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Address correspondence to Andrew S. Belmont, Dept. of Cell and Structural Biology, University of Illinois, Urbana-Champaign, B107 CLSL, 601 S. Goodwin Ave., Urbana, IL 61801. Tel.: (217) 244-2311. Fax: (217) 244-1648. E-mail: asbel{at}uiuc.edu
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Abstract |
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Key Words: mitotic chromosome; large-scale structure of chromatin; scaffold-associated regions; transgenes; chromosome scaffold
* Abbreviations used in this paper: DHFR, dihydrofolate reductase; dSAR, double SAR construct; MAR, matrix-associated region; MTX, methotrexate; SAR, scaffold-associated region.
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Introduction |
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Experiments aimed at identifying the role of specific sequences have been constrained by experimental limitations associated with FISH for mapping the locations of specific DNA sequences within chromosomes. The harsh DNA denaturation conditions required for FISH lead to loss of the traditional chromosome scaffold. However, chromosomes extracted with 0.51.0 M univalent salt, producing a "partial halo/scaffold" preparation, show a relative enrichment of nontranscribed spacer sequences from the human ribosomal repeats within the scaffold fraction relative to transcribed sequences (Bickmore and Oghene, 1996). A more significant, preferential positioning of the Pax6 gene region to the edge of the chromosome scaffold with flanking genomic regions oriented into the "halo" fraction also was observed (Bickmore and Oghene, 1996).
Investigation of several chromosome loci by FISH at best have shown only a slight deviation from a random, lateral distribution within unextracted chromosomes (Baumgartner et al., 1991). Reduction in chromatid diameters in mammalian mitotic chromosome in regions containing integrated yeast chromosomes have been explained as the result of the increased SAR frequency in yeast DNA (McManus et al., 1994). Alternatively, a higher density of transcriptionally active regions with a resulting impairment in chromosome condensation has been proposed (McManus et al., 1994).
Here, we have attempted an approach, based on engineering chromosome regions with altered sequence content, which is motivated by two long-term goals. First, by labeling chromosome regions of variable size, we should be able to discern folding motifs within native chromosomes. Second, by experimentally manipulating the sequence composition of these engineered chromosome regions, we should be able to directly test the targeting of specific sequences and sequence requirements for normal chromosome condensation. In this paper, we describe the folding of mitotic chromosome regions, hundreds to thousands of kb in size, engineered to contain a large fraction of foreign DNA containing lac operator repeats and varying SAR density.
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Results |
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Fig. 1 outlines our experimental protocol. Two flanking SAR sequences from the human ßinterferon gene were used to flank the dihydrofolate reductase (DHFR)selectable marker. Both the "control" vector and the "double SAR" (dSAR) vector were transfected into CHO DG44 cells, containing a double deletion of the endogenous DHFR gene (Urlaub et al., 1986). After lac repressor immunostaining, the number of cells showing large spots was below 1% of the total cell pool of stable transformants. To enrich for cell clones with large inserts, fluorescence-activated cell sorting was used to select cells with high DHFR expression, using a cell permeable, fluorescein-labeled methotrexate (MTX) conjugate. MTX binds to DHFR, and binding is proportional to DHFR expression (Kaufman et al., 1978). The cell population corresponding to the highest 1% fluorescence intensity was sorted and subcloned by serial dilution.
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Two clones from the control vector (Con-1, Con-610) and two clones from the dSAR vector (dSAR-d11, dSAR-g12) were selected for detailed characterization and analysis. Southern blot analysis was used to estimate vector copy number in these stable cell clones. Different dilutions of DNA samples from these cell lines were digested with EcoRI, which cuts within the lac operator array, producing a 292-bp DNA band corresponding to a lac operator 8-mer (Robinett et al., 1996). As standards, varying amounts of the dSAR vector were digested as well, and were run on the gel. After blotting and hybridization to a lac operator DNA probe, integrated intensities of signals from each lane were plotted for different exposures. This analysis produced estimates of 1,000, 10, 1,000, and 300 copies for the Con-1, Con-610, dSAR-d11, and dSAR-g12 clones, respectively.
A rough estimate of the ratio of vector sequence to total DNA was calculated for two of the cell lines, dSAR-d11 and Con-1, with the largest labeled chromosome regions. Measurements of longitudinal lengths of inserts of 20 isolated chromosomes of the dSAR-d11 clone immunostained with lac repressor gave an average of 0.5 µm. Metaphase chromosomes from CHO DG44 cells have a DNA content of 70 Mbp per 1 µm length of chromosome arm (Li et al., 1998). Application of this to our cell lines yielded an estimate of 70 and 60% vector DNA content for Con-1 and dSAR-d11, respectively.
