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MICROSCOPY RESEARCH AND TECHNIQUE 36:301–312 (1997)
Imaging of BrdU-Labeled Human Metaphase Chromosomes
With a High Resolution Scanning Ion Microprobe
RICCARDO LEVI-SETTI,1 * JAN M. CHABALA,1 KONSTANTIN GAVRILOV,1 RAFAEL ESPINOSA III, 2
AND MICHELLE M. LE BEAU2
1The
Enrico Fermi Institute and Department of Physics, The University of Chicago, Chicago, IL 60637
of Hematology/Oncology, Department of Medicine, The University of Chicago, Chicago, IL 60637
2Section
KEY WORDS
chromosome banding-nucleoside SIMS mapping-sister chromatid exchangefragile site-Giemsa
ABSTRACT
Detailed maps of the A-T distribution within human mitotic chromosomes labeled
with BrdU are obtained with a high resolution scanning ion microprobe through the detection of
bromine by imaging secondary ion mass spectrometry (SIMS). Corresponding maps of the emission
loci of the molecular ion CN2 describe the overall DNA, RNA and protein distribution in the
chromosomes. Several chromosome preparations exhibit base-specific banding patterns (SIMSbands) which mimic the well known G- or Q-bands resulting from conventional staining methods for
optical microscopy. SIMS-bands are more noticeable in mitotic cells at the first cell cycle and after in
situ denaturation or Giemsa staining. Sister chromatid exchanges (SCE) at the second cell cycle and
beyond, occurring both spontaneously and promoted following cell culture exposure to the chemical
aphidicolin (an inhibitor of DNA replication), can be visualized readily from the relative label signal
intensities between sister chromatids. The comparison of base-specific label maps with CN2 maps,
in conjunction with the appearance of base-specific banding patterns, is informative about protein
survival and/or removal following different chromosome preparation protocols. In addition, the
resulting condensation state of the chromosomes can be appraised during SIMS analysis from the
sample topography (imaged via the collection of mass-unresolved secondary ions). We demonstrate
that imaging SIMS is a powerful complement to existing methods for the study of banding
mechanisms and for the elucidation of chromosome structure. The advantages of this novel
approach to the systematic and quantitative study of cytogenetic phenomena and methodologies are
still largely untapped. Microsc. Res. Tech. 36:301–312, 1997. r 1997 Wiley-Liss, Inc.
INTRODUCTION
The University of Chicago Scanning Ion Microprobe
(UC SIM) utilizes Secondary Ion Mass Spectrometry
(SIMS) imaging to map the distribution of elements and
several chemical compounds in soft and mineralized
biological material, at a typical image resolution of
30–40 nm (Levi-Setti, 1988). The instrument uses a
40–60 keV energy, focused heavy ion probe to scan the
surface of a material, leading to the emission of secondary ions which are constituents of the material itself.
These secondary ions are then collected and analyzed
by a mass spectrometer, and recorded to generate
mass-resolved images. Because elemental isotopes can
be detected by this approach, the method lends itself to
the use of isotope tracers that need not be radioactive.
High-resolution SIMS imaging microanalysis with the
UC SIM has already proven to be of unique value in a
number of biological applications (Chabala & LeviSetti, 1987; Levi-Setti, 1988). More recently, we applied
the SIMS methodology to the mapping of labeled nucleosides in human metaphase chromosomes (for a review,
see Levi-Setti and Le Beau, 1992), and in Drosophila
polytene chromosomes (Levi-Setti et al., 1994a). Here
we discuss recent advances in the study of human
metaphase chromosomes.
r 1997 WILEY-LISS, INC.
The technique was first tested using bromodeoxyuridine (BrdU, a thymidine analog) and 14C-thymidine as
labels, to explore the feasibility of studying sister
chromatid exchange (SCE) and fragile sites by this
method (Hallégot et al., 1989). SCEs result from a
spontaneous recombination, by which homologous DNA
sequences cross over between sister chromatids, and
are thought to be associated with various pathologies.
Their rate of occurrence may be promoted by chemicals
and may serve as an index of the mutagenic properties
of these chemicals. Fragile sites are loci where chromosomes are prone to break and undergo rearrangements,
including homologous and non-homologous recombination. In a few cases, these fragile sites are known to be
related to genetic abnormalities. However, their pathological role, genetic and molecular basis, and possible
connection with SCE, are generally not known. The
label signatures in these experiments were the secondary ion 81Br (free of the PO3 interference that affects the
other Br isotope at mass 79) and 28CN2 ( 14C14N), also
Received 5 January 1996; accepted in revised form 5 March 1996.
