The Barr Body is a Looped X Chromosome Formed by Telomere Association

Abstract

We examined Barr bodies formed by isodicentric human X chromosomes in cultured human cells and in mouse-human hybrids using confocal microscopy and DNA probes for centromere and subtelomere regions. At interphase, the two ends of these chromosomes are only a micron apart, indicating that these inactive X chromosomes are in a nonlinear configuration. Additional studies of normal X chromosomes reveal the same telomere association for the inactive X but not for the active X chromosome. This nonlinear configuration is maintained during mitosis and in a murine environment.

Full text

Proc.
Nati.
Acad.
Sci.
USA
Vol.
88,
pp.
6191-6195,
July
1991
Genetics
The
Barr
body
is
a
looped
X
chromosome
formed
by
telomere
association
(chromosome
structure/X-inactivation/centromere/interphase
cytogenetics/in
situ
hybridization)
CHERI
L.
WALKER*t,
COLYN
B.
CARGILE*,
KIMBERLY
M.
FLOY*,
MICHAEL
DELANNOYt,
AND
BARBARA
R.
MIGEON*§
Departments
of
*Pediatrics
and
*Cell
Biology,
The
Johns
Hopkins
University
School
of
Medicine,
Baltimore,
MD
21205
Communicated
by
Victor
A.
McKusick,
April
15,
1991
ABSTRACT
We
examined
Barr
bodies
formed
by
isodi-
centric
human
X
chromosomes
in
cultured
human
cells
and
in
mouse-human
hybrids
using
confocal
microscopy
and
DNA
probes
for
centromere
and
subtelomere
regions.
At
interphase,
the
two
ends
of
these
chromosomes
are
only
a
micron
apart,
indicating
that
these
inactive
X
chromosomes
are
in
a
nonlinear
configuration.
Additional
studies
of
normal
X
chromosomes
reveal
the
same
telomere
association
for
the
inactive
X
but
not
for
the
active
X
chromosome.
This
nonlinear
configuration
is
maintained
during
mitosis
and
in
a
murine
environment.
Barr
bodies
are
unique
chromatin
structures
formed
in
nuclei
of
the
mammalian
female
as
a
means
of
sex
chromosome
dosage
compensation.
First
identified
as
a
nucleolar
satellite
present
only
in
female
cells
(1),
the
Barr
body
represents
a
single
inactive
X
chromosome.
In
cultured
human
cells,
it
is
most
easily
identified
at
the
periphery
of
the
interphase
nucleus,
when
other
chromosomes
are
not
condensed.
Be-
cause
the
Barr
body
is
difficult
to
see
among
clumped
heterochromatin
in
interphase
mouse
fibroblasts,
and
be-
cause
the
silent
human
X
reactivates
more
frequently
in
rodent
than
human
cells,
Dyer
et
al.
(2,
3)
suggested
that
mouse
cells
may
not
form
proper
Barr
bodies.
Analysis
of
cultured
human
cells
by
electron
microscopy
(4)
or
in
situ
hybridization
(2)
places
the
Barr
body
adjacent
to
the
nuclear
envelope
in
75-80%
of
interphase
cells.
Comings
(5)
sug-
gested
that
inactive
X
chromosomes
attach
randomly
to
the
nuclear
membrane,
and
the
multiple
Barr
bodies
in
aneuploid
cells
are
widely
distributed
(6,
7).
Nuclear
matrix
attachment
sites
are
similar
for
the
active
and
inactive
X
chromosomes
(8).
Yet,
the
configuration
of
the
Barr
body
has
been
rela-
tively
unexplored.
DNA
hybridization,
in
situ
(9),
has
pro-
vided
a
powerful
method
to
examine
chromosomes
during
interphase,
revealing
an
orderly
arrangement
of
chromo-
somes
in
the
interphase
nucleus
(10-13)
and
tissue-specific
variation
(14,
15).
Using
such
methods
to
explore
the
human
inactive
X
chromosome,
we
find
that
the
Barr
body
consists
of
a
condensed
X
chromosome
in
a
nonlinear
configuration,
with
telomeres
in
close
proximity.
We
examined
the
Barr
body
in
interphase
and
mitotic
cells
using
fluorescent
probes
for
centromere
and
telomere
regions
of
human
X
chromosomes.
