Correlation between interphase
and metaphase chromosome
arrangements as studied by
laser-uv-microbeam experiments*
T.Cremer, H. Baumann, K. Nakanishi* and
C. Cremen
Institute of Anthropology and Human Genetics, University of
Heidelberg, D-6900 Heidelberg, FRG, 'Department of Pathology, Kyoto
Prefecture! University of Medicine, Kyoto, Japan and 'Institute of
Applied Physics I, University of Heidelberg, D-6900 Heidelberg, FRG
I n s p i t e o f many attempts a t r e s o l u t i o n , t h e problem o f the chromo­
some topography i n i n t e r p h a s e
significance
(Lewin,
nuclei
and
i t s possible
functional
1981; V o g e l and K r u g e r , 1983) has remained a
m a t t e r o f g r e a t c o n t r o v e r s y and u n c e r t a i n t y
(for reviews
s e e Co­
mings,1980; A v i v i & Feldman, 1 9 8 0 ) . I n p a r t i c u l a r , the r e l a t i o n s h i p
between metaphase and
still
interphase
arrangements
o f chromosomes i s
l a r g e l y unknown* λ s o l u t i o n o f t h i s problem seems e s p e c i a l l y
u r g e n t , s i n c e t h e s p a t i a l r e l a t i o n s h i p o f chromosomes a t metaphase
h a s been e x t e n s i v e l y s t u d i e d i n many s p e c i e s d u r i n g t h e l a s t
years.
twenty
R e c e n t l y , Bennett and coworkers have demonstrated a h i g h l y
o r d e r e d arrangement o f c e n t r o m e r e s i n metaphase p l a t e s o f c e r t a i n
plant
species (Bennett,
1983)
and s t i m u l a t e d
fresh interest
development o f t e c h n i q u e s
f o r mapping the arrangements o f
d u a l chromosomes d i r e c t l y
within
the interphase
nucleus.
i n the
indivi­
In this
paper we d e s c r i b e a laser-uv-microbeam approach which a l l o w s s e l e c ­
t i v e m i c r o i r r a d i a t i o n o f a s m a l l amount o f chromatin i n the i n t e r ­
phase n u c l e u s o f c u l t u r e d
cells
and s t u d y o f i t s
distribution in
chromosomes during t h e subsequent m i t o s i s . A l t e r n a t i v e l y , c h r o m a t i n
can be m i c r o i r r a d i a t e d d u r i n g m i t o s i s and i t s d i s t r i b u t i o n
can be
s t u d i e d i n the subsequent i n t e r p h a s e n u c l e u s .
* Supported by the Deutsche F o r s c h u n g s g e m e i n s c h a f t , Bonn. Or. K.
N a k a n i s h i was on a F e l l o w s h i p o f the A. von Humboldt S t i f t u n g .
Chromosomes today, volume 8
George Allen St Unwin 1984
204
Study of chromosome arrangements
TERRITORIAL ORGANIZATION OP CHROMOSOMES IN THE
OF CULTURED CHINESE HAMSTER CELLS
A prerequisite
to any
INTERPHASE NUCLEUS
meaningful conclusion
which might
be
drawn from studies of chromosome arrangements a t metaphase, on
the
arrangement of chromosomes i n the
as-
interphase
nucleus,
i s the
sumption that each chromosome continues to e x i s t i n the
nucleus as a s p a t i a l l y d i s t i n c t , and
tion, s t i l l
space.
The
hypothesis
coherent domain of
that
such
chromosome
(also c a l l e d chromosome domains) e x i s t
i n the
has
(1885)
been put
i t s decondensa-
r e l a t i v e l y compact e n t i t y . I n t h i s c a s e , each s i n g l e
chromosome would occupy a small and
nuolear
i n s p i t e of
interphase
forward already
by
Rabl
the
interphase
and
total
territories
nucleus
Boveri
Other models, however, proposed h a l f a century l a t e r ,
(1909).
have
still
s e r i o u s l y considered
the p o s s i b i l i t y that the chromatin f i b r e s of
euchromatic
regions
within
extensively
dispersed
individual
i n the
chromosomes
interphase
nucleus
might
become
(Comings,
1968;
Vogel and Schroeder, 1974). In case that the chromatin f i b r e , which
is
supposed
to
constitute
each
i n d i v i d u a l chromosome, would
extended throughout a major p a r t of the t o t a l nuclear
be
epace, each
chromosome would form a s s o c i a t i o n s with many other chromosomes a t
many s i t e s .
