ISSN 10227954, Russian Journal of Genetics, 2011, Vol. 47, No. 6, pp. 674–681. © Pleiades Publishing, Inc., 2011.
Original Russian Text © E.Z. Kvon, S.A. Demakov, I.F. Zhimulev, 2011, published in Genetika, 2011, Vol. 47, No. 6, pp. 765–773.
Transcriptional activity of a chromosome region is
one of the main markers of its functional significance.
It is generally accepted that all decompacted regions of
polytene chromosomes, like nucleoli, puffs, loose
bands, andinterbands are transcriptionally active .
However, unlike other transcriptionally active chro
mosome regions, the molecular genetic organization
of the interbands, as well as the levels of their expres
sion still remain unclear. Complications in the inter
band analysis are associated with the small interband
m, on average) and low DNA content (2 kb)
. In a number of studies it was demonstrated that
H uridine incorporated into interband regions .
Furthermore, antibodies against DNA:RNA hybrids
 and RNA polymerase II , as well as RNP parti
cles [5, 6] were localized in these regions. These results
suggested that interbands were likely to contain per
manently active genes. However, some evidence con
flicts with this proposal and suggests possible regula
tory role of the interband regions [7, 8]. So far, none of
currently existing hypotheses on the functional role of
interbands received definite experimental support.
An original approach, based on electron microscopic
analysis (EM) of transgenic constructs inserted into
polytene chromosomes enabled DNA cloning from
some interband regions [7, 9–11]. For two of these inter
band regions, 3C56/C7 and 61C7/C8, the molecular
limits were determined precisely. In the first case, reces
, associated with the deletion of about
900 bp, removed the interband and possibly some mate
rial from the ends of the neighboring bands 3C56 and
3C7, causing fusion of these . In the second case,
insertion of a
transposon into the 61C7/C8 interband
region led to the formation of a new band, providing
cloning of the interband sequence, using transposon as a
molecular probe . In addition, it was shown that in
new genetic environment, DNA fragments from the
interband regions mentioned, included into a unique
transposon, also formed the interband structures .
This enabled analysis of the interband regions using
In the present study, transcriptional activity of the
3C56/C7 and 61C7/C8 interband regions was exam
ined with the help of PCR on a cDNA template (OT
PCR). In the salivary glands of 3rd instar larvae the tran
scriptional level of the interbands examined was very low.
For the 3C56/C7 region the data were confirmed using
more exact, quantitative realtime PCR (RTPCR).
MATERIALS AND METHODS
RNA isolation and purification.
To tal RNA was iso
lated using the TRIsol reagent (Invitrogen), according
to the recommendations of the manufacturer. To
remove the DNA admixture from the RNA samples,
the later were treated with 40 units of DNAse I without
the RNAse activity (Roche) for 2 h at room tempera
ture in the DNAse I buffer. Then, extraction of RNA
with TRIsol reagent was repeated.
Amplification of the DNA fragment on the
RNA template was carried out using an AccessQuick RT
PCR System kit (Promega) according to the recommen
dations of the manufacturer. In each reaction 1
g of total
RNA an a pair of primers in final concentration of 1
was used. The reaction was run in a final volume of 50
Oligonucleotide primer pairs used (hereafter, 5'–3') were
: AGGGGTCCACACAGGT and
Chromatin Decompaction in the Interbands of
Chromosomes Does Not Correlate with High Transcription Level
E. Z. Kvon, S. A. Demakov, and I. F. Zhimulev
Institute of Chemical Biology and Fundamental Medicine, Russian Academy of Sciences, Novosibirsk, 630090 Russia
Received November 11, 2010
—The search for correlation between structural organization of particular chromosome regions and
their functions is among key directions of molecular cytogenetics. In this study, we used polytene chromo
as a convenient model for examining transcriptional activity of chro
momeres (bands) and interchromomeres (interbands) in eukaryotic interphase chromosomes. Using cloning
of the interband DNA sequences and determination of the molecular limits for some interbands, we analyzed
the transcriptional activity of these regions and compared the band and interband transcriptional activity in
polytene chromosomes. Our results showed the absence of correlation between the decompacted state of the
interbands examined and the levels of transcription observed in these regions.