*Correspondence to: Prof. Riccardo Levi-Setti, The Enrico Fermi Institute, The
University of Chicago, 5640 S. Ellis Avenue, Chicago, IL 60637, U.S.A.
Grant from: National Science Foundation, Grant number: BIR 9317959.
302
R. LEVI-SETTI ET AL.
Fig. 1. Schematized representation of the components of the
University of Chicago Scanning Ion Microprobe (UC SIM). By means
of a ‘‘switchyard’’ quadrupole, the secondary ion beam can be directed
either into an Extranuclear 300 RF quadrupole mass filter, or into a
modified Finnigan MAT 90 magnetic sector mass spectrometer. (From
Levi-Setti et al., 1994, reproduced by permission of The Royal Microscopical Society).
devoid of significant interferences and copiously emitted from DNA. Additional information concerning the
chemical structure of the chromosomes can be obtained
through mapping with the latter molecular ion in its
normal 26CN2 form, which accurately describes the
cumulative distribution of proteins, as well as of DNA
and RNA, providing detailed histological SIMS maps.
Subsequent studies using the above labels did succeed
in detecting the differential label staining expected for
sister chromatids, as well as possible examples of SCE
(Levi-Setti and Le Beau, 1992). It also became apparent
that the nucleoside distribution along the chromatids
was not uniform, and that banding patterns could be
recognized. In a few examples, these ‘‘SIMS bands’’
appeared similar to the well known patterns resulting
from the trypsin-Giemsa stain (G-bands) and the fluorescent quinacrine mustard stain (Q-bands). The SIMS
bands acquire particular significance, as will be discussed further below, because by this approach we hope
to shed light on the true biochemical nature of the
banding patterns observed with the more conventional
staining methods. This is an issue that has not yet been
resolved (Sumner, 1990) and that has been thought, in
rather unspecified terms, to involve DNA-protein interactions. The ability to differentiate isotope-tagged DNA
from proteins using the SIMS technique is particularly
relevant in regard to this hypothesis.
Although encouraging, our previous attempts to map
base-specific label distributions in human metaphase
chromosomes were frustrated by the relatively poor
transmission (,0.2%) of the RF quadrupole mass filter
incorporated in the UC SIM for SIMS analysis at that
time. The resulting detected counts were often insufficient to construct images with statistically useful de-
ION MICROPROBE IMAGING OF HUMAN CHROMOSOMES
303
Fig. 2. (a): 81Br2 SIMS map of a BrdU-labelled
mitotic cell, obtained by adding 7 single-pass
maps, 35 minutes overall scan time. Three chromosomes overlap an unrelated cell nucleus. The
image displays 9.1 3 105 counts. Sister chromatids are equilabeled, indicating that the cell is
undergoing the first mitosis following exposure to
BrdU. 45 µm full scale. (b): Same as the image in
(a), shown in inverted contrast to facilitate comparison with optical staining. (c): Magnified detail of (b), exhibiting banding patterns (SIMSbands). 2.0 3 105 counts displayed. 23 µm full
scale. (d): 26CN2 map of area in (c), from a
single-pass scan of 2.2 minutes duration. 1.2 3
106 counts displayed. The structure of the basespecific map in (c) is not visible in this map, which
includes the overall contribution from DNA, RNA
and proteins.
tail. In addition, difficulties were encountered in obtaining satisfactory spreading of mitotic cells on conducting
substrates (e.g., Au foils), which exhibit hydrophobic
behavior. The limitations resulting from low count
statistics have been largely overcome in the present
investigation, which takes advantage of a radical upgrade of the UC SIM. The instrument is now coupled to
a high performance magnetic sector mass spectrometer
and exhibits a better than fifty-fold increase in SIMS
sensitivity (Chabala et al., 1995; Levi-Setti et al.,
1994b). At the same time, a satisfactory protocol has
been developed to obtain consistently well-distributed
chromosome spreads on conducting substrates (LeviSetti et al., 1995). It is now feasible to pursue several of
the original goals of our program, concentrating here on
the SIMS analysis of alcohol-acid-fixed, BrdU-labelled
chromosomes. Here we present details of the appearance and occurrence of base-specific SIMS-banding in
relation to cell division and to in situ procedures such as
DNA denaturation and Giemsa staining. These procedures will be shown to deplete the protein content of the
chromosomes to some extent. The differential BrdU
labelling of sister chromatids as a function of the
number of cell divisions will be shown to enable the
routine detection of SCEs occurring in normal mitotic
cell cultures, and at an increased rate following exposure of the cultures to the chemical aphidicolin. The UC
SIM can also yield images of the surface topography of a
sample by collecting the overall ion-induced secondary
ion (ISI) emission from the sample; this feature will be
used to illustrate the morphological changes associated
with the different chromosome preparation procedures
mentioned above.
