THE
JOURNAL
Copyright
OF HISTOCHEMISTRY
© 1976 by The
AND
AUTOMATIC
Vol.
CYTOCHEMISTRY
Histochemical
Society,
DETECTION
Inc.
AND
LOCALIZATION
OF
24, No. 1. pp. 168-177, 1976 Printed in U.S.A.
SISTER
CHROMATID
EXCHANGES’ G. W. Research
ZACK,
J. A. SPRIET,
S. A. LATT,
G. H. GRANLUND
of Electronics, Massachusetts Institute (G.Z., J.S., G.G.,2 I.Y.); and Clinical Genetics Center, and the Department of Pediatrics,
Labomtory
02139
Boston,
Massachusetts
AND
I. T. YOUNG
of Technology, Cambridge, Division, Children’s Hospital Harvard Medical School, 02115 (S.L.)
Massachusetts Medical
Sister
chromatids of human metaphase chromosomes from cells which have replicated medium containing 5-bromodeoxyuridine exhibit unequal fluorescence when stained with the dye 33258 Hoechst. Sister chromatid exchanges occurring in these chromosomes are apparent as interchanges of brightly and dully fluorescing chromatids. A technique for detecting such exchanges by computer analysis of chromosome images has been developed and found to compare favorably with manual methods. The exchanges have been localized in the context of quinacrine banding patterns. twice
in
A sister
chromatid
exchange
is an interchange
genetic
material
between
replication
of ucts
at
homologous
changes,
loci
which
are
chromosomes, under control sensitive
are more conditions
indicators
some
damage
quency
(15),
without
some
morphology.
the
detected, as well
1 and
frequent and
crographs preferentially
of chromo-
band-interband
changes
in fre-
were
initially
which
de-
can
A
recently
on
(BrdU)
to
benzimidazole alternative tion
using
significantly proach
developed
the
technique
ability
quench
of the
dye means Giemsa enabled
fluorescence
stain
(10,
11,
resolution. sister
chromatid
bis-
as well incorpora18),
as
offers
This
in
that exchanges interbands or
indicate
inspection
detection
tid
exchanges.
(14).
of photomitend very
to occur close to
junctions. work the
applies computer Hoechst-fluorescence images
and
localization
to of sister
analysis and automate chroma-
AND METHODS
Human peripheral leukocytes were cultured at 37#{176}C in Eagle’s minimal essential medium (MBA) supplemented with 2 mM L-glutamine and 20% fetal calf serum (GIBCO) to which crude phytohemagglutinin was added. At the start of growth, 5-bromodeoxyuridine (BrdU) (0.09 mM), 5-fluorodeoxyuridine (0.4 tiM) and uridine (6 zM) were added to the cultures. After 70-72 hr (2 generations), colcemid (Ciba) was added to a final concentration of 0.1 zg/ml, and 2 hr later cells were harvested by centrifugation, suspended in 75 mM KC1 for 12 mm, fixed in at least two changes of 3:1 methanol-acetic acid and air-dried on glass microscope slides. The slides were stained first with quinacrine dihydrochloride at 20 mg/ml in H2O and mounted in pH 5.5 buffer (3), photographed and destained in 3:1 methanol-acetic acid. They were restained in 0.5 g/ml of 33258 Hoechst (8, 16) in 0.14 M NaCI-0.004 M KC1-0.01 M phosphate (pH 7) and mounted in 0.16 M sodium phosphate-0.04 M sodium citrate (pH 7) for photography. Chromosome fluorescence was observed in a Leitz
13),
of the
the
quinacrine
visual
MATERIALS
5-bromodeoxyuridine
33258 Hoechst of detecting BrdU
improved has
(12,
with
on
be
are closely spaced, which they can be
localized. based
to the banded pattern of is exhibited by the chro-
quinacrine-fluorescence
(21, 22), technique
they
relative that stained
based
The present techniques to
in chromo-
of autoradiography resolution of this with
Studies
(1)
when
than breaks extremely
markedly
efficiency
mosomes
are forms
especially if they as the precision with
Ex-
metaphase
increasing Exchanges
2).
in
of certain comparable
tected by means but the low spatial limits
(Figs.
detectable
to be localized fluorescence
prod-
ap-
exchanges
‘This work was supported by the National Institutes of Health under Grant 5P01GM1942803. S.L. was supported by research grants from the National Institute of General Medical Sciences (GM21121), The American Cancer Society (VC144 and Massachusetts Division, Inc.), and a U.S. Public Health Service Career Development Award (GM00122) from the National Institute of General Medical Sciences. 2 Also Associate Professor of Electrical Engineering, Linkoeping University, Linkoeping, Sweden. 168
Downloaded from jhc.sagepub.com at University of New England on June 13, 2015
SISTER chromo without chromatid
CHROMATID
chromosome with sister
some
sister exchange
L1::!I1
stained
bright
::1
After
one
j
z:
less
After
i1H; I I:
two
1.
Sister
BrdU
bright
divisions
in BrdU
of
two
chromatids
and
centromere
-
single
strand
of
DNA
in
chromatid
without
------
single
strand
of
DNA
in
chrornatid
with
Schematic representation in medium containing depending on whether
chromatid
in
lessbright-dull
representation
FIG.
division
I
i
generations chromatid,
exchange
chromatid
Hoechst
I
169
EXCHANGES
exchanges
result
BrdU BrdU
(bright)
(dull)
of sister chromatid exchanges. Chromosomes from cells grown two BrdU exhibit different 33258 Hoechst fluorescence intensity on each one or two DNA polynucleotide chains have been substituted with BrdU. in
sharp
reciprocal
interchanges
of
fluorescence
intensity
between
chromatids.
