1022-7954/01/3701- $25.00 © 2001
Russian Journal of Genetics, Vol. 37, No. 1, 2001, pp. 24–29. Translated from Genetika, Vol. 37, No. 1, 2001, pp. 29–35.
Original Russian Text Copyright © 2001 by Semeshin, Shloma, Belyaeva, Zhimulev.
Activation of genetic loci in polytene chromosomes
of Diptera is accompanied by local band decondensa-
tion leading to the formation of puffs . Variation in
the number of puff-forming bands and the extent of
their decompaction causes a great variety of puffs [2, 3].
In addition to typical “light” puffs, polytene chromo-
regions having more dense and sometimes lumpy struc-
ture. Investigations of puff organization are mostly con-
cerned with light puffs whereas the organization of
“dark” puffs remains poorly studied. The category of
dark puffs includes both small and large puffs. Small
puffs have a low decompaction level and a slightly
increased diameter; they permanently function in
ontogeny weakly incorporating
H-uridine [4, 5]. Large
puffs (e.g., 2B, 35B, 56E) contain heterogeneous and
partly dispersed material; they actively synthesize
RNA. Morphologically similar dark puffs were earlier
described in salivary gland polytene chromosomes in
the family Simuliidae  and in polytene chromosomes
. The authors explain this phenomenon
by the presence in the dark puff regions of intercalary
heterochromatin that can be involved in pufﬁng.
Although numerous works are focused on intercalary
heterochromatin (see  for review), its participation in
puff formation remains unclear.
The organization of polytene chromosome struc-
tures can be studied using morphological analysis of
transposed DNA fragments having known molecular
characteristics. In polytene chromosomes, these frag-
ments can form bands, interbands, and puffs [9, 10].
 presented results on transposition of
DNA fragments containing artiﬁcial gene
to region 65A of the 3L chromosome. As shown by
light microscopy, repetitive arrays of this insert form a
dense amorphous puff of small size, which is morpho-
logically similar to small puffs. Another suitable model
is polytene chromosome regions transformed by cDNA
) locus from the dark-puff
region 2B of the X chromosome .
To reveal factors that determine the morphology of
dark puffs, we conducted an electron microscopic (EM)
analysis of artiﬁcial and natural dark puffs.
MATERIALS AND METHODS
We used transformed strains of
taining 1, 2, 4, 13, and 16 copies of the
construct in the 65A region of chromosome 3L (Fig. 1a)
 and line
. In this construct, cDNA of
locus from the 2B puff, which is under the pro-
moter of the heat-shock gene
(Fig. 1b)  is
inserted in the 99B region of chromosome 3R. Heat
C at 10 min) was applied to third-instar larvae.
The effect of bands adjacent to the 2B puff on the structure
of this puff was studied using insertion
. In anal-
ysis of transformants, strain Oregon R was used as control.
EM analysis of puffs from other polytene chromosome
regions was conducted using strain Batumi L. Small puffs
were chosen for EM analysis according to the description
of pufﬁng stages (PSs)  and on the basis of
incorporation [4, 5] and immunoﬂuorescence intensity of
DNA–RNA hybrids [14, 15].
EM preparations were obtained as described earlier
. Sections of 0.12–0.15
m were prepared on an
LKB-IV ultramicrotome and analyzed using a JEM-100C
electron microscope at 80 kV.
Modeling of Dark Puffs Using
in Polytene Chromosomes of
V. F. Semeshin, V. V. Shloma, E. S. Belyaeva, and I. F. Zhimulev
Institute of Cytology and Genetics, Russian Academy of Sciences, Novosibirsk, 630090 Russia;
fax: (3832)33-12-78; e-mail: firstname.lastname@example.org
Received April 2, 2000
—Modeling of morphologically unusual “dark” puffs was conducted using
strains transformed by construct
, in which gene
is controlled by the promoter of the
In polytene chromosomes, insertions of this type were shown to form structures that
are morphologically similar to small puffs. By contrast, the
) locus, which normally pro-
duce a dark puff in the 2B region of the X chromosome, forms a typical light-colored puff when transferred to
the 99B region of chromosome 3R using
A comparison of transposon-induced puffs with those
appearing during normal development indicates that these puff types are formed via two different mechanisms.
One mechanism involves decompaction of weakly transcribed bands and is characteristic of small puffs. The
other mechanism is associated with contacts between bands adjacent to the pufﬁng zone, which leads to mixing
of inactive condensed and actively transcribed decondensed material and forming of large dark puffs.