In
addition
to
normal
X
chromo-
somes,
we
studied
isodicentric
X
chromosomes
(16),
which
form
bipartite
Barr
bodies
(16).
Always
inactive,
they
are
mirror
image
duplications
with
two
centromeres
(one
non-
functional)
and
with
two
identical
telomeres
(see
Fig.
1).
The
duplicate
centromeres
as
well
as
common
telomeres
and
their
longer
length
facilitate
structural
analysis.
To
compare
dis-
tance
between
hybridization
signals
with
relative
physical
length
we
examined
three
isodicentrics,
two
joined
by
their
long
arms
(3935
and
7213)
and
the
third
attached
at
the
short
arms
(411).
We
isolated
these
dicentric
chromosomes
from
their
normal
homologue
in
hybrid
cells
so
that
all
signals
would
come
from
the
dicentric
X
chromosome
and
to
exam-
ine
the
human
Barr
body
in
a
mouse
cell
environ.
Finally,
we
simultaneously
hybridized
centromere
and
subtelomere
probes
using
differential
labels
and
confocal
microscopy.
MATERIALS
AND
METHODS
Cell
Lines.
These
are
characterized
in
Table
1.
The
hybrids
derived
from
A9
mouse
fibroblasts
were
selected
in
hypo-
xanthine/aminopterin/thymidine
medium,
back
selected
in
6-thioguanine
to
eliminate
the
active
X;
to
retain
the
inactive
X,
the
silent
HPRT
locus
was
reactivated
by
5-azacytidine.
Inactive
X
hybrids
derived
from
tsA1S9T
mouse
cells
were
selected
directly
at
390C
for
activity
of
the
AJS9T
locus
at
Xpll
(17).
Preparation
of
Slides.
Interphase
cells.
Confluent
cells
in
LabTek
slide
chambers
were
fixed
in
methanol/acetic
acid
(3:1)
and
air
dried.
Mitotic
cells.
Logarithmic-phase
cells
were
treated
with
colcimide
(1
hr),
and
mitotic
cells
were
detached
by
shake-
off,
fixed
in
methanol/acetic
acid,
dropped
onto
slides,
and
air
dried.
Probes.
For
simplicity,
the
human
X-specific
probes
used
to
mark
the
centromeres
and
ends
of
the
short
and
long
arms
of
the
chromosome
are
called
XCen,
Xptel,
and
Xqtel,
respec-
tively.
Fig.
1
shows
the
location
of
sequences
homologous
to
these
probes
(labeled
XC,
29C1,
and
F8).
Xce,.
The
XC
probe
for
the
centromere
region
is
a
2-kilo-
base
(kb)
BamHI
fragment
(19)
homologous
to
alphoid
DNA;
under
stringent
conditions
XC
hybridizes
specifically
with
the
X
chromosome
(19).
Xp
el.
The
29C1
probe
used
to
mark
the
short
arm
(Xp)
telomere
is
a
1.8-kb
Pst
I
fragment
hybridizing
to
a
subtelo-
meric
sequence
located
about
20
kb
from
the
end
of
the
short
arms
of
X
and
Y
chromosomes
(20);
each
X
chromosome
has
3-10
tandem
copies.
Xqel.
The
F8c
probe
used
to
mark
the
long
arm
(Xq)
telomere
is
a
1.4-kb
EcoRI
fragment
containing
exon
26
of
the
blood-clotting
factor
VIII
locus
(21),
which
hybridizes
ex-
clusively
to
the
locus
in
Xq28,
near
the
telomere.
Nick-Translation.
XC
and
29C1
inserts
and
the
entire
F8c
plasmid
were
labeled
with
Biotin-11-dUTP
using
the
BRL
nick-translation
kit.
The
probes
were
purified
in
a
spin
column
containing
50
mM
Tris,
10
mM
EDTA,
and
0.1%
SDS
(pH
7.4)
and
stored
at
4°C
for
up
to
1
month.
Abbreviations:
DNP,
dinitrophenyl;
DAPI,
4',6-diamidino-2-
phenylindole.
tPresent
address:
Department
of
Molecular
Genetics,
Baylor
College
of
Medicine,
Houston,
TX
77030.
§To
whom
reprint
requests
should
be
addressed
at:
CMSC
10-04,
The
Johns
Hopkins
Hospital,
Baltimore,
MD
21205.
6191
The
publication
costs
of
this
article
were
defrayed
in
part
by
page
charge
payment.
