4
Chromosome Organization within the Nucleus
their arrangement in mitosis, and suggests
that the chromatin did not undergo ex-
tensive rearrangement within the nucleus
following decondensation in telophase. In
addition, the chromosomes occupied sep-
arate domains and were not intertwined.
Finally, chromosomes contacted the NE at
particular loci. These studies in polytene
chromosomes showed chromosomes were
organized according to two fundamental
organizational principles: speci±c contacts
with the NE and persistence of the mitotic
chromosome arrangement.
2.2
Fluorescence
In Situ
Hybridization (FISH)
Following the initial work in polytene
chromosomes, great interest was created
in testing whether the same conclusions
wou
ldho
ldinord
inarynonpo
lytenechro
-
mosomes during interphase. Measuring
interphase chromosome arrangements in
normal cells required FISH in which DNA
probes speci±c for a given locus are hy-
bridized onto intact nuclei and detected
using fluorescent probes. One potential
danger in using FISH is that the harsh
conditions needed for hybridization can of-
ten disrupt the organization of the nucleus.
This is particularly true of methods that in-
volve air-drying or acid treatments during
±xation, and it is always worthwhile, in as-
sessing the value of a given FISH analysis,
to examine the protocol in detail. It is likely
that some of the published studies that
have failed to observe reproducible nuclear
organization failed because of overly harsh
sample preparation that destroys the three-
dimensional structure. Certainly, any pro-
cedure in which nuclei are flattened or
spread will, by de±nition, destroy the three-
dimensional structure. Fortunately, the
study of nuclear organization by FISH has
been greatly facilitated by the development
of ±xation and hybridization procedures
that preserve the three-dimensional struc-
ture of the nucleus. Many such studies,
summarized below, have shown that the
two main results of the polytene studies,
namely, NE interactions and persistence
of mitotic arrangement, also hold true in
other cell types.
2.3
Interactions with the Nuclear Envelope
Many studies have used FISH to show
speci±c chromosome regions are localized
to the NE, which probably accounts for
the earlier observations using electron
microscopy that revealed multiple sites
of chromatin–NE contact in the nucleus.
In
most
cases,
we
do
not
yet
know
what
DNA
sequences
determine
this
interaction, although several studies have
now suggested that silenced chromatin
may confer NE attachment (see below).
Ap
r
i
o
r
i
, if a given locus is found
near the NE, this could either reflect
an
interaction
of
the
chromatin
with
the NE, or alternatively it might simply
reflect a nonrandom radial positioning
within the nucleus due to either the
intrinsic
folding
of
the
chromosomes
or perhaps due to exclusion from the
nuclear interior of loci that cannot interact
with some internal structure. However,
all available evidence suggests that the
localization of chromatin near the NE
involves an actual physical interaction
between chromatin and some component
of the NE. Polytene chromosomes have
been
clearly
shown
to
be
physically
stuck onto the NE at discrete sites. This
conclusion is also supported by studies
of interphase chromatin movement
in
vivo
in which loci near the NE tend to
move signi±cantly less than more internal
loci.
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