I f so,
chromosome
arrangements
at
metaphase
n a t u r a l l y y i e l d an i n s u f f i c i e n t p i c t u r e of t h e i r interphase
would
arran-
gements.
Laeer-uv-microirradiation
(>,»
257 nm)
has provided the p o s s i -
b i l i t y to put such models to a rigorous t e s t . Small p a r t s of i n t e r phase n u c l e i of cultured Chinese hamster c e l l s were m l c r o i r r a d i a ted. Obviously, the d i s t r i b u t i o n of
raicroirradiated
chromatin i n
the chromosomes of the subsequent metaphase c r i t i c a l l y depends
on
the a c t u a l arrangement of interphase chromosomes i n the m i c r o i r r a diated nuclear p a r t . In the case of a t e r r i t o r i a l o r g a n i z a t i o n , i t
i s predicted
that microirradiated chromatin i s r e s t r i c t e d to a
few
metaphase chromosomes, depending on the s i z e of the micr.oirradiated
nuclear segment. I n c o n t r a s t , extensive d i s p e r s i o n of the chromatin
f i b r e s from i n d i v i d u a l chromosomes should be
r i n g of m i c r o i r r a d i a t e d
i n d i c a t e d by
scatte-
chromatin across a correspondingly i n c r e a -
sing number of metaphase chromosomes.
Microirradiated
chromatin was
i d e n t i f i e d e i t h e r autoradiogra-
Τ. Cremer et al.
phically
takes
by
place
detection
during
the
of
unscheduled
course o f
l e s i o n s i n the m i c r o i r r a d i a t e d
Cremer
e t a l . , 1982a) or
detection
mainly
of
\ Ν
by
DNA
excision
nuclear
use
pyrimidine
of
synthesis
r e p a i r of
205
(UDS)
which
DNA
photo-
segment (Zoen et a l . , 1979;
antibodies
dimers
i n a DNA
specific
for
structure
the
which
b
F i g . I s P a i r s o f daughter n u c l e i were f i x e d i n a c e t i c acid/methan o l (1:3) 3h 45* a , 3h 25' c and 4h 25' e a f t e r m i c r o i r r a d i a t i o n o f
a s m a l l p a r t o f the metaphase p l a t e o f Chinese hamster c e l l s
(M3-1) . a,c,e N u c l e i
s t a i n e d w i t h DAPI. b,d,f immunocytochemical
l o c a l i z a t i o n of m i c r o i r r a d i a t e d chromatin ( a r r o w s ) . S t a i n i n g for
i n d i r e c t immunofluorescence microscopy was performed as p r e v i o u s l y
d e s c r i b e d (Cremer e t a l . , 1983). A n t i b o d i e s w i t h a high a f f i n i t y
for u v - i r r a d i a t e d DNA were r a i s e d independently i n r a b b i t s by Dr.
K. N a k a n i s h i and Dr. J . J . C o r n e l l s . Both a n t i s e r a y i e l d e d i d e n t i c a l
r e s u l t s . G o a t - a n t i - r a b b i t igG conjugated w i t h f l u o r e s c e i n e i s o t h i o c y a n a t e (FITC) was obtained from Nordic. For comparison n u c l e i were
c o u n t e r s t a i n e d w i t h DAPI, t h e r e a f t e r . By appropriate f i l t e r i n g , the
blue DAPI f l u o r e s c e n c e could e a s i l y be d i s t i n g u i s h e d from the green
immunofluorescence. Microphotopraphe were taken with a Z e i s s photomicroscope equipped w i t h e p i f l u o r e s c e n c e i l l u m i n a t i o n . Α m i r r o c ­
l i k e d i s t r i b u t i o n o f m i c r o i r r a d i a t e d chromatin i n each p a i r o f
daughter n u c l e i becomes more obvious when changes i n the p o s i t i o n s
o f the whole n u c l e i during the p o s t i n c u b a t i o n period are taken i n t o
c o n s i d e r a t i o n and n u c l e i are a c c o r d i n g l y r e p o s i t i o n e d .