This
article
must
therefore
be
hereby
marked
"advertisement"
in
accordance
with
18
U.S.C.
§1734
solely
to
indicate
this
fact.

Proc.
Natl.
Acad.
Sci.
USA
88
(1991)
Table
1.
Characteristics
of
cell
lines
Inactive
X
Cell
line*
Human
chromosome
content
chromosome
F411
46X,dic(X)(qter>p21.2::p21.2>qter)t
Dicentric
X
GM3935
46X,dic(X)(pter>q27.1::q27.1>pter)
Dicentric
X
7213
46X,dic(X)(pter>q26.3::q26.3>pter)
Dicentric
X
F411-A9
Dicentric
X;
five
autosomes
Dicentric
X
GM3935-A9
Dicentric
X;
five
autosomes
Dicentric
X
GM7213-tsA1
Dicentric
X;
four
autosomes
Dicentric
X
DB1214-A9
Normal
active
X;
one
autosome
None
GL-A9
Normal
inactive
X;
one
autosome
Normal
X
*The
first
three
entries
are
fibroblast
cell
lines;
the
remaining
entries
are
hybrids.
tBreakpoints
of
the
three
dicentric
chromosomes
were
defined
using
molecular
probes.
In
Situ
Hybridization.
Biotinylated
probes.
Slides
were
immersed
for
2
min
in
70%o
formamide/2x
SSC
(lx
SSC
=
0.15
M
sodium
chloride/0.015
M
sodium
citrate),
dehydrated
in
a
series
of
70-100%
ethanol,
and
air
dried.
Hybridization
was
as
described
by
Devilee
et
al.
(22)
(37TC
overnight)
using
80-90
ng
of
probe
per
slide
in
formamide
(60%
for
XC,
50%
for
29C1,
and
F8c).
For
hybridizations
with
XC,
the
slides
were
washed
(30
min,
200C)
in
2x
SSC/0.1%
SDS
and
then
washed
in
O.1x
SSC/0.1%
SDS
(30
min,
42°C).
The
third
wash
was
like
the
first.
Hybridizations
with
29C1
and
F8c
were
as
for
XC,
except
all
washes
were
in
2x
SSC/0.1%
SDS.
Slides
were
washed
in
a
fluorescein
buffer
of
3
M
NaCl/20
mM
Tris,
pH
8.
The
biotin
label
was
detected
as
described
by
Pinkel
et
al.
(23),
except
that
fluorescein
buffer
was
substituted
for
BN
buffer
in
all
solutions
and
washes.
Dinitrophenyl
(DNP)-labeled
probes.
XC
was
labeled
with
DNP
by
reaction
with
2,4-dinitrophenylbenzaldehyde
(DN-
BAL,
Aldrich)
as
described
by
Shroyer
et
al.
(24)
and
purified
in
a
spin
column
with
TE
buffer.
The
hybridization
mixture
(50%
formamide,
0.4
mg
of
salmon
sperm
DNA
per
ml,
2x
SSC,
and
60
ng
of
biotinylated
29C1
and
60
ng
of
DNP-labeled
XC
per
slide)
was
heated
to
70°C,
iced,
and
placed
under
coverslips.
For
double
labels,
slides
were
incubated,
rinsed,
and
labeled
with
fluorescein
as
for
single
probes.
After
the
last
wash
in
fluorescein
buffer,
100
ul
of
1:50
rabbit
anti-
DNP/5%
bovine
serum
albumin/and
0.04%
goat
serum
was
applied
(overnight,
4°C).
Slides
were
rinsed
three
times
in
29C
-
xc
w
0
*m
h
0
_-
p~l.2n]
.m
-.
!
-
v
~0
.m
q
26
0
m
FE
-
U.
-
q27
_
r.
m
v^6.
FE
Normal
X
F411
GM3935
GM7213
.2
FIG.
1.
Diagram
(Upper)
and
photographs
(Lower)
of
G-banded
isodicentric
X
chromosomes
showing
breakpoints
(in
italics)
and
location
of
sequences
homologous
to
probes
[Xptel
(v),
29C1;
Xcen
(e),
XC;
Xqtel
(*),
F8].
phosphate-buffered
saline,
treated
with
100
,Al
of
anti-rabbit
IgG
conjugated
to
a
Texas
red
label
in
PBS,
and
counter-
stained
with
propidium
iodide
in
phenylenediamine
antifade
solution.