Orthoplan microscope equipped with incident illumination using a 200-watt mercury light source, a UG-1 filter and TK 400 dichroic mirror for excitation and K 400 and K 460 filters for emission. Spreads were selected by a cytologist and photographs were taken through a x 100 achromat objective with an Orthomat II automatic camera on Tri-X film. A flying-spot scanner with variable resolution of up to 5200 samples/in was used to digitize the images on negatives to 64 gray levels with input to a 32,000 word PDP-9 computer. Individual chromosome images were defined interactively, with the computer operator specifying each chromosome’s end points, points of maximum width and centromere with a Sylvania DT-1 tablet. Peripheral equipment used included a Burroughs 18 million bit disc, a Computek 300 keyboard terminal, a 1024 x 1024 4-level CRT display, a 16-level video monitor system and a Versatec Matrix printerplotter.
DETECTION
OF
Hoechst-fluorescence detection
of
images
sister
feature
exploited
was
as abrupt
lateral
bright a
chromatids. to locate
chromatid
piecewise line
linear
segments
centromere. fined the uniform length by
at
except each
one-third.
chromatid
appearance
of
cx-
of bright
The computer was protransverse position of the
the
point
along
as
shown
in
scan
axis
consisted the
in 3.3% telomere,
the
Figure end
maximum-width scan length,
axis 3.
of the
of The two
points
and
points which
dewas
of the total chromosome where it was reduced
The
resulting
position
versus
Downloaded from jhc.sagepub.com at University of New England on June 13, 2015
for The
interchanges
connecting The transverse
used
exchanges.
the
at each
chromosome,
were
chromatid
changes and dull grammed
EXCHANGES
function axial
of bright distance
was
170
ZACK
1’-
-
‘
?:r
-
ET
AL.
It
WI!
#{149} -
t. 22 C.?
‘P
/
I.
I
1-
0 Co Co
. 5)0
,
so
:
Ia!I
.
‘I,’
5)5) #{149}0
11.
5)0
. 4)
I
LI
-eQ
.5)5)
.fi vs 5.. 54.’ a
T
.-
I
a.5) 112
F
F,
1)2
-5
a.0 5)
S.
SQ
.
4)
..-‘s
‘
E H-
4.#{149},_
I
‘‘
-
.5
‘1
L.-O
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5)
tc/)
SISTER
CHROMATID
bright
.4
chroaatid
brightn.s.
traniveri.
1-.
171
EXCHANGES
bright
section
chroatid
fL 3. Location
FIG.
of the maximum
bright chromatid. At each point along the chromosome the brightness is sampled of each such sampling corresponds to the center of the bright chromatid.
over
triplets
of sequential
noise
effects.
Since
transversely.
The
smoothed decrease greatly
oversampled
smoothing
did
The (p)
(20-80
not
derivative with
of bright
chromatid
respect
to
axial
the
(n).
As shown
in Figures
the
derivative
was
difference
The
results
length chromatids
making the unreliable. of the
exchange
are shown in Table I. The by the computer coincided by a cytologist in 93% results on chromosomes exchanges
in
against
response
,
each were
p
detection
cases. cells treatment
of axis
width present. in 2% end since often not
Iderivativi
I
of the scheme detected detected
Preliminary having many with
____________________________________
the
In our method we calculated Ip(i) - p(j) ,p(j) p(k) and Ip(k) - p(i) where i,j and k were consecutive points along the chromosome axis. The position value of the point not contained in the smallest of these differences was then set equal to the nearer of the other two position values. It is likely that a variety of algorithms would be satisfactory. a
chro.oso.e
a thres-
localization
exchanges with those
of the from to
at
was -
magnitude
an exchange was was not performed
of the chromosome’s the tips of the two coincident, chromatid
the
3)
+
of the chromosome
to determine whether The test for exchanges
quite bright
position (n)
p(n
compared
to one-third
the
distance
4-6,
to were
points/s), sensitivity.
by
equal
images
degrade
approximated
hold
values3
the
FIG. 4. Method of detecting a sister chromatid cxchange. The derivative of bright chromatid position with respect to axial distance is determined along the chromosome axis. The magnitude of the derivative is large in the region of a sister chromatid exchange. Consequently, the position of the maximum of any peak extending above a threshold is identified as the location of an exchange.
Downloaded from jhc.sagepub.com at University of New England on June 13, 2015
172
ZACK
CHPO1OSO1[
E’r AL.
NUNBEP
3
,__ :
r’’-
nr
T’THH
HH
.
T
LHFUr1’Trr
k.i1
4’_-_
.-..-------1_-
-.‘_i
‘S
S
!
.;
,
!r
-rS’FS,
,
5r’5r’F
t
FIG. 5. Example of a chromosome exhibiting sister chromatid exchanges. This chromosome was from a cell that underwent two cycles of BrdU incorporation. The chromosome was stained with quinacrine to show banding (bottom), destained and restained with 33258 Hoechst to show sister chromatid exchanges (top). The position of the bright chromatid and the derivative described in Figure 4 are shown and were used to localize the cxchanges
relative
bifunctional dicate
to the
integrated
alkylating
an
error
density
agent
rate
profile
Mitomycin
of about
of the
C in-
10%.
banding
pattern.
purposes
of this
bands were
LOCALIZATION Sister
chromatid
relative
exchanges
to profiles from
as shown some the
in Figure scan axis
width
exchange was
quinacrine 7
and
detection, not
were
of integrated
obtained
for
OF EXCHANGES
reduced
(2,
optical
localized density images
5). For each width was the
chromosame as
at the
that telomeres.
the For
defined
bands,
and arbitrarily
computer
aligned
orescence
images
regions
regions as
Hoechst by
between
of uncertainty in
and
Figure
8.
The
quinacrine
minimizing
the
flu-
cost
func-
tion
fluorescence
except
work,
(interbands)
scan the
error
= (difference
+ (difference
2 (difference Results Table
of the IIA
and
in left endpoint in right
in centromere localization
examples
Downloaded from jhc.sagepub.com at University of New England on June 13, 2015
locations)2
endpoint
locations)2
locations)2 process
are
shown
given
in
in Figure
are
9.