206
Study of chromosome
arrangements
represent the major DNA-photoproduct o f u v - i r r a d i a t i o n
(Cremer e t a l . ,
1983; Hens e t a l . , 1983). With
a t 257 nm
both methods o f
d e t e c t i o n we found that m i c r o i r r a d i t a t i o n o f a s m a l l p a r t o f t h e
interphase nucleus (approx. 5 % o f the t o t a l
different
lines
o f Chinese hamster
cells,
nuclear
resulted
area) i n
i n intensive
l a b e l l i n g of segments from a few chromosomes o n l y (3 t o 4 on t h e
average) , while
the majority
o f chromosomes
i n each
metaphase
spread (modal numbers 22 - 23) remained u n l a b e l l e d . I t i s important
to note that t h i s r e s u l t was obtained both a f t e r
microirradiation
of n u c l e i i n 61 (Cremer e t a l . , 1982a) and i n s-phaee (Hens e t a l . ,
1983) , i n d i c a t i n g t h a t a t e r r i t o r i a l o r g a n i z a t i o n o f chromosomes i s
maintained during subsequent s t a g e s o f the c e l l c y c l e . Furthermore,
i t was found that t h e number o f chromosomes i n which formation o f
s i s t e r chromatid exchanges
(SCEs) was induced a t a g i v e n u v - i n c i -
dent energy increased s i g n i f i c a n t l y with t h e s i z e o f t h e m i c r o i r r a diated
nuclear
area
(Raith
e t a l . , 1984). I n a d d i t i o n ,
chromosomes were m i c r o i r r a d i a t e d e i t h e r a t metaphase
mitotic
( s e e below) o r
a t anaphase (Hens e t a l . , 1983). I n the subsequently formed
inter-
phase n u c l e i t h e m i c r o i r r a d i a t e d chromatin was l i m i t e d t o a s m a l l
volume ( F i g . 1 ) . I n conclusion these microbeam experiments support
the concept that interphase chromosomes occupy d i s t i n c t domains o f
the interphase nucleus.
MIRROR-LIKE DISTRIBUTION OF CHROMATIN IN PAIRS OF DAUGTHER NUCLEI
M i c r o i r r a d i t a i o n o f metaphase
chromosomes i n l i v i n g
Chinese
hamster c e l l s allowed us t o t e s t a h y p o t h e s i s which was proposed
for t h e f i r s t
time by Theodor
Boveri
(1888; 1909). B o v e r i
stated
that arrangements o f chromosomes i n p a i r s o f daughter n u c l e i
would
occur i n a m i r r o r - l i k e fashion. T h i s h y p o t h e s i s was based on o b s e r v a t i o n s made during the f i r s t
cell
cycles
of f e r t i l i z e d
eggs o f
A s c a r i s megalocephala. B o v e r i found that t h e arrangements o f prophase chromosomes were mirror l i k e i n each p a i r o f daughter
cells
d e r i v e d from t h e same m i t o t i c event, while they appeared v a r i a b l e
to a l a r g e extent i n c e l l s from d i f f e r e n t m i t o t i c e v e n t s . By means
of immunofluorescence microscopy we have v i s u a l i z e d
the d i s t r i b u -
t i o n o f m i c r o i r r a d i a t e d chromatin i n a sample o f 27 p a i r s o f daugh-
Τ. Cremer et al.
ter
n u c l e i obtained a f t e r m i c r o i r r a d i t ion o f a s m a l l p a r t o f
metaphase p l a t e
of
the
(see P i g .
