URIDINE-5-H
OF THE
3 RADIOAUTOGRAPHY
HUMAN
DAVID
SEX
CHROMATIN
BODY
E. C O M I N G S
From the Department of Medicine, Division of Medical Genetics, University of Washington, Seattle
ABSTRACT
To obtain an estimate of the rate of R N A synthesis by the heterochromatic sex chromatin
body, human female fibroblasts were labeled with uridine-5-H 3 and radioautographed.
The number of grains over the sex chromatin body was compared with the n u m b e r of
grains over a comparable area ofeuchromatin. The ratio was 0.37. When corrected for the
higher content of D N A per unit area in heterochromatin, the maximum rate of R N A synthesis by the D N A of the sex ehromatin body was approximately 18% of the rate of R N A
synthesis by a comparable amount of euchromatin DNA. The rate of R N A synthesis by
the sex chromatin body did not increase significantly with partial despiralization of this
chromatin at prophase.
The hypothesis that in each female m a m m a l i a n
cell one X chromosome is randomly inactivated
(1, 2) explains a great number of phenomena
related to the different behavior in males and
females of X-linked genes. This inactive X
chromosome is detected in the interphase cell as
the sex chromatin body (3). It is generally assumed
that this heterochromatic X chromosome is
inactive in R N A synthesis. This assumption is
based, in part, on the observation that heterochromatin in other organisms is largely inactive
in the synthesis of R N A (4-6). There are, however,
a number of factors which suggest that the inactive X chromosome may synthesize some R N A :
(a) some X-linked loci may not be inactivated
(7-9); (b) the abnormalities seen in X X Y and
in X X X X Y individuals may be due to incomplete inactivation of the X chromosomes;
(c) in mice there appears to be a gradient of X
inactivation (10); (d) since heterochromatin may
undergo D N A replication without uncoiling (11,
12), it is possible that some R N A synthesis may
also occur; (e) electron microscopy of the sex
chromatin body reveals it to be considerably less
condensed than metaphase chromosomes (13), a
feature compatible with some synthesis of R N A ;
(f) isolated repressed chromatin is capable of
some R N A synthesis (14); and (g) R N A may
function as a repressor molecule (15) and thus be
a necessary part of the inactivated chromosome.
Thus, although there are valid reasons both for
suspecting that in humans the inactive X chromosome is completely inactive, as well as for believing
it to be capable of some R N A synthesis, there has
been no direct attempt to quantitate its capacity
for R N A synthesis. In the present study, to attempt
an estimate of this capacity, cultured h u m a n
female fibroblasts were labeled with tritiated
uridine, radioautographed, and the number of
grains over the sex chromatin body was compared
with the number of grains over a comparable area
of euchromatin.
MATERIAL
AND METHODS
Human fibroblasts, obtained from a normal, 46 XX,
11 yr old girl, were cultured on cover slips in Eagle's
medium with 10% fetal calf serum, 1-glutamine,
sodium pyruvate, nonessential amino acids, penicillin, and streptomycin. The cells were in culture for
4 months and were in their 13th transfer. Following
transfer with trypsin, during the log phase of cell
growth, uridine-5-H 3, 50 /zc/ml (specific activity
437
F m v R s 1 a T h e n u m b e r of grains over the sex ehromatin body (in this case, 0) is compared with the
n u m b e r of grains (3) in an identical area shifted a constant distance counterclockwise along the nuclear
membrane.
FIGURE 1 b Pre- a n d postradioautography of a uridine-5-H3-1abeled fibroblast.
FIGVRE 1 C T h e decrease in the n u m b e r of grains over the sex chromatin body (arrow) persists into prophase. X 1500.
20 c / m ~ N u c l e a r - C h i c a g o Corporation, Des Plaines,
Illinois) was a d d e d to t h e culture. After 5 m i n t h e
culture m e d i u m was p o u r e d off a n d the cells were
fixed for 30 rain in several c h a n g e s of 1-'3 glacial
acetic acid a n d m e t h a n o l . U r i d i n e - 5 - H 3 was used
since a n y conversion to thymidylic acid removes t h e
H 8 label a n d prevents incorporation into D N A by
this route (16). However, because t h e r e is some incorporation of label into D N A by conversion to
deoxycytidine (17), the ceils were treated with deoxyribonuclease, 100 ~ g / m l in 5.0 mM MgSO4, phosp h a t e buffer p H 6.7, at 37°C for 2 hr. H3-thymidine labeled control slides d e m o n s t r a t e d t h a t D N A was
completely removed. T h e cells were stained in acetoorcein. T o avoid selection bias, the cells were photog r a p h e d before r a d i o a u t o g r a p h y . 37 cells containing
a distinct sex c h r o m a t i n body located at the nuclear
periphery, 14 cells c o n t a i n i n g neither a sex c h r o m a tin b o d y n o r other h e t e r o c h r o m a t i n , a n d 23 cells in
p r o p h a s e were p h o t o g r a p h e d with a Zeiss Photoscope. T h e cells were covered with K o d a k A R 10
fine g r a i n stripping film (18), exposed for 40 days,
a n d developed in K o d a k 19b developer. T h e ceils
p h o t o g r a p h e d before r a d i o a u t o g r a p h y were located
by coordinates a n d r e p h o t o g r a p h e d by phase-contrast microscopy. 30 additional p o s t r a d i o a u t o g r a p h y
chromatin-positive cells were p h o t o g r a p h e d . Ribonuclease, 1 5 0 / z g / m l (Sigma), p H 6.7 at 37¢C for 2
hr, r e m o v e d all radioactivity.