4',6-Diamidino-2-phenylindole
(DAPI)
Staining.
Cells
fixed
on
slides,
pretreated
with
RNase,
were
stained
(10
min)
with
DAPI
in
150
mM
NaCl/10
mM
Tris/1%
Triton
X-100/0.1%
bovine
serum
albumin,
and
Mowial
solution
was
placed
under
the
coverslip
before
sealing.
Analysis.
Single
label.
Slides
were
examined
microscopi-
cally
for
number
and
position
of
signals.
The
two
signals
from
the
two
ends
(or
two
centromeres)
of
isodicentric
chromo-
somes
were
usually
in
the
same
focal
plane
(those
that
were
not
could
not
be
measured).
In
contrast,
if
also
present,
the
signal
from
the
normal
X
chromosome
was
often
in
another
focal
plane.
The
distance
between
signals
was
determined
from
photomicrographs
(1200x
magnification);
distance
was
measured
from
the
center
of
one
signal
to
the
center
of
the
other.
In
some
cases,
measurements
were
made
directly
from
superimposed
confocal
images,
using
the
"length"
program,
and
were
similar
to
those
obtained
from
photomicrographs.
Double
label.
Signals
obtained
by
labeling
XpteI
with
biotin
and
XCIN
with
DNP
were
analyzed
using
a
Nikon
Optiphot
microscope
mounted
to
a
laser-scanning
confocal
imaging
system
(Bio-Rad
MRC
500).
We
obtained
z
and
xz
series
from
computer-assisted
images
taken
simultaneously
from
two
channels.
The
images
were
subtracted,
one
from
the
other
to
eliminate
cross
signals,
and
then
merged.
RESULTS
Signals
in
Control
Cells.
The
mean
percent
of
cells
with
at
least
one
signal
was
37
(range,
17-76)
with
Xcen,
25
(range,
16-34)
with
Xptel,
and
13
with
Xqtel
probes.
For
all
probes,
and
in
all
cell
lines,
the
signals
were
discrete.
Cells
from
normal
males
and
females
labeled
with
XCC"
showed
the
expected
X
dosage.
The
number
of
Xptel
signals
was
similar
in
both
sexes
reflecting
the
locus
on
two
X
chromosomes
in
females
and
on
X
and
Y
chromosomes
in
males;
most
often
the
two
signals
were
in
separate
parts
of
the
cell,
consistent
with
separate
domains
for
the
two
X
chromosomes
in
females
and
the
X
and
Y
chromosomes
in
males
(25,
26).
Hybrids
with
only
a
normal
human
active
X
chromosome
(DB1214-
tsAlS9)
had
only
a
single
signal
with
either
XCCR
(Fig.
2A)
or
Xptel
in
>90%
of
labeled
cells.
Signals
in
Interphase
Cells
with
Isodicentric
Chromosomes.
Cells
with
three
discrete
XCCn
signals
were
seen
in
all
three
dicentric
cell
lines.
The
signal
for
the
normal
X
is
not
always
seen
in
photographs
as
it
may
not
be
in
the
same
focal
plane
as
the
dicentric
signals.
Fig.
2D
shows
a
cell
with
three
signals,
including
two
that
are
close.
This
characteristic
close
double
signal
(in
a
single
focal
plane)
was
seen
in
41%
of
cells
with
an
XCen
signal
(shown
in
Figs.
2
C
and
E
and
3:
Xcen).
Often
peripheral,
the
double
signal
resembles
the
bipartite
Barr
body
formed
by
these
chromosomes
in
interphase
nuclei
(16)
(visualized
with
DAPI
in
Fig.
2B).
Two
close
Xptel
signals
were
also
seen
in
37%
of
labeled
cells
(Fig.
3:
Xpte)
and
in
cells
labeled
with
Xqtel
(Fig.
2G).
Double
Signals
Originate
from
Isodicentric
Chromosomes.
As
it
is
absent
in
control
fibroblasts,
this
characteristic
double
signal
is
not
due
to
replicated
or
diffuse
signals.
Double
signals
were
seen
in
hybrid
cells
with
a
dicentric
chromosome
but
no
normal
X
chromosome
(66%
of
cells
labeled
with
XCen
and
37%
of
cells
labeled
with
XptcI);
in
cells
with
two
signals,
the
two
were
invariably
close.