l for examples). To
t e s t the
chromatin d i s t r i b u t i o n i n each p a i r , the
207
the
similarity
daughter
nuclei
were p r o j e c t e d over each o t h e r i n a mirror l i k e fashion and p o s i t i o -
F i g . 2s
M i c r o i r r a d i a t i o n of interphase n u c l e i at two s i t e s o f
C h i n e s e hamster c e l l s (M3-1) was performed s i x hours a f t e r r e l e a s e
o f the c e l l s from a thymidine b l o c k (2 mM) . Chromosome p r e p a r a t i o n s
were performed i n s i t u as p r e v i o u s l y described (Zorn e t a l . , 1979)
a f t e r a p o s t i n c u b a t i o n period o f 4 hours i n the presence of c a f f e i ­
ne (2 mM). a,c,e I n t e r p h a s e n u c l e i and metaphase spreads s t a i n e d
w i t h DAPI. b,d,e Imrounocytochemical l o c a l i z a t i o n of m i c r o i r r a d i a t e d
chromatin. (For d e t a i l s see legend to F i g . 1) Arrows i n d i c a t e
corresponding s i t e s o f m i c r o i r r a d i a t e d chromatin i n a,b, c,d and
e , f . a,b M i c r o i r r a d i a t i o n a t two
nuclear
s i t e s c l o s e to each
o t h e r . c,d Metaphase spread obtained a f t e r m i c r o i r r a d i a t i o n of the
n u c l e u s i n the preceding i n t e r p h a s e a t two
s i t e s c l o s e t o each
o t h e r . e f Metaphase spread obtained a f t e r m i c r o i r r a d i a t i o n of the
i n t e r p h a s e nucleus a t two s i t e s f a r apart from each other as demon­
s t r a t e d by two adjacent n u c l e i . Notably, two s i t e s of i n d i r e c t
immunofluorescence corresponding w i t h two s i t e s of p a r t i a l chromo­
some s h a t t e r i n g (PCS) (Cremer e t a l . , 1983) can be detected i n the
two metaphase s p r e a d s . I n c o n t r a s t , a f t e r m i c r o i r r a d i a t i o n of the
i n t e r p h a s e nucleus a t one s i t e i n d i r e c t immunofluorescence c o r r e s ­
ponding t o one s i t e o f PCS was g e n e r a l l y observed i n the subsequent
metaphase (Hens e t a l . , 1983).
r
208
ned
Study of chromosome arrangements
i n a way
that y i e l d e d an o v e r l a p of both the
total
nuclear
areas and the immunoflourescent areas as complete as p o s s i b l e . For
comparison, 27 p a i r s of non-daughter n u c l e i o f s i m i l a r
s i z e were
tested from the same sample. I n support of B o v e r i ' s h y p o t h e s i s ,
found that the extent to which the Iramunofluorescent
we
areas o v e r l a p ­
ped was s i g n i f i c a n t l y l a r g e r (by a f a c t o r of approx. 2) i n p a i r s of
daughter n u c l e i . (K. Nakanishi & T. Cremer, i n preparation) .
Interphase
%
30
20
10
Uhu.
00.1
05
3
1.0
%« Metaphase
30
20+
10
15
dη
00,1
%•
3020·
10
η «95
JTHU05
1.0
15
d
2J0 n
C
3
<0>
30
20
10
b
00.1
Interphase
%
3^0,34
η =104
η
05
1.0
Metaphase
00,1
05
10
15
d
n
e
U
5
=127
2Ό 2.3 3.1
3^1,04
η =88
15
d
2.0
n
F i g . 3: Comparison o f r e l a t i v e d i s t a n c e s d i n interphase a,c and
metaphase b,d between the two s i t e s o f m i c r o i r r a d i a t e d chromatin i n
Chinese hamster c e l l s (M3-1) ( f o r further d e t a i l s compare F i g . 2) .
I n each interphase nucleus and metaphase spread the d i s t a n c e d
between the c e n t e r s of the two immunofluorescent s i t e s correspon­
ding to the two s i t e s of m i c r o i r r a d i a t e d chromatin was determined.