The Mbp size range of integration regions and high number of vector copies make it difficult to construct a physical map of the insertion sites. To probe the length of the uninterrupted vector arrays inside insert regions, we cut genomic DNA with restriction enzymes with no sites within either of the plasmid vectors, using pulse-field gel electrophoresis and Southern blotting for analysis. Assuming normal, nonrepetitive DNA flanking the vector insertion sites, use of a frequent cutter restriction enzyme should produce flanking, genomic sequences only several kb in size, negligible relative to a vector concatemer hundreds of kb in size. If an insert region consisted of multiple copies of vector DNA only, we would predict single bands hundreds of kb to tens of Mbp in size.
One cell line, Con-610, produced clear evidence for large, contiguous vector concatemers (Fig. 2). Two distinct bands, the first >150 kbp and the second >200 kbp in size were observed after both NheI and EcoRV digests, and two closely sized bands between 200 and 250 kb in size were observed after MluI digestion. The remaining clones showed mixtures of high mol wt fragments with smaller fragments. The MluI digest produced fragments hundreds of kb to several Mbp in size from the dSAR-g12, dSAR-d11, and Con-1 clones (Fig. 2 C). However, this finding was not confirmed by NheI and EcoRV digests. Even though high molecular DNA vector fragments were observed for these three clones, their fraction in the NheI digest is less compared with the MluI digest. For the EcoRV digest, most of the insert DNA can be cut into fragments of 50 kbp, corresponding to only two or three copies of the vector. Vector fragment lengths of several hundred kbp and longer represented only a small fraction of the hybridization signal.
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DNA halo analysis of vector sequences
To characterize our cell lines further, we mapped the location of vector sequences within mitotic halos. Previously, biochemical assays were used to demonstrate association of SAR/MAR sequences with nuclear scaffolds or matrices, or, in a small number of cases, with mitotic scaffolds (Mirkovitch et al., 1988; Laemmli et al., 1992; Saitoh and Laemmli, 1994). In these experiments, no data addressed how SAR sequences were distributed within undigested halos, and localization to scaffold fractions was based on biochemical fractionation rather than microscopic visualization of spatial localization. Moreover, because the proportion of SAR sequences in the matrix versus supernatant (halo) fraction has frequently been normalized by DNA concentration rather than cell number, scaffold sequences that show substantial enrichment in the scaffold fraction may partition in absolute terms predominately to the halo fraction (Bode and Maass, 1988). Attempts to locate endogenous DNA sequences within mitotic halos have been hindered by disruption of the mitotic halo during the FISH DNA denaturation step. Here, we have used lac repressor staining, thus allowing examination of intact halos. No consistent trend was observed. Vector sequences from large chromosome transgene insertion sites all showed scaffold enrichment, independent of SAR content. Smaller transgene insertion sites showed modest or no scaffold enrichment, for the vector with SARs, with the small Con-610 insert region from a vector without SARs showing depletion from the central "scaffold region."
Mitotic halos were prepared from isolated mitotic chromosomes using either high salt or LIS extraction. The scaffold fraction of vector DNA was defined as the high intensity DAPI staining located in the central halo area, which retained the general shape of the unextracted chromosome. The measured width of this DAPI-rich core is approximately one half to two thirds that expected for the native chromatid, and compares well with the observed width of axial condensin immunofluorescence staining observed within native chromosomes and mitotic halos (Maeshima and Laemmli, 2003). Consistent with previous papers (Bickmore and Oghene, 1996), traditional FISH conditions led to the loss of the central scaffold structure (Fig. 3, AC). In contrast, staining with purified lac repressor, followed by antibody detection, or direct labeling with purified GFPdimer lac repressor fusion protein did not change the appearance of scaffold and halos. Similar results were obtained with both methods of halo production. The large insert from the dSAR-d11 cell line consistently showed the strongest concentration of lac operator staining over the chromosome scaffold (Fig. 3, JL). The large insert in the Con-1 cell line showed strong staining appearing qualitatively uniform across the halo and scaffold regions (Fig. 3, PR), and the large insert in the dSAR-g12 cell line showed an increased association at the scaffold edges (Fig. 3, GI). In contrast, the insert region from Con-610 (Fig. 3, MO) and the small inserts regions for the dSAR-d11 and dSAR-g12 cell lines (Fig. 3, DF) instead showed a large fraction of staining extending as fibers or loops well into the halo region, without significant accumulation near the chromosome scaffold.
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Chromosomes have normal diameter at the insertion site with SAR sequences distributed throughout the chromosome width
At light microscopy resolution, metaphase chromosomes within intact metaphase plates had a normal chromosomal width at the insert sites (Fig. 5). Staining was performed on fixed cells not expressing lac repressor by using purified lac repressor, followed by immunostaining with anti-lac repressor antibodies (Fig. 5, columns 14). For cells expressing GFPdimer lac repressor, cells were fixed and stained with DAPI (Fig. 5, column 5). In both cases, repressor staining was uniform across the entire chromosome width for large insert sites. The chromosome width at these insertion sites, as defined by repressor staining, was the same as the width throughout the entire chromosome visualized by DAPI staining. In chromosomes with small insert sites, repressor-labeled spots were smaller than a full chromosome width; however, spots in different labeled chromosomes appeared at all positions, peripheral as well as interior, across the entire chromosome width.