As
expected,
two
sets
of
paired
(four)
signals
were
common
in
mitotic
cell
hybrids
(Fig.
4).
That
the
close
double
signals
were
rare
in
cells
lacking
dicentric
chromosomes
and
frequent
in
cells
with
only
dicentric
chromosomes
indicates
they
come
from
6192
Genetics:
Walker
et
al.

Proc.
Natl.
Acad.
Sci.
USA
88
(1991)
6193
3935
3935
hybrid
3935
hybrid
--
-
FIG.
2.
(A)
Control
hybrids
with
normal
active
X
chromosome
(interphase)
showing
single
XCef
signal.
(B-D)
The
411
fibroblasts
(interphase)
stained
with
DAPI
(B)
to
show
bipartite
Barr
bodies
and
labeled
with
XCef
to
mark
centromeres
(C
and
D).
(E-G)
The
411
hybrid
cells
labeled
with
Xce,
in
interphase
(E)
and
mitosis
(F).
(G)
Same
hybrids
labeled
with
Xqtel.
Note:
The
variable
distance
be-
tween
X~e,
signals
(C
vs.
D)
resembles
that
seen
with
DAPI
in
B.
The
signal
for
the
normal
X
not
seen
in
C
is
seen
in
D.
(x750.)
the
two
centromeres
(or
the
two
telomeres)
of
the
dicentric
chromosomes.
Interphase
Position
of
Isodicentric
X
Centromeres.
The
distance
between
the
two
centromeres
varied
among
cell
lines,
ranging
in
fibroblasts
from
0.9
to
2.2
,um
(Table
2).
Unexpectedly,
it
was
greater
in
the
411
chromosome,
joined
by
the
short
arms,
than
in
3935
and
7213
chromosomes
with
centromeres
separated
by
the
long
arms
(Fig.
1).
Interphase
Position
of
Isodicentric
X
Telomeres.
The
two
signals
from
chromosomes
labeled
with
Xptel
were
surpris-
ingly
close;
the
distance
was
like
that
between
centromeres,
both
about
1
,m
(Table
2).
When
both
telomeres
of
411
were
labeled
with
Xqtel,
the
distance
between
them
(1.0
±
0.3
,m)
was
less
than
expected
for
a
chromosome
of
its
size
and
was
considerably
less
than
between
centromeres
(2.3
±
0.7
,m)
(Table
2).
Isodicentric
Human
X
Chromosomes
in
Hybrid
Cells.
Hy-
brid
cells
also
let
us
examine
the
human
inactive
X
chromo-
some
in
a
foreign
environment.
In
hybrids
derived
from
mouse
A9
cells
by
5-azacytidine
treatment,
the
two
Xcen
signals
were
as
close
as
in
human
parent
cells
(2.2
vs.
2.3
,um
and
1.3
vs.
1.3
,um
for
411
and
3935,
respectively)
(Table
2
and
Fig.
3:
Xcen).
However,
the
centromeres
were
significantly
further
apart
in
the
7213
hybrid
(derived
from
mouse
tsAl
cells)
than
in
parent
human
cells
(2.6
,m
vs.
0.9
Am,
P
<
0.0005).
The
Xptel
signals
were
only
slightly
farther
apart
in
hybrids
than
in
parent
cells
(1.5
vs.
1.1,
P
<
0.005,
and
1.6
vs.
1.1,
P
<
0.0005,
for
3935
and
7213,
respectively)
(Table
2
and
Fig.
3:
Xpel).
Mitotic
Cells.
To
examine
the
inactive
X
chromosome
in
mitosis,
cells
from
mitotic
shake-offs
were
fixed
without
hypotonic
treatment.
One
caveat
is
that
if
sister
chromatids
are
still
joined,
signals
due
to
replication
of
the
sequence
at
one
telomere
(signal
on
each
sister
chromatid)
are
not
easily
distinguished
from
those
at
two
telomeres.
However,
this
ambiguity
can
be
resolved
when
the
two
chromatids
disjoin
72I3
7213
hybrid
7213
hybrid
FIG.
3.
Biotinylated
signals
for
xcen
(Left)
and
Xptel
(Right)
in
7213
and
3935
hybrids
and
parental
human
cells,
as
indicated.
(x
750.)
in
anaphase,
seen
in cells
with
four
signals
(i.e.,
Fig.
4).