MATERIALS AND METHODS
Sample Preparation
Because of the irreversible structural incorporation
of the BrdU label into DNA, the standard culture and
specimen preparation techniques employed for optical
microscope observation are adequate for SIMS analysis. However, the requirements that the substrates be
conducting to prevent electrical charging, and at the
same time wettable by the mitotic cell fixative, necessitates the development of a special methodology. The
chromosome samples were obtained from human peripheral blood lymphocytes of healthy individuals, cultured
in BrdU-containing medium following the protocol previously described (Levi-Setti and Le Beau, 1992). Relevant to the present context is the incubation time (72
hours), which allows, on the average, three cell divisions to occur. After arresting the cells in mitosis with
Colcemid (0.05 µg/ml for 45 minutes, leading to a wide
range of chromosome condensation levels), the cultures
were treated with hypotonic KCl (0.075 M, 8 minutes)
followed by 4–6 changes of fixative (absolute methanol:
glacial acetic acid, 3:1). This method of fixation is
known to remove some of the histone (primarily H1)
and non-histone proteins from the chromosomes (Sumner, 1990). However, estimates of this loss vary over a
wide range. As will be shown below, our observations
suggest that a substantial amount of these proteins is
304
R. LEVI-SETTI ET AL.
Fig. 3. (a): Sum of 8 81Br2 SIMS scans totaling 36 minutes, 9.8 3
106 counts, 38 µm vertical full scale. The label intensity ratio between
sister chromatids of this BrdU-labeled cell averages close to the
expected 1:1/2 ratio for second division. (b): Magnified detail of (a),
showing structure in the packing of chromatin, but no recognizeable
banding. 3.6 3 105 counts, 19 µm full scale. (c): Same as in (a), shown
in inverted contrast. Three SCEs are marked by pointers. (d): Another
detail of the same chromosome spread, exhibiting 2 additional SCEs.
3.5 3 105 counts, 19 µm full scale.
still present after the above exposure to acid fixative.
Instead of using glass slides as substrate, as is routine
for optical microscope observation, the cell suspensions
were dropped onto glass coverslips that have been
lightly Au-coated and treated with a non-ionic surfactant (Triton X-100, 0.1%), and air dried. This procedure
ensured uniform wetting of the substrate by the cell
suspension until dry, a feature that is essential to
obtaining well distributed chromosomes which are free
of deformity. For SIMS analysis, the samples were
further coated with a thin, sputter-deposited layer of
gold.
Several variants to the above standard preparation
protocol were introduced, both at the culture level and
in situ, to explore the chemical basis of a number of
cytogenetic phenomena and procedures: 1) To promote
the occurrence of SCE and the expression of fragile
sites, some of the BrdU-containing cultures were exposed to the chemical aphidicolin (0.4 µM) for 24 hours
at 37°C (Rassool et al., 1992). 2) Toward elucidating
aspects of the staining process, some of the samples
were Giemsa and trypsin-Giemsa stained, following
standard procedures. 3) To explore the chemical changes
associated with DNA denaturation, some of the samples
were subjected to the procedures for fluorescent in situ
hybridization (FISH), except exposure to the DNA
probe, according to the formamide/SSC protocol of
Rowley et al. (1990).
ION MICROPROBE IMAGING OF HUMAN CHROMOSOMES
305
Fig. 4. (a): 81Br2 SIMS map of BrdU-labelled
mitotic cell cultured in the presence of aphidicolin,
shown in inverted contrast. The average number
of SCEs/cell in this sample is 2.5 greater than for a
normal culture. Sum of 9 single-pass maps, totaling 39.3 minutes. 3.7 3 105 counts, 47 µm full
scale. (b): The same cell as in (a), imaged with
26CN2. Some structure, due to the condensation
pattern of the chromatin, is detectable here. 3.0 3
106 counts from a single scan in 2.2 minutes. (c):
Detail of another aphidicolin-treated cell, exhibiting 5 SCEs. 1.8 3 105 81Br2 counts acquired in 4
single-pass scans totaling 17 minutes. 17 µm full
scale. (d): 26CN2 map corresponding to the area in
(c), revealing a break (possibly due to a fragile
site) in the q arm of the left chromatid of the
uppermost left chromosome. This occurs at the
location of an SCE, as shown in (c). 5.1 3 106
counts from a single scan in 2.2 minutes.