438
T o evaluate R N A synthesis of the sex c h r o m a t i n
body in relation to n u c l e a r e u c h r o m a t i n , the prer a d i o a u t o g r a p h y p h o t o g r a p h s were projected, a n d
the sex c h r o m a t i n was outlined on paper. T h e radioa u t o g r a p h e d cell was t h e n projected; the outline of
t h e sex c h r o m a t i n b o d y was superimposed on t h e
r a d i o a u t o g r a p h e d c h r o m a t i n body; a n d the n u m b e r
of grains within the outline were counted. T h e outline was t h e n m o v e d a constant interval (5 c m on t h e
projected image) counterclockwise along the n u c l e a r
m e m b r a n e , a n d t h e n u m b e r of grains within t h e
outline was a g a i n counted. This gave t h e n u m b e r of
grains over t h e sex c h r o m a t i n b o d y a n d the n u m b e r
of grains over a c o m p a r a b l e area of e u c h r o m a t i n
selected w i t h o u t bias (Fig. 1 a). Similar counts were
m a d e for the ceils p h o t o g r a p h e d only after radioautography.
RESULTS
T h e results o b t a i n e d f r o m cells p h o t o g r a p h e d
before r a d i o a u t o g r a p h y
a n d f r o m cells p h o t o g r a p h e d o n l y after r a d i o a u t o g r a p h y a r e c o m p a r a b l e a n d a r e s h o w n in T a b l e I A. T h e r a t i o of
t h e n u m b e r of g r a i n s over t h e sex c h r o m a t i n b o d y
to t h e n u m b e r of g r a i n s o v e r a c o m p a r a b l e a r e a
of e u c h r o m a t i n w a s 0.37. T h e d i s t r i b u t i o n of
g r a i n s is s h o w n in Fig. 2.
THE JOUnNAL OF CELL BIOLOGY • VOLUME ~8, 1966
X Sex chromatin
25
X ~
0 Adjacent euchromatin
X
No. 20
~~0
15
....
Ox
x
of
10
/
"o,
,
i
t
ceils
\
/
5
/
\
t
0-._.0..
X ~ x
o
®
FIGURE ~ T h e distribution of the n u m bers of grains over the sex e h r o m a t i n
body and over a comparable area of euchromatin in cells labeled with uridine-5H 3.
\
~--
0
1
2
3
'"n
4
~
6
7
No. of grains
TABLE
I
Uridine-5-HLLabeled Human Female Fibroblasts
A
Grains over the sex chromatin body in relation to grains over a comparable adjacent area
Grains over sex chromatin
(SC)
Avg
grain
count
No.
Cells p h o t o g r a p h e d
radioautography
Ceils
photographed
radioautography
SD
Grains over
comparable adjacent
area (AA)
Avg
grain
count
Ratio*
SC/AA
SO
before
37
0.946
0.94
2.54
1.42
0.372
after
30
1.03
1.13
2.76
1.50
0.373
B Total numbers of grains over nuclei of cells with sex chromatin, of cells without sex chromatin, and of
cells in prophase
Cells w i t h sex c h r o m a t i n
Cells w i t h o u t sex c h r o m a t i n
Cells in p r o p h a s e
No.
Avg grain count
sD
37
14
23
337
320
354
160
104
137
* SC g r a i n c o u n t s i g n i f i c a n t l y d i f f e r e n t f r o m A A g r a i n c o u n t s P < 0.001.
Since t h e s u p p r e s s o r o f R N A s y n t h e s i s in t h e
h e t e r o c h r o m a f i n m i g h t be a diffusible factor, t h e
d e n s i t y of g r a i n s a r o u n d t h e s e x c h r o m a t i n w a s
compared with that around the comparable area
of e u c h r o m a t i n . A l t h o u g h s o m e of t h e sex c h r o m a t i n b o d i e s s h o w e d a d e c r e a s e in t h e n u m b e r o f
p e r i p h e r a l g r a i n s , in o t h e r s this d e c r e a s e w a s n o t
s e e n a n d w a s n o t sufficiently c o m m o n to be
significant.