For
all
three
isodicentrics,
the
mean
distance
between
double
signals,
whether
telomeric
or
centromeric,
was
consistently
about
1
,um.
Therefore,
at
mitosis,
when
chromosomes
are
most
condensed,
the
two
ends
(which
may
be
as
close
as
the
replicated
chromatids-i.e.,
Fig.
4C)
are
only
slightly
closer
than
in
interphase.
The
distance
between
centromeres
changed
little
from
interphase
to
mitosis
for
the
3935
chro-
mosome
(1.3
vs.
1.1
,um,
P
c
0.05)
but
decreased
significantly
for
the
411
chromosome
with
centromeres
separated
by
the
short
arms
(2.3
vs.
1.1,
P
2
0.0005).
Relationship
of
Centromere
and
Telomere
Analyzed
by
Simultaneous
Hybridization
with
Xcen
and
Xpt'
Probes.
When
the
3935
chromosome
was
doubly
labeled
[DNP-labeled
X'en
conjugated
with
Texas
red
and
biotin-labeled
Xptel
conju-
gated
with
fluorescein
isothiocyanate
(FITC)-avidin]
the
four
signals
obtained
were
close
but
difficult
to
resolve
with
the
compound
microscope.
Using
confocal
microscopy,
we
could
superimpose
Texas
red
and
FITC-avidin
signals
and
optically
section
the
cell
from
top
to
bottom
along
the
z
axis
by
0.5-,um
intervals
to
examine
the
three-dimensional
rela-
tionships.
Signals
were
often
at
the
periphery
of
the
nucleus
and
most
often
near
the
top,
perhaps
an
ascertainment
bias
favoring
brighter
signals.
Fig.
SA
shows
that
all
four
signals
were
adjacent.
Although
cells
were
acid
fixed,
we
could
detect
differences
in
the
depth
of
signals.
Telomere
signals
(green)
came
into
view
before
those
from
the
centromeres
(red),
suggesting
that
telomeres
were
closer
to
the
nuclear
membrane;
this
was
supported
by
an
xz
image
(optical
section
in
the
vertical
plane),
which
also
revealed
a
greater
distance
from
centromere
to
telomere
than
between
the
two
cen-
tromeres
or
the
two
telomeres
(Fig.
SB).
However,
the
span
Genetics:
Walker
et
al.

Proc.
Natl.
Acad.
Sci.
USA
88
(1991)
*
FIG.
4.
Replicating
Barr
bodies
in
cells
with
the
3935
chromosome.
(A)
Replicated
bipartite
Barr
body
in
DAPI-stained
fibroblast.
(B
and
C)
Signals
from
replicating
isodicentric
chromosomes
in
mitotic
cells
from
hybrid,
labeled
with
Xptel
(B)
and
Xcen
(C).
(x750.)
between
XCIN
and
Xptel
signals
was
not
large
compared
to
the
widely
separated
signals
seen
in
hybrids
with
the
normal
human
active
X
chromosome
(data
not
shown).
Table
2.
Distance
between
double
signals
resulting
from
in situ
hybridization
of
biotinylated
probes
Phase
of
Distance,*
Cell
line
Probe
cell
cycle
,um
n
Active
normal
X
DB
hybrid
XpteI
+
Xqtel
Interphase
10.4
±
5.3
10
Inactive
normal
X
G1
hybrid
XptcI
+
Xqtel
Interphase
1.5
±
0.3
22
Inactive
dicentric
X
F411
Xcen
Interphase
2.2
±
1.7
24
411-hybrid
Xcen
Interphase
2.3
±
0.7
28
411-hybrid
Xcen
Mitosis
1.1
±
0.3
35
411-hybrid
Xqtel
Interphase
1.0
±
0.3
20
GM3935
Xcen
Interphase
1.3
±
0.5
23
3935-hybrid
Xcen
Interphase
1.3
±
0.5
16
3935-hybrid
Xcen
Mitosis
1.1
±
0.3
22
GM3935
Xptel
Interphase
1.1
±
0.3
28
3935-hybrid
Xptel
Interphase
1.5
±
0.7
20
3935-hybrid
Xptel
Mitosis
1.1
±
0.3
36
GM7213
Xcen
Interphase
0.9
±
0.3
21
7213-hybrid
Xcen
Interphase
2.6
±
0.4
41
7213-hybrid
Xcen
Mitosis
1.1
±
0.2
10
GM7213
Xptel
Interphase
1.1
±
0.4
24
7213-hybrid
Xptel
Interphase
1.6
±
0.4
27
7213-hybrid
Xptel
Mitosis
1.0
±
0.2
13
n,
Number
of
cells
analyzed.