Scanning Ion Microprobe
An exploded schematic of the recently upgraded UC
SIM, in the configuration employed in this investigation (magSIMS), is shown in Figure 1. It differs from
that originally described (Levi-Setti et al., 1986) by its
coupling with a high performance magnetic sector mass
spectrometer (modified Finnigan MAT 90) for SIMS
analysis. The primary column is unchanged, relative to
previous descriptions, and for the present work was set
to focus a 6 pA Ga1 ion beam, extracted from a liquid
metal ion source, to a spot 35 nm wide. The ejected
secondary ions are collected and energy-analyzed by a
spherical 90° electrostatic sector. The secondary ion
transport system has been entirely redesigned, and the
overall collection and transmission efficiency in the
magSIMS mode is now estimated at ,20%, at a mass
resolution m/Dm , 550, independent of mass. Compared with the former RF quadrupole mass filter
performance, where the transmission efficiency dropped
by a factor of 5 with increasing mass, at a mass
resolution m/Dm , 200, this represents an improvement in overall detection efficiency by factors of 20–100
over the periodic table of the elements (Chabala et al.,
1995). To ensure even transmission of the SIMS transport system while the point of primary ion impact is
scanned over the field of view, the secondary ion beam is
synchronously descanned (dynamic emittance matching) so as to be constantly redirected toward the center
of the entrance slit of the magnetic spectrometer.
Image Acquisition and Resolution
The SIMS signals are detected by an active film
electron multiplier (ETP AF820, pulse counting) capable of accepting count rates up to 50 MHz. Images,
containing 512 3 512 picture elements in a square
raster from single scans, are stored in digital memory
for subsequent analysis with a KONTRON IMCO image processing system. Mitotic cells are located by
scanning the samples using the 81Br2 SIMS signal
displayed on a CRT, at fields of view 160 µm wide.
Analytical maps are then recorded from single pass
scans at fields of view of width in the range of 20–50 µm,
to encompass the entire mitotic cell. The 6 pA Ga1
probe erodes the chromosomes very slowly: We estimate that about 300 scans, 40 3 40 µm2 in size, at a
dwell time of 1 µs/pixel, are needed to completely erode
a set of BrdU-labelled chromosomes prepared by the
standard preparation procedure above. Counting rates
cannot exceed 256 counts/pixel, the limit imposed by
the 8 bit image processor memory, to avoid image
saturation. This limit is generally reached for the
abundantly emitted CN2 in a single pass scan at a
dwell time of 1 µs/pixel. However, for the 81Br2 label,
the sum of 5–10 such single pass maps for a particular
mitotic cell is needed (added together after registration
of the images to compensate for occasional minor
specimen drift) before the computer saturation limit is
reached. Such stacks of sequential images effectively
provide an in-depth view of elemental distributions
(SIMS tomography) and may be used for 3D image
306
R. LEVI-SETTI ET AL.
Fig. 5. (a) 81Br2 map of a BrdU-labelled mitotic cell stained with Giemsa. Here the Br content of eosin overshadows the label, and originates recognizeable Giemsa banding patterns of
equal intensity for both sister chromatids. 9.0 3
106 counts from a single scan in 2.2 minutes. 40
µm full scale. (b) 26CN2 map of the area in (a). The
bright halo surrounding the chromosomes is attributed to the thiazines residue from Giemsa.
The apparent lack of emission from the chromosome interior is a photographic reproduction artifact; the signal intensity within chromatids in
fact equals that of unstained chromosomes. 2 3
107 counts from a single scan in 2.2 minutes. (c)
Another example of a BrdU-labelled mitotic cell
stained with Giemsa. 7.7 3 106 81Br2 counts from
5 single-pass scans in 44 minutes. 47 µm full
scale. (d) Magnified detail of (c) showing well
defined SIMS bands. 2.1 3 106 counts, 25 µm full
scale.
reconstruction. To emphasize relevant features and to
smooth the graininess of low signal regions of the
images, gray levels may be minimally averaged using
lowpass routines as needed, without affecting the image resolution. The relationship between probe size and
image resolution as a function of signal count statistics
and pixel spacing (related to the magnification and
raster size) has been extensively studied (Levi-Setti et
al., 1987). The image resolution is defined in terms of an
edge spread function, measuring the sharpness of the
boundary separating two regions of different signal
density. Experimentally, this quantity is obtained from
line scans across images of sharp boundaries. Only for
signal density differences across an edge in excess of
,10 counts/pixel does the edge spread function approach the probe size. Here, this condition is usually
satisfied for the CN2 images and for the sum of several
81Br2 maps, provided that the probe spacing in the
raster be less than the probe diameter (for a raster of
512 3 512 and a probe diameter of 35 nm, the field of
view must be 18 µm wide or less). At lower magnifications, the edge spread function approaches, instead, the
spacing between pixels, which can be greater than the
probe size. The images presented here were taken from
areas 20 to 50 µm wide, leading to an estimate of the
edge spread function, for high counts/pixel regions, in
the range of ,40–100 nm.