T h e r e w a s n o s i g n i f i c a n t d i f f e r e n c e ( P = 0.65)
b e t w e e n t h e total n u m b e r o f g r a i n s o v e r t h e n u c l e i
of cells c o n t a i n i n g sex c h r o m a t i n a n d t h e total
n u m b e r of g r a i n s o v e r t h e n u c l e i of cells w i t h o u t
s e x c h r o m a t i n ( T a b l e I B).
T o e v a l u a t e t h e possibility t h a t , w i t h p a r t i a l
d e s p i r a l i z a t i o n o f t h e sex c h r o m a t i n b o d y d u r i n g
p r o p h a s e , this c h r o m a t i n m i g h t b e c o m e active in
R N A synthesis, 23 cells in p r o p h a s e w e r e also
p h o t o g r a p h e d before r a d i o a u t o g r a p h y . I n t h o s e
cells w h i c h s h o w e d a n i d e n t i f i a b l e p r e s u m p t i v e
DAVID E. COMINGS Urldlne-5-H 3 Radioautography
439
heterochromatic X chromosome, the n u m b e r of
grains overlying it was again less t h a n t h a t over
adjacent c h r o m a t i n (Fig. 1 c). T h e total n u m b e r
of grains over the nuclei of prophase cells was not
significantly different (P = 0.55) from t h a t over
the nuclei of interphase cells (Table I B), indicating t h a t the suppression of R N A synthesis
which occurs d u r i n g mitosis (19, 20) has not yet
b e g u n at prophase.
DISCUSSION
Even t h o u g h the sex c h r o m a t i n body is located at
the t h i n n e d - o u t edge of the nucleus, it is possible
t h a t some e u c h r o m a t i n m a y lie above or below it.
Electron micrographs of the sex c h r o m a t i n body
show it to be liberally interspersed with euchrom a t i n material (13). W h e t h e r this is euchromatic
X c h r o m a t i n material or autosomal c h r o m a t i n
is not known. Since some n o n - X e u c h r o m a t i n is
p r o b a b l y present in the area of the sex c h r o m a t i n
body, a n d because tritium has a beta track of u p
to 5 #, the ratio of 0.37 for grains over the sex
c h r o m a t i n body in relation to grains over a comparable adjacent area of e u c h r o m a t i n is a m a x i m a l
figure. Since the a m o u n t of D N A in heteroc h r o m a t i n is approximately twice t h a t in a comp a r a b l e area of e u c h r o m a t i n (21), t h e m a x i m u m
rate of synthesis of R N A b y sex c h r o m a t i n D N A
becomes approximately 18% of the rate of R N A
synthesis by a comparable a m o u n t of e u c h r o m a t i n
DNA. Again, since some autosomal e u c h r o m a t i n
m a y be present in this area, the true percentage
m a y range a n y w h e r e between 0 a n d 18%.
I n relation to the distribution of grain counts
over the sex c h r o m a t i n body (Fig. 2), the lack of a
bimodal curve with most counts zero a n d with a
smaller n u m b e r of counts of 2 or more grain~
suggests t h a t w h a t e v e r activity is present is not
due to the synthesis by a few sex c h r o m a t i n bodies
of significant a m o u n t s of R N A a n d the absence of
synthesis by others in a different portion of the
cell cycle. A m i n o r degree of bimodality could,
however, be missed. T h e existence of some cells
with fairly high grain counts over the sex chrom a t i n body suggests t h a t the lower limit of R N A
synthesis m a y be greater t h a n zero.
T h e absence of a significant difference between
the total n u m b e r of grains over the nuclei of cells
without a sex c h r o m a t i n body a n d the total
n u m b e r of grains over the nuclei of cells with a
sex c h r o m a t i n body suggests t h a t cells without sex
c h r o m a t i n are not merely dying or degenerate
cells.
Due to the conditions of the experiment,
utilizing a short pulse of a high c o n c e n t r a t i o n of
uridine-5-H 3, R N A with a more r a p i d rate of
t u r n o v e r would be preferentially labeled. Studies
are in progress to detect the possible synthesis of
a stable R N A as a n integral p a r t of the sex
c h r o m a t i n body.
This study was supported by G r a n t H E 3091 from
the United States Public Health Service.
Received for publication 8 November 1965.
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DAVID E. Co~x~Gs
Uridine-g-H 3 Radioautography
441