*Mean
±
SD.
Interphase
Position
of
Telomeres
in
Normal
X
Chromo-
somes.
Because
either
Xp
or
Xq
telomere
of
isodicentrics
was
capable
of
telomere
association,
we
analyzed
hybrids
having
normal
X
chromosomes,
using
a
mixture
of
Xptel
and
Xqtel
probes.
The
mean
distance
between
these
signals
in
hybrids
with
the
wild-type
active
X
chromosomes
was
10.4
±
5.3
Aum,
with
a
wide
range
from
3
to
22
,um
(DB
hybrid
in
Table
2).
In
striking
contrast,
the
two
signals
were
close
(1.5
±
0.3
Aum)
in
hybrids
with
only
the
normal
inactive
X
chromosome,
showing
proximity
of
long-
and
short-arm
telomeres
(Fig.
6A
and
Table
2,
G1
hybrid).
DISCUSSION
Evidence
That
the
Inactive
X
Chromosome
Forms
a
Loop
Structure
with
Telomeres
Associated.
These
studies
show
that
the
inactive
X
chromosome,
whether
isodicentric
or
struc-
turally
normal,
is
not
a
linear
structure.
The
studies
with
single
probes
show
that
the
two
ends
of
these
chromosomes
are
too
close
together
for
linear
structures.
Because
we
only
measured
distance
between
two
discernible
signals
and
our
probes
were
subtelomeric,
the
distance
between
telomeres
may
be
even
less
than
measured.
In
any
event,
based
on
interphase
studies
of
Lawrence
et
al.
(27,
28),
Trask
et
al.
FIG.
5.
Confocal
images
of
3935
hybrid
simultaneously
labeled
with
Xptel
and
Xen.
(A)
z
series
image
showing
superimposed
telomere
(green)
and
centromere
(red)
double
signals.
Nucleus
visualized
with
propidium
iodide.
(x1700.)
(B)
Superimposed
xz
vertical
images
of
the
same
cell,
taken
through
telomere
and
through
centromere
signals,
showing
close
proximity
of
the
two
centromere
signals
(seen
as
two
red
parallel
lines)
and
the
greater
distance
from
centromere
to
telomere
(green
signal).
(x6000.)
FIG.
6.
Telomere
association
of
the
normal
inactive
X
chromo-
some.
(A)
G1-A9
hybrid
labeled
with
XptcI
and
XqtcI
showing
proximity
of
signals
from
the
two
ends
of
the
chromosome.
(B)
Characteristic
bends
in
all
three
inactive
X
chromosomes
(arrows)
in
a
hypotonic-treated
metaphase
from
a48,
XXXX
human
cell.
(x750.)
6194
Genetics:
Walker
et
al.

Proc.
Natl.
Acad.
Sci.
USA
88
(1991)
6195
FIG.
7.
Diagram
showing
one
simple
model
that
fits
our
obser-
vations
of
dicentric
(A
and
B)
and
normal
(C)
inactive
X
chromo-
somes.
The
model
in
B
accounts
for
the
greater
variability
observed
for
centromere
signals.
(28),
and
others
[reviewed
by
Trask
et
al.
(29)
and
Manuelidis
and
Cher
(30)],
the
1-,um
distance
between
inactive
X
telo-
meres
is
roughly
equivalent
to
one
or
two
megabases
of
interphase
DNA.
This
is
only
slightly
greater
than
the
700-nm
fiber
that
characterizes
metaphase
chromosomes
and
is
con-
siderably
less
than
the
>10-,um
length
of
chromosome
1
(smaller
than
the
smallest
isodicentric
chromosome
ana-
lyzed)
measured
by
electron
microscopy
in
an
acid-fixed
prometaphase
preparation.
In
fact,
at
interphase,
the
mean
distance
between
telomeres
of
the
normal
active
X
is
10-fold
greater
(10.4
±
5.3
Am;
Table
2,
DB
hybrid,
Xptel
plus
XqtIc).
The
small
distance
between
centromeres
of
dicentrics
means
that
these
chromosomes
must
be
enfolded
so
that
centromeres
are
also
adjacent.