SISTER
Exchanges gories
were on
the
assigned
basis
the computer-generated files. In many cases the
bands
interband one
category
each an
and
compared
the
various
did
to allow is made
of the
fact
of a junction
are
as shown
to that
of exchanges predicted
by
found a simple
CHROMOSOME
occurrence
density
interband
and
in
into
band,
that
regions tively,
of about as shown
differs
from
and
in Table can
be
in its
chromatid
model
spect
NUMBER
to the
center
of
a
fractionation junction
An be
center
each
of
adjacent of
and
ap-
the
interband
39%, 22% and 39%, respecin Figure 10. This subdivision
previously inclusion
considered
models
of a junction
exchanges to axial
assumed between
the
results
chromosome
of exchanges.
profile
sinusoidal
band
into
20) distribution
random
proximately each If
lumped
on
integrated
density pronot resolve
173
EXCHANGES
based of
decisions.
are
consists
results
cate-
measurements
regions
use
region
interband,
IIB. The
enough junction
telomere
the
integrated profiles
the
clearly and
to
of manual
CHROMATID
position
occur on the
region. randomly chromosome,
(9, 19, If sister with
reone
9
HOECHST
,-.‘-..-.-._-
-.‘,-.
OF BRIGHT
POSITION
PEP
I
IVAT
lYE
IHTEGPRTED
Cu I Mcii:
FIG. 6. Example of a twisted chromosome without ance is similar to that of a chromosome with a sister chromatid does not change sufficiently rapidly that threshold. As a result, no exchange is detected.
CHROMATID
DENSITY
PROFILE
i Hi:
a sister chromatid chromatid exchange, its derivative with
exchange. The but the lateral respect to axial
Downloaded from jhc.sagepub.com at University of New England on June 13, 2015
photographic appearposition of the bright distance exceeds the
174
ZACK
would
expect
tion
the
same
of exchanges
interbands. ered
When
separately,
A utomatic
39%-22%-39%
among the the
Detection
bands, three
data
do
distribujunctions
regions not
ET
are differ
AL.
cantly and
6.0),
consid-
grouped
signifi-
cantly
from
distribution
(x’
when
interbands
and
together,
the
but
this
= 5.0,
(x2
I
Chromatid
Exchanges#{176}
Exchanges present#{176}and detected by computer Exchanges present but not detected by computer Exchanges not present but mistakenly detected by computer
72
4 -
b
Sixty-two chromosomes from As determined by a cytologist.
bright
quinacrin.
X2o.95,2
junctions
do
differ
are signifi-
3.8).
X20.95,1
DISCUSSION
TABLE
of Sister
The
automated
exchanges
a
data
= 0.9,
eight
cells.
has
Precise
localization
respect
to
because
of the
afforded
by integrated
crine
through
This use
these
often
poor
is
would
may
reliable. with
more
difficult
delineation
density
obtained limitation
chromatid and
exchanges
patterns
of Giemsa which
of sister
to be simple of
banding
fluorescence
negatives. 11, 17, 18),
fluorescence
detection proven
of bands
profiles
of quina-
from photographic be circumvented
staining
techniques
allow
direct
scanning
(10, of
(band)
dull
quinscrine
fluorescence
(interband)
rateddensityprofile
FIG. 7. Generation of integrated density profiles. The optical density of the photographic negative is integrated across the chromosome image at each point along the axis to produce the integrated density profile. Regions of bright quinacrine fluorescence correspond to peaks in the integrated density profile, while regions of dull fluorescence correspond to valleys in the profile.
integrated
density
end
FIG. 8. Operational definition and junctions are defined along density profile. Each telomere transition
of band, interband and band-interband the chromosome axis as corresponding (end) is composed of an interband
profile
-
junction regions. Bands, interbands to thirds of the height of the integrated and a junction region, but the point of
is unknown.
Downloaded from jhc.sagepub.com at University of New England on June 13, 2015
SISTER
CHROMATID TABLE
Localization Decisi
A. Three-category
of Sister
II Chromatid
No Decision
Band
9
Poorly
Junction
8
Telomerec
Interband
a
Sixty-two
C
Consists
d
Combined
a
decision
Integrated
density
from
profile
does
of a junction
and
an
junction,
interband
eight
differentiated
Possible
bandsb
31 12
12
Band Interband
chromosomes
Exchanges11
on Possible
decision
B. Two-category
175
EXCHANGES
orjunction11
9 32
Poorly
differentiated
bandsb
31
cells.
not have
clearly
defined
peaks
and valleys
in the vicinity
ofthe
exchange.
interband.
and
telomere
:.:-:
data
from
part
A.
-
:
.i
.
S
.f+Th’ r+Th\
-*
ct*OMQS#{226}PENUPIER
1.4
FIG. 9. Examples of sister chromatid exchanges exhibited by chromosomes 5, 7, 8 and 14. Exchanges, which are visible in the Hoechst fluorescence images, have been detected as illustrated in Figure 4 and localized relative to integrated density profiles ofquinacrine banding patterns. The Number 5 chromosome illustrates the detection of two closely-spaced exchanges. The exchange exhibited by the Number 7 chromosome was not identified as occurring in a band, junction or interband because of the poor differentiation of the regions in the integrated density profile. In the Number 8 chromosome, an exchange has been detected in a telomeric region, which contains both a junction and an interband. The Number 14 chromosome exhibits an exchange in an interband.
Downloaded from jhc.sagepub.com at University of New England on June 13, 2015
176
ZACK
ET
AL.
The not
data
examined
inconsistent
to date
with
the
changes occur preferentially gions or close to band-interband simple
model
against
compared
assumed
exchanges
with
chromosome. would assume
IS,
I
11.3
#{149}xp.cted: data:
9
6.4
11.3
8
12
29 29
model
I
I
39%
#{149}zpected:
16.0
25.0
41
9
32
41
data:
FIG. 10. Comparison of exchange locations with a random distribution assuming a locally sinusoidal shape for the integrated density profile. According to the model, the fractions of chromosome lengths corresponding
to band,
39%,
22%
junction
and
percentages
39%,
are
and
interband
respectively.
independent
regions
Note
of period
that
condensed
are
or amplitude
predict
small
amount
ments
concerning
tid
to axial
position
a greater
exchanges
of data. the
linearly
sity.