BrdU Labeling and Its Detection
BrdU, incorporated in the chromosomes as a thymidine analog, is the carrier of the Br label that is
detected by SIMS. Br is present in its two isotopic
components 79Br and 81Br in their natural abundances,
50.54% and 49.46% respectively, and is predominantly
emitted as a negative ion. As previously shown (Hallégot et al., 1989), PO32 represents a major interfering
molecule at mass 79 (,39%), that could not be separated from 79Br2 at the limited mass-resolving power
(Dm/m , 200) of the RF quadrupole mass filter used in
previous studies. We have shown (Levi-Setti et al.,
1994b) that the magSIMS upgraded version of the UC
SIM can indeed separate the two species at mass 79 (at
Dm/m , 3,400), at the expense however of a reduced
SIMS system transmission. In view of the much improved detection sensitivity of magSIMS for 81Br2
(,1%) compared with that reported previously (0.01%
from Levi-Setti and Le Beau, 1992), we opted as an
unwarranted effort the use of the potential additional
contribution at mass 79.
As is well known, and reviewed in detail by Levi-Setti
and Le Beau (1992), the relative amounts of label that
are incorporated within sister chromatids from the
BrdU-containing culture medium varies with cell cycle,
because the label is only introduced in newly-synthesized DNA during chromosome replication, starting
from unlabeled and one parental DNA. Thus at the first
cell cycle, each chromatid contains one labeled and one
parental (unlabeled DNA) strand: the sister chromatids
carry the same quantity of label, so that the relative
label amounts stand in the ratio 1/2:1/2 (taking as 1 the
content of fully labelled DNA). It follows from combinatorial arithmetics that after two S phases, one chromatid will carry the label in both DNA strands, the other
only in one, and the label ratio between sister chroma-
ION MICROPROBE IMAGING OF HUMAN CHROMOSOMES
307
Fig. 6. (a) 81Br2 map of a BrdU-labelled mitotic
cell after DNA denaturation with FISH protocol.
6.7 3 105 counts from 7 single-pass scans, 31
minutes, 36 µm full scale. (b) 26CN2 map of the
area in (a), showing fine condensation structure
not visible in undenatured samples. 2.4 3 106
counts from single scan in 2.2 minutes. (c) Detail
of another denatured mitotic cell, with markers
pointing at 3 SCEs. 1.9 3 105 counts, 7 single-pass
scans in 31 minutes, 24 µm full scale. (d) 26CN2
map of the area in (c), showing coiling of the
chromatid fibers. 4.0 3 106 counts from single
scan, 4.4 minutes.
tids will be 1:1/2. At the third cell division, ratios 1:1
and 1:1/2 should occur with equal probability. Because
the SIMS signal is proportional to the concentration of
the element being detected, it is possible to identify the
number of divisions of a particular mitotic cell in the
presence of BrdU from the measured ratio of Br label
intensities between sister chromatids. Importantly, at
the second division and beyond, where one chromatid
may carry twice the amount of label as the other, it is
possible to detect SCEs by simple inspection of the
SIMS images. In the present experiment the cells were
cultured for the average time (72 hours) necessary for 2
3-cell cycles to occur; therefore, examples of all 3 above
occurrences are represented in the results to follow.
It should be emphasized that the above predictions
are valid, provided the entire label content of the
chromosomes is recorded. This would require the total
sputter erosion of the sample, a goal that is seldom
attainable. Because one of our 40 3 40 µm2 SIMS scans
probes only the outermost layers of the samples (,6
monolayers), the condensation form of the chromosomes and the spatial distribution of the proteins may
affect the actual surface density of DNA detected by the
probe. As a consequence, the ratios of label intensities
between chromatids that we measure may exhibit
substantial variation in individual scans, approaching
the predicted values only after averaging over a large
number of chromosome maps.
RESULTS
Several mitotic cells were located and analyzed for
each of the sample categories. Representative examples
of 81Br2 and 26CN2 SIMS maps are presented here for
several of these categories. A few color-coded correlative
maps are shown in the attached color plate.