The
simplest
nonlinear
model
to
fit
our
observations
is
a
loop
(modeled
in
Fig.
7).
As
the
telomere
signals
are
always
close,
whereas
the
distance
between
cen-
tromeres
is
variable
among
the
three
dicentrics,
the
base
of
the
loop
is
at
the
telomeres; the
centromeres
may
be
closer
(Fig.
7A)
or
farther
apart
(Fig.
7B).
Supporting
the
looped
structure
are
the
characteristic
bends
in
the
proximal
long
arm
of
normal
inactive
X
chromosomes
observed
in
metaphase
preparations
(31).
Fig.
6B
shows
a
human
metaphase
with
four
normal
X
chromosomes
in
which
all
three
inactive
X
chromosomes
are
bent.
That
the
telomeres
are
not
closer
together
is
probably
due
to
disruption
of
telomere
association
by
hypotonic
treatment
used
to
prepare
metaphases.
Relationship
Between
Chromosome
Configuration
and
Tran-
scription.
Similar
location
of
signals
in
hybrid
and
human
cells
for
411
and
3935
chromosomes
indicates
that
cell
environment
alone
does
not
determine
chromosome
configuration.
How-
ever,
the
position
of
centromeres
may
be
affected
by
tran-
scriptional
activity.
Selection
of
the
7213
hybrid
required
activity
of
the
A1S9T
locus,
not
far
from
the
centromere,
and
greater
transcriptional
activity
of
this
gene
might
be
respon-
sible
for
the
greater
distance
between
Xen
signals
in
hybrid
cells.
Similarly,
the
relatively
greater
distance
between
cen-
tromeres
of
the
411
chromosome
in
human
and
hybrid
cells
(>2
,um)
could
be
due
to
expression
of
some
loci
on
the
intervening
short
arm
(17).
In
both
cases,
this
distance
de-
creases
considerably
in
mitotic
cells,
when
euchromatic
(tran-
scribed)
regions
condense,
as
expected,
if
in
fact,
transcrip-
tional
activity
influences
position
of
centromeres.
The
Barr
Body
in
Mitosis.
Although
centromeres
of
the
isodicentric
chromosomes
are
closest
in
mitosis,
the
distance
between
the
telomeres
changes
relatively
little
from
inter-
phase
to
mitosis.
That
the
distance
between
telomeres
in
mitotic
cells
is
also
1
gm
(Table
2
and
Fig.
4)
suggests
that
the
loop
structure
is
maintained
during
mitosis.
Significance
of
Telomere
Association.
Although
both
telo-
meres
of
the
inactive
X
chromosome
are
close
to
the
nuclear
membrane,
we
have
no
evidence
of
membrane
attachment.
There
is
some
electron
microscopic
evidence
for
a
network
of
filaments
emanating
from
the
membrane
toward
the
Barr
body
(3),
and
in
meiosis
telomeres
are
frequently
reversibly
associated
with
the
nuclear
envelope
(18,
32).
Yet,
the
proximity
of
telomeres
in
mitotic
cells
(in
absence
of
nuclear
membrane)
suggests
that
the
nuclear
envelope
is
not
required
to
maintain
telomere
association.
Hinton
(33)
showed
that
terminal
adhesions
between
nonhomologous
ends
of
poly-
tene
chromosomes
were
inherent
in
the
nature
of
the
telo-
mere.
The
common
DNA
sequence
(telomere)
at
the
ends
of
all
human
chromosomes
could
predispose
to
this
kind
of
association;
however
our
observations
that
the
telomeres
of
the
active
X
chromosome
are
not
close
suggest
that
associ-
ation
of
the
two
ends
of
the
inactive
X
chromosome
is
not
a
general
characteristic
of
interphase
chromosomes
and
may
be
a
unique
attribute
of
inactive
chromosomes.
How
telom-
ere
association
occurs
and
what
role
the
looped
configuration
of
the
chromosome
plays
in
silencing
transcription
of
the
inactive
X
chromosome
are
subjects
for
further
study.
We
thank
Joyce
Axelman
for
maintaining
fibroblast
and
hybrid
cell
cultures
and
Dr.
Laura
Manuelidis
for
advice
on
preparing
the
manuscript.
This
work
was
supported
by
National
Institutes
of
Health
Grant
HD05465.
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