This
fact,
sion
of
integrated
thirds for uncertainty
coupled
with density
the
inten-
arbitrary
profile
purposes, interpretation
divi-
peaks
into
distribution more elegant
than
work.
treatment could
be
peaks
of the
assigning
exchanges
junction
categories.
of
can
be made
additional
The authors his assistance
thank Mr. in generating
files
shown
simple
model
used
in
that
such
a
that,
although with
distributions,
only could
as regions increase to
band,
the
5.
6. 7.
the
in bands. difficulty of
interband,
and
greater
statisti-
with data
statechroma-
will
be required.
in this
Monson the
H. Hayes plots and
for pro-
publication. CITED
1972
interbands not
definitive of sister
1. Caspersson T, Lindsten J, Lomakka G, Moller A, Zech L: The use of fluorescence techniques for the recognition of mammalian chromosomes and chromosome regions. Int Rev Exp Pathol 11:2,
Caspersson,
difficulty is
of ex-
ACKNOWLEDGMENT
Gaussian
as coincident
model with re-
3. Czaker R: Banding patterns and late replication in HeLa cells. Humangenetik 19:135, 1973 4. Granlund GH: The use of distribution functions to describe integrated profiles of human chromosomes, Chromosome Identification. Edited by T
providing a much description of
present defined
Gaussian
be defined asymmetry
the
One
would
bands
terms
functions (4-6), mathematical
patterns
present
in
the
1970
introduces an of results
localization.
analyzed
of on
2. Caspersson T, Zech L, Johansson C, Modest Ed: Identification of human chromosomes by DNAbinding fluorescent agents. Chromosoma 30:215,
operational definition of bands and could be made if integrated density were
banding
prematurely
to fluorescence
classification about the
Improved interbands profiles
of
and junctions were defined optical density, which is
proportional
of exchange
use
were
number
Before locations
cal confidence,
of
(23).
interbands of integrated
not
could This
by
chromosomes
Bands, in terms
the
or
reThe
data
distribution
LITERATURE
slides,
the
Perhaps a more realistic a random distribution
would
these
the sinusoid. A random distribution ofsister chromatid exchanges thus would result in 39% occurring in bands, 22% in junctions and 39% in interbands, or a total of 61% in either junctions or interbands.
microscope
ex-
interband junctions.
random
respect
found that
changes in bands than in interbands. The data would thus exhibit an even more pronounced nonrandom character. The results presented here represent only a
total
61
in
which a
been
spect to position along the DNA of a chromatid. Since the concentration of chromosomal DNA appears to be low in regions of constrictions (7), which correlate with interband regions, such a
total
22%
have
hypothesis
8.
9.
L Zech.
Academic
Press,
New
York,
1973, p 85-87 Granlund GH: The use of distribution functions to describe integrated density profiles of human chromosomes. J Theor Biol 40:573, 1973 Granlund GH: Statistical analysis of chromosome characteristics. Pattern Recog 6:115, 1974 Heneen WK, Caspersson T: Identification of the chromosomes of rye by distribution patterns of DNA. Hereditas 74:259, 1973 Hilwig I, Gropp A: Staining of constitutive heterochromatin in mammalian chromosomes with a new fluorochrome. Exp Cell Res 75:124, 1972 Holmberg M, Jonasson J: Preferential location of X-ray induced chromosome breakage in the R-
Downloaded from jhc.sagepub.com at University of New England on June 13, 2015
SISTER
10.
11.
12.
13.
14.
15.
CHROMATID
bands of human chromosomes. Hereditas 74:57, 1973 Kim MA: Chromatid exchanges and heterochromatin alteration of human chromosomes with BrdU labelling demonstrated by benzimidazol fluorochrome and Giemsa stain. Humangenetik 25:175, 1974 Korenberg J, Freedlender E: Giemsa technique for detection ofsister chromatid exchanges. Chromosoma 48:355, 1974 Latt SA: Microfluorometric detection of deoxyribonucleic acid replication in human metaphase chromosomes. Proc Natl Acad Sci USA 70:3395, 1973 Latt SA: Microfluorometric analysis of deoxyribonucleic acid replication kinetics and sister chromatid exchanges in human chromosomes. J Histochem Cytochem 22:478, 1974 Latt SA: Localization of sister chromatid exchanges in human chromosomes. Science 185:74, 1974 Latt SA: Sister chromatid exchanges, indices of human chromosome damage and repair: detection by fluorescence and induction by Mitomycin C. Proc Natl Acad Sci USA 71:3162, 1974
EXCHANGES 16. Loewe
177
H,
Urbanietz
J:
Basisch
2,6-bis-benzimidazolderivate,
17.
18.
substitutiente
eine
neue
chemo-
therapeut isch akt ive korperklasse . Arznei m Forsch 24:1927, 1974 Miller OJ, Miller DA, Warburton D: Application of new staining techniques to the study of human chromosomes. Prog Med Genet 9:1, 1973 Perry P, Wolff S: New Giemsa method for the differential staining of sister chromatids. Nature 251:156,
19. Seabright pattern
1974
of
karyotype.
M: High induced
resolution exchanges
Chromosoma
studies in the
40:333,
20.
on the human
1973
Seabright M: Noninvolvement of the human X chromosome in X-ray induced exchanges. Cytogenet Cell Genet 12:342, 1973 21. Taylor JH: Sister chromatid exchanges in tntium-labeled chromosomes. Genetics 43:515, 1958 22. Taylor JH, Woods PS, Hughes WL: The organization and duplication of chromosomes as revealed by autoradiographic st udies using tnt ium-labeled thymidine. Proc Natl Acad Sci USA 43:122, 1957 23. Unakul W: Giemsa banding in prematurely condensed chromosomes obtained by cell fusion. Nature
(New
Biol)
242:106,
Downloaded from jhc.sagepub.com at University of New England on June 13, 2015
1973
JOURNAL
Copyright
OF HISTOCHEMISTRY
© 1976 by The
AND
AUTOMATIC
Vol.