N u c l e i and metaphase spreads were considered as e l l i p s e s and the
diameters of the two major axes a and b were a l s o measured. To
avoid any p r e j u d i c e , evaluation o f metaphase spreads was performed
by a person without information on the p a r t i c u l a r type of m i c r o i r r a d i a t i o n . To account for v a r i a t i o n s i n s i z e o f n u c l e i and metaphase
spreads d was divided by 1/2-{ab i n each c a s e . 1/2f3? corresponds t o
the radius o f a c i r c l e of the same area, a) Frequency d i s t r i b u t i o n
o f d i n a sample o f 104 interphase n u c l e i w i t h two s i t e s o f microi r r a d i a t i o n c l o s e to each other. Nuclei were s i t u a t e d c l o s e t o the
metaphase spreads evaluated i n b (compare F i g . 2 a,b) . b) Frequency
d i s t r i b u t i o n of d i n 95 metaphase spreads obtained 4-6 hours a f t e r
m i c r o i r r a d i a t i o n of n u c l e i i n 4419 c e l l s a t two s i t e s as d e s c r i b e d
i n a (compare F i g . 2 c,d) . c) Frequency d i s t r i b u t i o n o f d
in a
sample o f 127 interphase n u c l e i s i t u a t e d c l o s e to metaphase spreads
evaluated i n d and m i c r o i r r a d i a t e d a t two s i t e s f a r apart from each
other, d)Frequency d i s t r i b u t i o n o f d
i n 88 metaphase spreads
obtained 4-6 hours a f t e r m i c r o i r r a d i a t i o n o f the interphase nucleus
i n 4350 c e l l s as described i n c (compare F i g . 2 e , f ) . Note t h a t the
mean values of d i n metaphase spreads s t i l l r e f l e c t the d i s t a n c e s
between the two s i t e s of m i c r o i r r a d i a t i o n i n the interphase nuc­
l e u s . The d i f f e r e n c e between d i n a,c (interphase n u c l e i ) and b,d
(metaphase spreads) i s h i g h l y s i g n i f i c a n t (PCO.001)
n
n
n
n
n
n
n
Τ. Cremer et al. 209
STATIC ARRANGEMENT OP CHROMOSOME TERRITORIES IN INTERPHASE NUCLEI
OF CULTURED CHINESE HAMSTER AND HUMAN FIBROBLASTOID CELLS
Theodor Boveri
(1909) was a l s o the f i r s t t o p r e d i c t t h a t t h e
chromosome t e r r i t o r i e s would be r a t h e r r i g i d l y f i x e d during
inter­
phase. Notably, c e l l c y c l e t r a n s i t times i n the developing
egg of
Ascaris
1
megalocephala
from which B o v e r i s hypothesis
was d e r i v e d
are r a t h e r short and many decades l a t e r the p o s s i b i l i t y o f e x t e n s i ­
ve a l t e r a t i o n s i n chromosome order during subsequent stages o f the
c e l l c y c l e was s t i l l
taken
among o t h e r s for other
i n t o c o n s i d e r a t i o n a s one p o s s i b i l i t y
c e l l types
(Vogel and Schroeder,
1974) . I n
c a s e o f a dynamic i n t e r p h a s e chromosome arrangement, s t u d i e s o f
metaphase chromosome arrangements could a t best provide
on
information
i n t e r p h a s e chromosome arrangements a t a l a t e stage of the c e l l
c y c l e . We, t h e r e f o r e , decided t o r e i n v e s t i g a t e t h i s problem both i n
f i b r o b l a s t o i d Chinese hamster c e l l s
and
i n human f i b r o b l a s t o i d
level
cells
approx. 30) . Interphase
(CHL)
(Flow
(Crenter e t a l . , 1982a,b)
2000; population
doubling
n u c l e i were m i c r o i r r a d i a t e d a t two
s i t e s and c e l l s were e i t h e r f i x e d immediately t h e r e a f t e r or allowed
to grow for an a d d i t i o n a l postincubation period
(20 and 40 hours i n
c a s e o f C H L - c e l l s and 10 and 20 hours i n c a s e o f human c e l l s ) .
Microirradiated
chromatin
was made
visible
a s described
above
e i t h e r by U D S - l a b e l l i n g or by immunofluorescent l a b e l l i n g . Our data
are c o n s i s t e n t with
B o v e r i ' s e a r l y hypothesis.
