established. Two of the insertions of the UAS-SuUR+
constructs were localized in polytene chromosome regions 59DE and 47A,
respectively (V.S., unpublished) [UAS(59DE) and UAS(47A)]. The main part of
the work was done using the UAS(59DE) line. Experiments on
reversibility of the swellings, antibodies localization and
[3H]uridine incorporation were performed on the UAS(47A) line.
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Results |
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The Sgs3 transgenic promoter, as well as the genomic Sgs3
gene, are expressed only in salivary gland cells of the third instar larvae,
beginning at mid instar (about 100 hours after oviposition) and continuing
until pupariation (120 hours of larval development or 0 hours prepupa) (for a
review, see Biyasheva et al.,
2001). Specific changes appear in polytene chromosomes during the
period of activity of the Sgs3 promoter and, as a consequence, there
is the period of SuUR+ ectopic expression in the
Sgs3-GAL4/+ UAS-SuUR+/+ larvae and prepupae. In young larvae,
actively feeding and moving in the media (this developmental stage corresponds
to 100-114 hour larvae), characteristic capsules appear in polytene chromosome
bands (Fig. 2A). In older
larvae migrating on tube walls (114-120 hours) some of these capsules convert
into swellings of tremendous sizes (Fig.
2B). The size and number of the swellings are maximal in 4-8 hour
prepupae. In the SuUR Sgs3-GAL4 UAS-SuUR+ larvae and
prepupae the swellings are bigger than in the transgenic strain with normal
endogenous SuUR genes. The localization of the capsules and swellings
in chromosomes is very specific (see below) and highly reproducible (see
mapping in Figs 2 and
3). In total, about 190 bands
demonstrate capsule or swelling formation in polytene chromosomes
(Table 1). These swellings look
like the puffs known in polytene chromosomes for decades (for a review, see
Zhimulev, 1999
). Nevertheless
they strongly differ from puffs, in at least, four aspects, which are listed
below.
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Such an important change in the structure of numerous bands may result in
changes in polytene chromosome function. In normal larvae more than 120 puffs
are activating and inactivating during this period in a cascade of changing
gene activity (Ashburner et al.,
1974). Among many thousands of salivary gland nuclei analyzed
after SuUR overexpression we could not find ecdysone-inducible puffs.
The exceptions were a few nuclei in which the chromosomes contained very small
puffs at the earliest ecdysone inducible sites 74E-75B.
The SuUR protein in wild-type larvae is localized at a limited (113) number
of sites (Makunin et al.,
2002). But after even a short period of overexpression in young
larvae, it appeared in practically all visible polytene chromosome bands.
(Fig. 1D,E). Swellings as a
rule have not yet developed at this stage; however, in places where they will
soon appear, small cavities free of the antibodies are visible (74A, 75C and
81F in Fig. 1D). At the stage
when the swellings reach their maximal size, staining of IH and PH with
antibodies is not revealed (Fig.
8A).
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Discussion |
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It is possible that the effects of additional doses of the SuUR protein are
determined by the similarity of SuUR to SWI2/SNF2
(Makunin et al., 2002), a
member of a protein family capable of remodelling chromatin complexes. For
this, SWI2/SNF2 has an ATP hydrolysing function. The SWI/SNF complex can alter
histone-DNA interactions in the nucleosome. High concentraions of SWI/SNF
complex can disrupt a synthetic nucleosome core
(Wolffe and Guschin, 2000
). As
shown recently, null mutation of the ISWI gene, a highly conserved
member of the SWI2/SNF2 family, affects both cell viability and gene
expression and causes striking alterations in the structure of the male X
chromosome (Deuring et al.,
2000
). Mutations of other gene, JIL-1, coding for tandem
chromosomal kinase, leads to dramatic changes in banding pattern
(Wang et al., 2001
). We could,
therefore, propose that overexpression of SuUR results in changes of
chromatin packaging specifically in all types of heterochromatin and results
in swellings. These changes appear to be reversible, and after lowering the
temperature, heterochromatic regions condense again, swellings disappear and
chromosomes acquire an almost normal morphology. These effects are probably
related to adaptation of the Ga14-UAS system to high temperature
(Brand et al., 1994
). This
means that chromosomes are able to restore normal structure and functions and
swelling formation does not cause irreversible changes in chromosome
structure. Most interesting was the finding that after gene overexpression,
the SuUR protein itself is not revealed within the swollen heterochromatic
regions where it normally resides. At the same time DNA is easily visible in
the swellings after staining with Hoechst 33258. It is possible that the SuUR
protein and other proteins dissociate from chromatin. A case of such
dissociation occurs when under the influence of the E(z) mutation,
some of proteins of the PcG complex dissociate from chromosomes
(Rastelli et al., 1993
).
Other cases of global changes of properly heterochromatin structure are
known as well. For example, specific puffs appeared in regions of PH in
polytene chromosomes of Glyptotendipes barbipes (Chironomidae) larvae
developing at 18°C (Keyl,
1963) or in Chironomus thummi thummi after long
maintenance of larvae in a solution of Actinomycin D
(Kiknadze, 1965
;
Valeyeva et al., 1979
).
Heterochromatin of Drosophila melanogaster mitotic chromosomes looks
decondensed in a mus-101ts mutant at 29°C
(Gatti et al., 1983
). The
mus-101 gene encodes a member of the superfamily of proteins
containing the BRCT domain, which is implicated in DNA repair and cell
checkpoint control (Yamamoto et al.,
2000
). It shares homology with human TopBP1 protein, which is
associated in vitro with DNA topoisomerase IIß and with the fission yeast
Rad4/Cut5 protein required for repair, replication and checkpoint control. So
this gene is probably involved in processes of chromatin reorganization, and
its action can influence heterochromatin condensation.
However, structures resembling the swelling described in this paper were not found before. These modifications of chromosome structure specifically appear in chromosome regions binding SuUR protein and demonstrating late replication in the endocycle.
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Acknowledgments |
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