CYTOCHEMISTRY
Histochemical
Society,
DETECTION
Inc.
AND
LOCALIZATION
OF
24, No. 1. pp. 168-177, 1976 Printed in U.S.A.
SISTER
CHROMATID
EXCHANGES’ G. W. Research
ZACK,
J. A. SPRIET,
S. A. LATT,
G. H. GRANLUND
of Electronics, Massachusetts Institute (G.Z., J.S., G.G.,2 I.Y.); and Clinical Genetics Center, and the Department of Pediatrics,
Labomtory
02139
Boston,
Massachusetts
AND
I. T. YOUNG
of Technology, Cambridge, Division, Children’s Hospital Harvard Medical School, 02115 (S.L.)
Massachusetts Medical
Sister
chromatids of human metaphase chromosomes from cells which have replicated medium containing 5-bromodeoxyuridine exhibit unequal fluorescence when stained with the dye 33258 Hoechst. Sister chromatid exchanges occurring in these chromosomes are apparent as interchanges of brightly and dully fluorescing chromatids. A technique for detecting such exchanges by computer analysis of chromosome images has been developed and found to compare favorably with manual methods. The exchanges have been localized in the context of quinacrine banding patterns. twice
in
A sister
chromatid
exchange
is an interchange
genetic
material
between
replication
of ucts
at
homologous
changes,
loci
which
are
chromosomes, under control sensitive
are more conditions
indicators
some
damage
quency
(15),
without
some
morphology.
the
detected, as well
1 and
frequent and
crographs preferentially
of chromo-
band-interband
changes
in fre-
were
initially
which
de-
can
A
recently
on
(BrdU)
to
benzimidazole alternative tion
using
significantly proach
developed
the
technique
ability
quench
of the
dye means Giemsa enabled
fluorescence
stain
(10,
11,
resolution. sister
chromatid
bis-
as well incorpora18),
as
offers
This
in
that exchanges interbands or
indicate
inspection
detection
tid
exchanges.
(14).
of photomitend very
to occur close to
junctions. work the
applies computer Hoechst-fluorescence images
and
localization
to of sister
analysis and automate chroma-
AND METHODS
Human peripheral leukocytes were cultured at 37#{176}C in Eagle’s minimal essential medium (MBA) supplemented with 2 mM L-glutamine and 20% fetal calf serum (GIBCO) to which crude phytohemagglutinin was added. At the start of growth, 5-bromodeoxyuridine (BrdU) (0.09 mM), 5-fluorodeoxyuridine (0.4 tiM) and uridine (6 zM) were added to the cultures. After 70-72 hr (2 generations), colcemid (Ciba) was added to a final concentration of 0.1 zg/ml, and 2 hr later cells were harvested by centrifugation, suspended in 75 mM KC1 for 12 mm, fixed in at least two changes of 3:1 methanol-acetic acid and air-dried on glass microscope slides. The slides were stained first with quinacrine dihydrochloride at 20 mg/ml in H2O and mounted in pH 5.5 buffer (3), photographed and destained in 3:1 methanol-acetic acid. They were restained in 0.5 g/ml of 33258 Hoechst (8, 16) in 0.14 M NaCI-0.004 M KC1-0.01 M phosphate (pH 7) and mounted in 0.16 M sodium phosphate-0.04 M sodium citrate (pH 7) for photography. Chromosome fluorescence was observed in a Leitz
13),
of the
the
quinacrine
visual
MATERIALS
5-bromodeoxyuridine
33258 Hoechst of detecting BrdU
improved has
(12,
with
on
be
are closely spaced, which they can be
localized. based
to the banded pattern of is exhibited by the chro-
quinacrine-fluorescence
(21, 22), technique
they
relative that stained
based
The present techniques to
in chromo-
of autoradiography resolution of this with
Studies
(1)
when
than breaks extremely
markedly
efficiency
mosomes
are forms
especially if they as the precision with
Ex-
metaphase
increasing Exchanges
2).
in
of certain comparable
tected by means but the low spatial limits
(Figs.
detectable
to be localized fluorescence
prod-
ap-
exchanges
‘This work was supported by the National Institutes of Health under Grant 5P01GM1942803. S.L. was supported by research grants from the National Institute of General Medical Sciences (GM21121), The American Cancer Society (VC144 and Massachusetts Division, Inc.), and a U.S. Public Health Service Career Development Award (GM00122) from the National Institute of General Medical Sciences. 2 Also Associate Professor of Electrical Engineering, Linkoeping University, Linkoeping, Sweden. 168
Downloaded from jhc.sagepub.com at University of New England on June 13, 2015
SISTER chromo without chromatid
CHROMATID
chromosome with sister
some
sister exchange
L1::!I1
stained
bright
::1
After
one
j
z:
less
After
i1H; I I:
two
1.
Sister
BrdU
bright
divisions
in BrdU
of
two
chromatids
and
centromere
-
single
strand
of
DNA
in
chromatid
without
------
single
strand
of
DNA
in
chrornatid
with
Schematic representation in medium containing depending on whether
chromatid
in
lessbright-dull
representation
FIG.
division
I
i
generations chromatid,
exchange
chromatid
Hoechst
I
169
EXCHANGES
exchanges
result
BrdU BrdU
(bright)
(dull)
of sister chromatid exchanges. Chromosomes from cells grown two BrdU exhibit different 33258 Hoechst fluorescence intensity on each one or two DNA polynucleotide chains have been substituted with BrdU. in
sharp
reciprocal
interchanges
of
fluorescence
intensity
between
chromatids.