Distances
between
the two s i t e s o f m i c r o i r r a d i a t e d chromatin changed only s l i g h t l y i n
C H L - c e l l s (Cremer e t a l . , 1982 a and appeared p r a c t i c a l l y
constant
i n human c e l l s (K. Nakanishi & τ . Cremer, i n preparation) .
CORRELATION BETWEEN CHROMOSOME POSITIONS IN INTERPHASE AND
METAPHASE
The
nucleus
arrangement o f chromosome t e r r i t o r i e s
i s expected
t o d i f f e r from t h e i r order
i n the i n t e r p h a s e
i n metaphase prepa­
r a t i o n s . F a c t o r s which d i s r u p t the interphase order i n c l u d e conden­
sation
o f chromosomes during
prophase
and chromosome
i n v o l v e d i n the metaphase p l a t e formation.
movements
I n a d d i t i o n , techniques
r o u t i n e l y used t o o b t a i n metaphase spreads s u i t a b l e for karyotyping
(including
s p i n d l e p o i s o n s , hypotonic
treatment
and spreading o f
210
Study of chromosome arrangements
chromosomes) may
destroy the chromosome arrangement i n the
metaphase p l a t e to an
extent which
is difficult
intact
to c o n t r o l . I t
should a l s o be noted t h a t chromosomes i n a metaphase spread
are
arranged i n a plane, while t h e i r r e s p e c t i v e t e r r i t o r i e s were a r r a n ged
i n the threedimensional
s t r u c t u r e of the preceding
interphase
nucleus. I n s p i t e of these l i m i t a t i o n s a s t a t i s t i c a l e v a l u a t i o n of
d i s t a n c e s between metaphase chromosomes has been attemped by many
authors, and c o n f l i c t i n g r e s u l t s have been p u b l i s h e d . Therefore,
found
i t important
reflect,
i f only
to
i n v e s t i g a t e whether
to a l i m i t e d
such
e x t e n t , the d i s t a n c e s between
r e s p e c t i v e chromosome t e r r i t o r i e s
we
d i s t a n c e s would
i n the preceding
the
interphase. I f
such a c o r r e l a t i o n could indeed be demonstrated even for metaphase
spreads obtained by routine preparation procedures, the c a s e o f the
metaphase p l a t e a s a s u b s t i t u t e for the i n v e s t i g a t i o n of i n t e r p h a s e
chromosome arrangements could
conditions which do
especially
under
not d i s r u p t i t s n a t u r a l arrangement by
be
strenghtened,
crude
spreading techniques.
U n t i l r e c e n t l y , the p o s s i b l i t y to study such a c o r r e l a t i o n
restricted
to a
few
cases
i n which
heterochromatic
regions
was
of
p a r t i c u l a r chromosomes could be studied i n m i t o s i s as w e l l as i n
interphase (e.g. Pera & Schwarzacher,
t h i s c o r r e l a t i o n we
nuclei
of
Chinese
1970) · As a g e n e r a l t e s t
have m i c r o i r r a d i a t e d two
hamster
cells
sites
(M3-1); e i t h e r
apart from each other. M i c r o i r r a d i a t e d chromatin
to
study
c l o s e to or
i t s r e s p e c t i v e p o s i t i o n i n metaphase
further experimental
details).