BrdU-Labeled Chromosomes
A partial mitotic cell resulting from the first division
is shown in Figure 2. Forty-three chromosomes are well
separated. Figure 2a is a 81Br2 map where each detected count is displayed to form a bright image,
reminiscent of fluorescent staining microscopy. Figure
2b is an inverted contrast version of the same, to
facilitate comparison with Giemsa-stained samples seen
by transmission in the optical microscope. We note first
that the well-separated chromatids of each chromosome have similar average image density, as expected
from equal incorporation of BrdU in the first cell
division. This is quantitatively confirmed by measuring
the average signal intensity in each chromatid, relative
to that within the cell nucleus, taken as a calibration
standard. This ratio is close to 1/2 of a similar ratio
measured for the brighter chromatid of chromosomes
from the second cell division (where both DNA strands
are labeled), indicating that we observe here only one
labeled strand. We also note that, although the label
density is essentially continuous for most chromatids,
SIMS bands are discernable, suggestive of Q-bands in
Figure 2a, or G-bands in 2b, although perhaps not as
sharply defined. These banding patterns are more
apparent in the magnified detail of Figure 2c. Also to be
noted is the homologous appearance of the SIMS bands
for sister chromatids, as expected for homologous label
distributions (identified here as A–T sequences). The
308
R. LEVI-SETTI ET AL.
Fig. 7. Topographic images of mitotic cells,
obtained with mass-unresolved secondary ions. (a)
and (b) show 2 different condensation states of
BrdU-labelled chromosomes, 37 and 39 µm full
scale respectively. (c) Collapsed appearance of
chromosomes after denaturation in 70% formamide 4 3 SSC, 70°C, following FISH protocol,
36 µm full scale. (d) Highly condensed and resolved chromatid appearance after Giemsa staining, 47 µm full scale.
overall chromosome morphology revealed by these maps,
together with the SIMS banding patterns, identifies
most of the chromosomes at first sight. Finally, Figure
2d shows a 26CN2 map, corresponding to the detail of
Figure 2(c). Here the structure present in the 81Br2
maps is generally absent, the images of sister chromatids often merge together. This masking must be attributed to the remainder of base pairs and proteins
convoluted with the A–T pairs. The brighter border of
the chromosome images is attributable to edge effects
(artifacts of the SIMS technique), not present in the
label images, also suggesting a protein coating of the
DNA.
Figure 3 shows 81Br2 SIMS maps of a partial metaphase cell from the second cell division, in both normal
and inverted contrast. Here the differential label content of sister chromatids is clearly visible, and measured, on the average, to be in the expected ratio 1:1/2.
This leads to the detectability, by inspection, of several
examples of SCEs, pointed out by markers. The image
intensity for the chromatids with only one labeled DNA
strand, comparable to that of the chromatids in Figure
2, appears smaller than the latter due to photographic
reproduction constraints. The SIMS banding patterns
evident in the singly-labeled chromatids of Figure 2 are
replaced in the doubly labeled chromatids of Figure 3
by an essentially continuous knotted structure, suggestive in some details of the coiled structure of the
chromatid fiber of metaphase chromosomes discussed
by, e.g., Gasser et al. (1986). The average rate of
occurrence of SCEs, measured from several mitotic
cells belonging to the same cell culture from an indi-
vidual healthy donor (control), is approximately 4.3/
cell, or about 9.4% of the chromosomes. This figure does
not include apparent SCEs occurring at the centromere
(often included in the count), which cannot be distinguished from a simple twist in the chromosome arms.
BrdU Labeling Plus Aphidicolin Treatment
Exposure to aphidicolin (Rassool et al., 1992) of
cultures from the same peripheral blood sample as the
control results in a marked increase in the average rate
of occurrence of SCEs: about 10.3/cell, or 23% of the
chromosomes, ,2.5 times that for the control. Examples of these mitotic cells are shown in Figure 4. The
81Br2 map of the metaphase cell of Figure 4a exhibits 5
SCEs and 7 centromeric twists. The identification of
these features is facilitated by comparison with the
CN2 map of Figure 4b, which characterizes the chromosome appearances and allows a precise localization of
the centromeres. A higher magnification detail of another mitotic cell is shown in Figure 4c and d. Here 5
SCEs and 1 twist can be identified, the uppermost left
SCE occurring at the site of a break in the left chromatid (a possible fragile site induced by aphidicolin)
visible in the CN2 map.
BrdU Labeling Plus Giemsa
The Br content of eosin (C20H6Br4Na2OS) in Giemsa
(a mixture of eosin with the thiazines methylene blue
and azure B) was found to overshadow (by at least a
factor of 10) the BrdU label, masking the differentiation
in label content of sister chromatids. Nonetheless it is
ION MICROPROBE IMAGING OF HUMAN CHROMOSOMES
309
Fig. 8. Comparison of optical microscope images of trypsin-Giemsa stained mitotic cells, with
base-specific SIMS maps from BrdU-labelled cells.