Orthoplan microscope equipped with incident illumination using a 200-watt mercury light source, a UG-1 filter and TK 400 dichroic mirror for excitation and K 400 and K 460 filters for emission. Spreads were selected by a cytologist and photographs were taken through a x 100 achromat objective with an Orthomat II automatic camera on Tri-X film. A flying-spot scanner with variable resolution of up to 5200 samples/in was used to digitize the images on negatives to 64 gray levels with input to a 32,000 word PDP-9 computer. Individual chromosome images were defined interactively, with the computer operator specifying each chromosome’s end points, points of maximum width and centromere with a Sylvania DT-1 tablet. Peripheral equipment used included a Burroughs 18 million bit disc, a Computek 300 keyboard terminal, a 1024 x 1024 4-level CRT display, a 16-level video monitor system and a Versatec Matrix printerplotter.
DETECTION
OF
Hoechst-fluorescence detection
of
images
sister
feature
exploited
was
as abrupt
lateral
bright a
chromatids. to locate
chromatid
piecewise line
linear
segments
centromere. fined the uniform length by
at
except each
one-third.
chromatid
appearance
of
cx-
of bright
The computer was protransverse position of the
the
point
along
as
shown
in
scan
axis
consisted the
in 3.3% telomere,
the
Figure end
maximum-width scan length,
axis 3.
of the
of The two
points
and
points which
dewas
of the total chromosome where it was reduced
The
resulting
position
versus
Downloaded from jhc.sagepub.com at University of New England on June 13, 2015
for The
interchanges
connecting The transverse
used
exchanges.
the
at each
chromosome,
were
chromatid
changes and dull grammed
EXCHANGES
function axial
of bright distance
was
170
ZACK
1’-
-
‘
?:r
-
ET
AL.
It
WI!
#{149} -
t. 22 C.?
‘P
/
I.
I
1-
0 Co Co
. 5)0
,
so
:
Ia!I
.
‘I,’
5)5) #{149}0
11.
5)0
. 4)
I
LI
-eQ
.5)5)
.fi vs 5.. 54.’ a
T
.-
I
a.5) 112
F
F,
1)2
-5
a.0 5)
S.
SQ
.
4)
..-‘s
‘
E H-
4.#{149},_
I
‘‘
-
.5
‘1
L.-O
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5)
tc/)
SISTER
CHROMATID
bright
.4
chroaatid
brightn.s.
traniveri.
1-.
171
EXCHANGES
bright
section
chroatid
fL 3. Location
FIG.
of the maximum
bright chromatid. At each point along the chromosome the brightness is sampled of each such sampling corresponds to the center of the bright chromatid.
over
triplets
of sequential
noise
effects.
Since
transversely.
The
smoothed decrease greatly
oversampled
smoothing
did
The (p)
(20-80
not
derivative with
of bright
chromatid
respect
to
axial
the
(n).
As shown
in Figures
the
derivative
was
difference
The
results
length chromatids
making the unreliable. of the
exchange
are shown in Table I. The by the computer coincided by a cytologist in 93% results on chromosomes exchanges
in
against
response
,
each were
p
detection
cases. cells treatment
of axis
width present. in 2% end since often not
Iderivativi
I
of the scheme detected detected
Preliminary having many with
____________________________________
the
In our method we calculated Ip(i) - p(j) ,p(j) p(k) and Ip(k) - p(i) where i,j and k were consecutive points along the chromosome axis. The position value of the point not contained in the smallest of these differences was then set equal to the nearer of the other two position values. It is likely that a variety of algorithms would be satisfactory. a
chro.oso.e
a thres-
localization
exchanges with those
of the from to
at
was -
magnitude
an exchange was was not performed
of the chromosome’s the tips of the two coincident, chromatid
the
3)
+
of the chromosome
to determine whether The test for exchanges
quite bright
position (n)
p(n
compared
to one-third
the
distance
4-6,
to were
points/s), sensitivity.
by
equal
images
degrade
approximated
hold
values3
the
FIG. 4. Method of detecting a sister chromatid cxchange. The derivative of bright chromatid position with respect to axial distance is determined along the chromosome axis. The magnitude of the derivative is large in the region of a sister chromatid exchange. Consequently, the position of the maximum of any peak extending above a threshold is identified as the location of an exchange.
Downloaded from jhc.sagepub.com at University of New England on June 13, 2015
172
ZACK
CHPO1OSO1[
E’r AL.
NUNBEP
3
,__ :
r’’-
nr
T’THH
HH
.
T
LHFUr1’Trr
k.i1
4’_-_
.-..-------1_-
-.‘_i
‘S
S
!
.;
,
!r
-rS’FS,
,
5r’5r’F
t
FIG. 5. Example of a chromosome exhibiting sister chromatid exchanges. This chromosome was from a cell that underwent two cycles of BrdU incorporation. The chromosome was stained with quinacrine to show banding (bottom), destained and restained with 33258 Hoechst to show sister chromatid exchanges (top). The position of the bright chromatid and the derivative described in Figure 4 are shown and were used to localize the cxchanges
relative
bifunctional dicate
to the
integrated
alkylating
an
error
density
agent
rate
profile
Mitomycin
of about
of the
C in-
10%.
banding
pattern.
purposes
of this
bands were
LOCALIZATION Sister
chromatid
relative
exchanges
to profiles from
as shown some the
in Figure scan axis
width
exchange was
quinacrine 7
and
detection, not
were
of integrated
obtained
for
OF EXCHANGES
reduced
(2,
optical
localized density images
5). For each width was the
chromosame as
at the
that telomeres.
the For
defined
bands,
and arbitrarily
computer
aligned
orescence
images
regions
regions as
Hoechst by
between
of uncertainty in
and
Figure
8.
The
quinacrine
minimizing
the
flu-
cost
func-
tion
fluorescence
except
work,
(interbands)
scan the
error
= (difference
+ (difference
2 (difference Results Table
of the IIA
and
in left endpoint in right
in centromere localization
examples
Downloaded from jhc.sagepub.com at University of New England on June 13, 2015
locations)2
endpoint
locations)2
locations)2 process
are
shown
given
in
in Figure
are
9.