Both
far
served a s a probe
preparations
i n d i r e c t immunofluorescence microscopy ( F i g s . 2 and 3; s e e
for
of
i n interphase
interphase
by
legends
nuclei
and
metaphase preparations c o n s i s t e n t l y r e v e a l e d two d i s t i n c t s i t e s of
immunofluorescent s t a i n i n g
( F i g . 2 ) . D i s t a n c e s between
s i t e s were measured i n enlarged photographic
with
regard
spreads
to
the
(see legend
varying
diameters
of
these
two
p r i n t s and c o r r e c t e d
nuclei
and
metaphase
to F i g . 3) . The r e s u l t s of these measurements
show that the mean d i s t a n c e s between the two s i t e s of m i c r o i r r a d i a ted
chromatin
i n the
interphase nucleus
are
reflected
by
their
corresponding d i s t a n c e s i n the subsequent metaphase spread. Another
series
of experiments
was
performed
in essentially
except t h a t metaphase p l a t e s were f i x e d
with a c e t i c
the same
way
acid/methanol
Τ. Cremeret al. 211
( I i 3 ) without c o l c h i c i n e pretreatment and hypotonic shock. P r e l i m i ­
nary d a t a of t h i s a e r i e s f u r t h e r support the concept that chromoso­
me order
i n t h e undisturbed metaphase p l a t e might indeed
the r e l a t i v e p o s i t i o n s o f chromosome t e r r i t o r i e s
interphase
these
nucleus
experiments
to a considerable extent. A f u l l
will
be p u b l i s h e d
elsewhere
reflect
i n the preceding
account o f
(H. Baumann, T.
Cremer, N. Bulbuc & J . J . C o r n e l l s , i n preparation) .
Fig. 2 c-f clearly
shows s h a t t e r i n g o f metaphase chromosomes
corresponding with the s i t e s of immunofluorescent
termed t h i s e f f e c t
et a l . ,
1983)
s t a i n i n g . We have
a s p a r t i a l chromosome s h a t t e r i n g
(PCS)
(Creraer
and suggest t h a t i t might a t l e a s t p a r t l y be due t o
an
undercondensation
in
t u r n c o u l d e x p l a i n why s e v e r a l chromosomes m i c r o i r r a d i a t e d a t
o f m i c r o i r r a d i a t e d chromosome segments. T h i s
one s i t e o f t h e i n t e r p h a s e nucleus were o f t e n c l o s e l y a s s o c i a t e d a t
one
s i t e o f PCS i n the subsequent
metaphase
(Hens e t a l . , 1983).
S h a t t e r i n g r e s u l t e d from the s y n e r g i s t i c e f f e c t o f c a f f e i n e (2mM)
on
chromosome damage
1983) . When c a f f e i n e
i n microirradiated
was omitted
chromosomal damage was much l e s s
cells
(Creraer
eta l . ,
from the post incubation medium
pronounced. However, immunofluo­
r e s c e n t l a b e l l i n g i n t h e s e metaphase spreads g e n e r a l l y was too weak
for
an unequivocal
i d e n t i f i c a t i o n o f the m i c r o i r r a d i a t e d
chroma­
t i n . P o s s i b l y , a n t i g e n i c s i t e s become masked when m i c r o i r r a d i a t e d
chromosomes condense normally i n the absence of c a f f e i n e .
MAPPING OF INTERPHASE POSITIONS OF INDIVIDUAL CHROMOSOMES
In
a r e c e n t l y p u b l i s h e d s e r i e s o f experiments
(Cremer e t a l . ,
1982 b) , the i n t e r p h a s e nucleus o f C H L - c e l l s was m i c r o i r r a d i a t e d a t
one s i t e e i t h e r a t i t s c e n t r a l p a r t or a t i t s perhiphery. Metaphase
spreads w i t h PCS obtained a f t e r
p o s t i n c u b a t i o n with c a f f e i n e (0.5
mM) were karyotyped. No s i g n i f i c a n t d i f f e r e n c e was observed between
the f r e q u e n c i e s o f damage obtained
f o r i n d i v i d u a l chromosomes a t
e i t h e r s i t e o f m i c r o i r r a d i a t i o n . I n t e r e s t i n g l y , w h i l e an average o f
5-6
damaged chromosomes per metaphase was found i n these e x p e r i ­
ments, t h e frequency o f j o i n t damage o f homologous chromosomes was
low, arguing a g a i n s t somatic p a i r i n g o f homologuos chromosomes a s a
frequent
event
i n this
cell
type. I n s p i t e o f their
limitation,
212
Study of chromosome
arrangements
these data demonstrate the f e a s i b i l i t y o f t h e microbeam approach
for mapping the p o s i t i t o n s o f i n d i v i d u a l chromosomes i n t h e i n t e r ­
phase nucleus.
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