(a) and (b) optical microscope, 50 and 33 µm full
scale respectively. (c) 81Br2 SIMS map, second
division, sum of 16 single pass maps recorded for
SIMS tomography, 5.6 3 105 counts, 70 minutes,
39.1 µm full scale. (d): 81Br2 SIMS map, third
division, sum of 9 single-pass scans, 6.7 3 105
counts, 39.3 minutes, 32 µm full scale.
still useful to investigate the chemical changes associated with Giemsa staining using SIMS. The 81Br2 maps
of 2 Giemsa-stained chromosome spreads are shown in
Figure 5a and c. Figure 5b is a CN2 map corresponding
to (a), and Figure 5d a magnified detail of 5c. Several
features stand out in these SIMS maps. First, the
apparent absence of CN2 emission from the chromosomes in 5b is a visual artefact of the photoreproduction. In fact, the measured CN2 signal intensity within
the chromosomes corresponds closely to that measured
for e.g., Figures 2d or 4d in the absence of Giemsa
staining, although the chromatids appear better separated and closely mimic the 81Br2 images in the present
case. This comparison to non-stained samples suggests
that some of the proteins not closely packed with DNA
may have been removed by staining, and that the
original histone proteins-DNA complex may either have
been left untouched, or partially replaced by the nitrogen-carrying components of Giemsa (thiazines). Second, CN2 originates from a bright halo surrounding the
chromosomes, with signal intensity about double that
of the chromosome interior. This halo can be attributed
to the residues of thiazines (eosin contains no nitrogen)
left by capillarity after in situ Giemsa staining. More
importantly, the Br2 distribution, in this case representing the eosin distribution, shows transverse bands, not
surprisingly similar to the Giemsa patterns. We shall
return to these observations, related to the banding
mechanism of optical stains, in the discussion section.
BrdU Labeling Plus DNA Denaturation
Two examples of mitotic cells subjected to the FISH
procedure (without DNA probe) are shown in Figure 6.
Both originate from the third cell division, because of
the presence of chromosomes exhibiting 1:1 and 1:1/2
label ratios between sister chromatids in the 81Br2
SIMS maps (Figs. 6a and c). Two SCEs are marked by
pointers in Figure 6c. The label intensities within the
chromatids are comparable to those measured for nondenaturated samples. Of particular interest are the
corresponding CN2 maps of Figure 6b, and especially
that of Figure 6d. In contrast to the rather uniform
maps for CN2 previously shown for non-denaturated
samples (see Fig. 2d and 4d), these maps exhibit
remarkable structure and evidence of a coiling arrangement of the chromatid fiber. Perhaps unexpectedly, the
CN2 signal within the chromatids is about twice as
intense as that for non-denaturated chromosomes, seemingly in contradiction with the evidence that much of
the protein coating, normally widening the slender
structure of the coiled chromatin, has been removed by
the denaturation process. We propose an explanation
for this apparent discrepancy by referring to the condensation state of the chromosomes, revealed by their
topography. This is shown in Figure 7, where Figures
7a and b show the appearance of two different presentations of mitotic cells among our samples, prior to any
further in situ procedure; Figure 7c shows the result of
denaturation, and Figure 7d that of Giemsa staining.
The denatured chromosomes in Figure 7c appear flattened with a thin raised rim, suggestive of the collapsed
chromosome state reported (Gormley and Ross, 1972;
Sumner, 1990) following treatment with hot 23 SSC,
part of the ASG (acetic/saline/Giemsa) staining method
of Sumner et al. (1971). We can surmise that the
intensity of the SIMS signal is a function of the aspect
Figure 9.
ION MICROPROBE IMAGING OF HUMAN CHROMOSOMES
ratio of the chromatin exposed to the primary probe. A
collapsed state would then favor a better secondary ion
yield than a more compacted normal state, or a contracted state such as that indicated as a result of
Giemsa staining in Figure 7d.
DISCUSSION
The images presented in the previous section are
illustrative of the direct information that high resolution SIMS imaging can provide concerning chromosomal structure and composition. In conjunction with
BrdU labeling, the method enables the detection of
SCEs at the base-specific level in images of comparable
contrast and resolution to those obtained with fluorescent dye techniques. Depth compositional information
from sequential SIMS mapping (although not exploited
in this presentation) is also available to explore the
base-specific 3D structure of the chromosomes. In conjunction with the label maps, the corresponding images
taken with the CN2 secondary ions provide complementary information about the overall chromosome morphology that may occasionally be valuable. Thus, in one
example shown (Figures 4c and d), this comparison has
made it possible to detect a break in one chromatid
(possibly due to a fragile site), coincident with the occurrence of an SCE. Furthermore, the difference between
these two sets of images, representing respectively the
DNA core and the overall DNA-protein complex, reveals
the level of protein digestion due to the fixation process and
subsequent in situ procedures. At the same time, images of
the surface topography indicate the condensation state
of the chromosomes under investigation.