SISTER
Exchanges gories
were on
the
assigned
basis
the computer-generated files. In many cases the
bands
interband one
category
each an
and
compared
the
various
did
to allow is made
of the
fact
of a junction
are
as shown
to that
of exchanges predicted
by
found a simple
CHROMOSOME
occurrence
density
interband
and
in
into
band,
that
regions tively,
of about as shown
differs
from
and
in Table can
be
in its
chromatid
model
spect
NUMBER
to the
center
of
a
fractionation junction
An be
center
each
of
adjacent of
and
ap-
the
interband
39%, 22% and 39%, respecin Figure 10. This subdivision
previously inclusion
considered
models
of a junction
exchanges to axial
assumed between
the
results
chromosome
of exchanges.
profile
sinusoidal
band
into
20) distribution
random
proximately each If
lumped
on
integrated
density pronot resolve
173
EXCHANGES
based of
decisions.
are
consists
results
cate-
measurements
regions
use
region
interband,
IIB. The
enough junction
telomere
the
integrated profiles
the
clearly and
to
of manual
CHROMATID
position
occur on the
region. randomly chromosome,
(9, 19, If sister with
reone
9
HOECHST
,-.‘-..-.-._-
-.‘,-.
OF BRIGHT
POSITION
PEP
I
IVAT
lYE
IHTEGPRTED
Cu I Mcii:
FIG. 6. Example of a twisted chromosome without ance is similar to that of a chromosome with a sister chromatid does not change sufficiently rapidly that threshold. As a result, no exchange is detected.
CHROMATID
DENSITY
PROFILE
i Hi:
a sister chromatid chromatid exchange, its derivative with
exchange. The but the lateral respect to axial
Downloaded from jhc.sagepub.com at University of New England on June 13, 2015
photographic appearposition of the bright distance exceeds the
174
ZACK
would
expect
tion
the
same
of exchanges
interbands. ered
When
separately,
A utomatic
39%-22%-39%
among the the
Detection
bands, three
data
do
distribujunctions
regions not
ET
are differ
AL.
cantly and
6.0),
consid-
grouped
signifi-
cantly
from
distribution
(x’
when
interbands
and
together,
the
but
this
= 5.0,
(x2
I
Chromatid
Exchanges#{176}
Exchanges present#{176}and detected by computer Exchanges present but not detected by computer Exchanges not present but mistakenly detected by computer
72
4 -
b
Sixty-two chromosomes from As determined by a cytologist.
bright
quinacrin.
X2o.95,2
junctions
do
differ
are signifi-
3.8).
X20.95,1
DISCUSSION
TABLE
of Sister
The
automated
exchanges
a
data
= 0.9,
eight
cells.
has
Precise
localization
respect
to
because
of the
afforded
by integrated
crine
through
This use
these
often
poor
is
would
may
reliable. with
more
difficult
delineation
density
obtained limitation
chromatid and
exchanges
patterns
of Giemsa which
of sister
to be simple of
banding
fluorescence
negatives. 11, 17, 18),
fluorescence
detection proven
of bands
profiles
of quina-
from photographic be circumvented
staining
techniques
allow
direct
scanning
(10, of
(band)
dull
quinscrine
fluorescence
(interband)
rateddensityprofile
FIG. 7. Generation of integrated density profiles. The optical density of the photographic negative is integrated across the chromosome image at each point along the axis to produce the integrated density profile. Regions of bright quinacrine fluorescence correspond to peaks in the integrated density profile, while regions of dull fluorescence correspond to valleys in the profile.
integrated
density
end
FIG. 8. Operational definition and junctions are defined along density profile. Each telomere transition
of band, interband and band-interband the chromosome axis as corresponding (end) is composed of an interband
profile
-
junction regions. Bands, interbands to thirds of the height of the integrated and a junction region, but the point of
is unknown.
Downloaded from jhc.sagepub.com at University of New England on June 13, 2015
SISTER
CHROMATID TABLE
Localization Decisi
A. Three-category
of Sister
II Chromatid
No Decision
Band
9
Poorly
Junction
8
Telomerec
Interband
a
Sixty-two
C
Consists
d
Combined
a
decision
Integrated
density
from
profile
does
of a junction
and
an
junction,
interband
eight
differentiated
Possible
bandsb
31 12
12
Band Interband
chromosomes
Exchanges11
on Possible
decision
B. Two-category
175
EXCHANGES
orjunction11
9 32
Poorly
differentiated
bandsb
31
cells.
not have
clearly
defined
peaks
and valleys
in the vicinity
ofthe
exchange.
interband.
and
telomere
:.:-:
data
from
part
A.
-
:
.i
.
S
.f+Th’ r+Th\
-*
ct*OMQS#{226}PENUPIER
1.4
FIG. 9. Examples of sister chromatid exchanges exhibited by chromosomes 5, 7, 8 and 14. Exchanges, which are visible in the Hoechst fluorescence images, have been detected as illustrated in Figure 4 and localized relative to integrated density profiles ofquinacrine banding patterns. The Number 5 chromosome illustrates the detection of two closely-spaced exchanges. The exchange exhibited by the Number 7 chromosome was not identified as occurring in a band, junction or interband because of the poor differentiation of the regions in the integrated density profile. In the Number 8 chromosome, an exchange has been detected in a telomeric region, which contains both a junction and an interband. The Number 14 chromosome exhibits an exchange in an interband.
Downloaded from jhc.sagepub.com at University of New England on June 13, 2015
176
ZACK
ET
AL.
The not
data
examined
inconsistent
to date
with
the
changes occur preferentially gions or close to band-interband simple
model
against
compared
assumed
exchanges
with
chromosome. would assume
IS,
I
11.3
#{149}xp.cted: data:
9
6.4
11.3
8
12
29 29
model
I
I
39%
#{149}zpected:
16.0
25.0
41
9
32
41
data:
FIG. 10. Comparison of exchange locations with a random distribution assuming a locally sinusoidal shape for the integrated density profile. According to the model, the fractions of chromosome lengths corresponding
to band,
39%,
22%
junction
and
percentages
39%,
are
and
interband
respectively.
independent
regions
Note
of period
that
condensed
are
or amplitude
predict
small
amount
ments
concerning
tid
to axial
position
a greater
exchanges
of data. the
linearly
sity.