Before discussing our observations that may be relevant to the mechanism of chromosome banding, it is
Fig. 9. (a) 81Br2 SIMS map of portion of a BrdU-labelled mitotic
cell. Sister chromatids are equilabeled, indicating first cell division
where only one DNA strand of each chromatid is labeled. The image is
shown on a pseudocolor scale which emphasizes a banded structure
due to base-specific (A-T) condensation (blue:zero signal; yellow–orange:
maximal signal). A coiled structure of the chromatin fibers is also
apparent in several chromosomes. (b) 81Br2 SIMS map of a BrdUlabelled mitotic cell, where the label intensity ratio between sister
chromatids averages close to the expected 1:1/2 ratio for second cell
division. Several occurrences of sister chromatid exchange (SCE) can
be detected in this micrograph. (c) False color superposition of a 81Br2
SIMS map (A-T pair distribution) of a BrdU-labelled mitotic cell
(green), with the corresponding 26CN2 map (blue). The latter includes
the overall contribution from DNA, RNA and proteins. This correlative
presentation emphasizes the occurrence of SCEs. (d) Portion of
BrdU-labelled mitotic cell cultured in the presence of the chemical
aphidicolin. The average rate of occurrence of SCEs in this sample is
2.5 times that of a normal culture. The image is a color-coded
superposition of 81Br2 (green) and 26CN2 (blue) SIMS maps. Four
SCEs are pointed out by arrows. In this example, the 26CN2 distribution extends beyond the chromatid outline, possibly due to residues of
nuclear material. (e) Portion of another BrdU-labelled mitotic cell
cultured in the presence of the chemical aphidicolin. 81Br2 is color
coded in white, 26CN2 in blue. Here the latter map helps identify a
break in the p-arm of the left chromatid in the chromosome located in
the NW corner. This break occurs in conjunction with an SCE and is
probably due to a fragile site. Several additional examples of SCEs are
clearly identifiable in this correlative image. (f ) Correlative superposition of 81Br2 (white) and 26CN2 (blue) SIMS maps for a BrdU-labeled
mitotic cell stained with Giemsa. Here the Br content of eosin
overshadows the label, and originates recognizeable Giemsa banding
patterns of equal intensity for both sister chromatids. The bright halo
surrounding the chromosomes is attributed to the thiazine residue
from Giemsa.
311
appropriate to compare in Figure 8 the optical micrographs of trypsin-Giemsa stained chromosomes (Figure
8a and b), with base-specific SIMS maps of BrdUlabeled chromosomes (Figure 8c and d). As previously
noted in regard to Figure 3, fully-labeled chromatids
show an essentially continuous label (or A-T pair)
distribution, with only occasional suggestion of banding, never differentiated at a level comparable to the
Giemsa bands. Even after denaturation, the correlation
between SIMS and Giemsa banding (see Figure 8) for
fully-labeled chromatids is at best rather weak. Only in
monofilarly labeled chromosomes, such as those in
Figure 2, banding suggestive of the Giemsa patterns,
that can be associated with possible regions of A-T
enrichment are discernable. These observations militate against attributing Giemsa banding exclusively to
patterns of base-specific condensation, at least in compacted metaphase chromosomes such as those examined in the present survey. The pattern of overall
chromosomal condensation becomes visible instead in
the CN2 maps of denatured chromosomes. Again however, this pattern, although structured, appears not
selectively differentiated. In view of the Br content of
eosin, the detection of Giemsa-like bands in the Br2
maps of Giemsa-stained chromosomes, at an intensity
level that is higher than that provided by BrdU labeling, simply reinforces the notion that eosin is associated with G-positive bands. By and large, these observations are consistent with the discussion of G-banding
summarized by Sumner (1990). Hints of the possible
role of divalent cations are found in the literature and
are also referred to by this author. We are presently
investigating the distribution of Ca and Mg in our
samples.
It should be noted that our observations on denaturated chromosomes represent a successful preliminary
to the localization of specific labelled probes, to develop,
in analogy with FISH, a methodology that should be
called SISH (for SIMS in situ hybridization). This goal
seems attainable using, e.g., BrdU-labelled probes (with
chromosomes not carrying the same label), or probes
labelled with other thymidine analogs such as iododeoxyuridine or fluorodeoxyuridine. By a similar approach, using stable isotope labels such as, e.g., deuterium it seems feasible to aim at the localization of
regulatory proteins, in conjunction with SISH, for
specific gene sequences. Further investigations along
these lines, with the UC SIM, are being formulated at
this time.
ACKNOWLEDGMENTS
This report is based on work supported by the
National Science Foundation under grant BIR 9317959.
We wish to thank Dr. Pamela L. Strissel for informative
discussions.
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