This
fact,
sion
of
integrated
thirds for uncertainty
coupled
with density
the
inten-
arbitrary
profile
purposes, interpretation
divi-
peaks
into
distribution more elegant
than
work.
treatment could
be
peaks
of the
assigning
exchanges
junction
categories.
of
can
be made
additional
The authors his assistance
thank Mr. in generating
files
shown
simple
model
used
in
that
such
a
that,
although with
distributions,
only could
as regions increase to
band,
the
5.
6. 7.
the
in bands. difficulty of
interband,
and
greater
statisti-
with data
statechroma-
will
be required.
in this
Monson the
H. Hayes plots and
for pro-
publication. CITED
1972
interbands not
definitive of sister
1. Caspersson T, Lindsten J, Lomakka G, Moller A, Zech L: The use of fluorescence techniques for the recognition of mammalian chromosomes and chromosome regions. Int Rev Exp Pathol 11:2,
Caspersson,
difficulty is
of ex-
ACKNOWLEDGMENT
Gaussian
as coincident
model with re-
3. Czaker R: Banding patterns and late replication in HeLa cells. Humangenetik 19:135, 1973 4. Granlund GH: The use of distribution functions to describe integrated profiles of human chromosomes, Chromosome Identification. Edited by T
providing a much description of
present defined
Gaussian
be defined asymmetry
the
One
would
bands
terms
functions (4-6), mathematical
patterns
present
in
the
1970
introduces an of results
localization.
analyzed
of on
2. Caspersson T, Zech L, Johansson C, Modest Ed: Identification of human chromosomes by DNAbinding fluorescent agents. Chromosoma 30:215,
operational definition of bands and could be made if integrated density were
banding
prematurely
to fluorescence
classification about the
Improved interbands profiles
of
and junctions were defined optical density, which is
proportional
of exchange
use
were
number
Before locations
cal confidence,
of
(23).
interbands of integrated
not
could This
by
chromosomes
Bands, in terms
the
or
reThe
data
distribution
LITERATURE
slides,
the
Perhaps a more realistic a random distribution
would
these
the sinusoid. A random distribution ofsister chromatid exchanges thus would result in 39% occurring in bands, 22% in junctions and 39% in interbands, or a total of 61% in either junctions or interbands.
microscope
ex-
interband junctions.
random
respect
found that
changes in bands than in interbands. The data would thus exhibit an even more pronounced nonrandom character. The results presented here represent only a
total
61
in
which a
been
spect to position along the DNA of a chromatid. Since the concentration of chromosomal DNA appears to be low in regions of constrictions (7), which correlate with interband regions, such a
total
22%
have
hypothesis
8.
9.
L Zech.
Academic
Press,
New
York,
1973, p 85-87 Granlund GH: The use of distribution functions to describe integrated density profiles of human chromosomes. J Theor Biol 40:573, 1973 Granlund GH: Statistical analysis of chromosome characteristics. Pattern Recog 6:115, 1974 Heneen WK, Caspersson T: Identification of the chromosomes of rye by distribution patterns of DNA. Hereditas 74:259, 1973 Hilwig I, Gropp A: Staining of constitutive heterochromatin in mammalian chromosomes with a new fluorochrome. Exp Cell Res 75:124, 1972 Holmberg M, Jonasson J: Preferential location of X-ray induced chromosome breakage in the R-
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SISTER
10.
11.
12.
13.
14.
15.
CHROMATID
bands of human chromosomes. Hereditas 74:57, 1973 Kim MA: Chromatid exchanges and heterochromatin alteration of human chromosomes with BrdU labelling demonstrated by benzimidazol fluorochrome and Giemsa stain. Humangenetik 25:175, 1974 Korenberg J, Freedlender E: Giemsa technique for detection ofsister chromatid exchanges. Chromosoma 48:355, 1974 Latt SA: Microfluorometric detection of deoxyribonucleic acid replication in human metaphase chromosomes. Proc Natl Acad Sci USA 70:3395, 1973 Latt SA: Microfluorometric analysis of deoxyribonucleic acid replication kinetics and sister chromatid exchanges in human chromosomes. J Histochem Cytochem 22:478, 1974 Latt SA: Localization of sister chromatid exchanges in human chromosomes. Science 185:74, 1974 Latt SA: Sister chromatid exchanges, indices of human chromosome damage and repair: detection by fluorescence and induction by Mitomycin C. Proc Natl Acad Sci USA 71:3162, 1974
EXCHANGES 16. Loewe
177
H,
Urbanietz
J:
Basisch
2,6-bis-benzimidazolderivate,
17.
18.
substitutiente
eine
neue
chemo-
therapeut isch akt ive korperklasse . Arznei m Forsch 24:1927, 1974 Miller OJ, Miller DA, Warburton D: Application of new staining techniques to the study of human chromosomes. Prog Med Genet 9:1, 1973 Perry P, Wolff S: New Giemsa method for the differential staining of sister chromatids. Nature 251:156,
19. Seabright pattern
1974
of
karyotype.
M: High induced
resolution exchanges
Chromosoma
studies in the
40:333,
20.
on the human
1973
Seabright M: Noninvolvement of the human X chromosome in X-ray induced exchanges. Cytogenet Cell Genet 12:342, 1973 21. Taylor JH: Sister chromatid exchanges in tntium-labeled chromosomes. Genetics 43:515, 1958 22. Taylor JH, Woods PS, Hughes WL: The organization and duplication of chromosomes as revealed by autoradiographic st udies using tnt ium-labeled thymidine. Proc Natl Acad Sci USA 43:122, 1957 23. Unakul W: Giemsa banding in prematurely condensed chromosomes obtained by cell fusion. Nature
(New
Biol)
242:106,
Downloaded from jhc.sagepub.com at University of New England on June 13, 2015
1973