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The above observations were based on light microscopy. To examine the labeled chromosome regions at higher resolution, cell lines not expressing lac repressor were studied with EM. Mitotic cells were collected by shakeoff after a 3-h nocodazole block and were deposited on coverslips before fixation with formaldehyde. Precise localization of the inserts was provided by lac repressor staining followed by pre-embedding immunogold staining and silver amplification of the gold-labeled secondary antibody. For all insert regions in all cell clones, chromosome diameters were similar to surrounding chromosome regions (Fig. 6). Moreover, large insertion sites showed uniform gold staining across the cross-sectional width of the chromosome.
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Our lac repressor immunogold staining allows ultrastructural analysis of specific chromosome regions within unextracted, metaphase chromosomes. Serial thin sections from dSAR-d11 cells support this "half band, variable orientation" hypothesis (Fig. 8). Both types of orientations, inferred from the light microscopy results, were observed. In cases where a full-width immunogold-labeled band was observed spanning the entire chromatid width, the major volume of stained region was only observed in several (45) of the nominally 40-nm thick serial sections. The remaining serial sections demonstrated that the stained region only occupied approximately one half the chromatid diameter. In cases where a single spot per chromatid was observed, the labeled region occupied only approximately one half the chromatid width, but appeared in roughly twice the number of adjacent sections (710). Fig. 8 (IL) shows an example with these two different orientations on the two sister chromatids, where the band-like staining (Fig. 8 I) appears in four sections and the spot-like staining (Fig. 8, J and K) appears in 10 sections. Note that the chromatid diameter is 500 nm. The length of the labeled band, corresponding to tangential sections, is slightly less than the chromatid diameter, typically in the 400-nm range. The width is in the 200300-nm range. Therefore, the band corresponds to a feature with a roughly 2:1 aspect ratio. Therefore, these section numbers agree well with the expected size of these labeled regions, taking into account as well that the chromosomes are always going to be at some angle to the section so that the sections are never exactly transverse or perpendicular to the band or spot.
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Significantly, both half-band inserts from the independent dSAR-d11 and g12 cell lines show the same characteristic 250-nm width, with examples of banded staining with sharply defined, parallel edges (Fig. 8 A), providing strong evidence for folding of a 250-nm-diam chromatid subunit to form the metaphase chromatid.
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Discussion |
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Normal chromosome condensation with abnormal DNA sequence composition
Despite estimated vector composition ranging from 6070%, we observed normal chromosome diameters for all of our clones. This argues for a relative independence of mitotic chromosome condensation to underlying sequence. In one of our clones, we obtained vector concatemers exceeding 200 kb in length; larger than the average loop size in radial loop models, yet normal chromosome diameter was again observed. Our results parallel similar findings in the literature(Monroe et al., 1992; McNally et al., 2000). In insect cells, large integrated arrays of vector sequence are observed after transfection, with stable inserts approaching the size of chromosome arms (Monroe et al., 1992), yet normal chromosome widths are observed in mitotic spreads. In mammalian cells, integration sites containing 2 Mbp vector concatemers again showed normal chromosome diameters in mitotic spreads when hybridized to vector sequence (McNally et al., 2000). We cannot exclude that in all of these examples, nonvector DNA integrated between the vector repeats is present, which maintains normal chromosome architecture. However our results imply that there is certainly a high degree of tolerance for large variations in DNA sequence and spacing.
In contrast, we have observed highly reproducible positioning of specific sequences with respect to the chromosome axis in a gene-amplified chromosome region (Dietzel and Belmont, 2001). Our observation of normal chromosome condensation with abnormal sequence composition does not imply that wild-type chromosomes necessarily show sequence-independent folding and positioning. Wild-type chromosomes might show highly reproducible folding and positioning of DNA sequences with this reproducibility serving other or additional purposes than chromosome condensation.
Hierarchical versus radial loop models of mitotic chromosome structure
Our work demonstrating the distribution of the 5' and 3' human ßinterferon gene SARs throughout the normal chromatid diameter is inconsistent with simple radial loop models in which most SAR sequences are targeted to an axial chromosome structure. Moreover, our results raise serious questions concerning the original experimental basis for radial loop models. Specifically, our results show that the disposition of vector sequences within classic chromosome halo preparations do not necessarily parallel their distribution within unextracted chromosomes. For instance, the large insert within the dSAR-d11 cell line shows significant enrichment of vector sequences in halo preparations, yet no axial vector distributional bias is observed within native chromosomes. Given these observations, one necessarily has to reexamine the functional significance that has been assigned to the observed preferred distribution of certain sequences within mitotic halos versus scaffolds with respect to the structure of native chromosomes.
If chromosomes compact through a radial loop organization, then the concept of a strict, one to one anchoring of SAR sequences to a well-defined, central axis needs modification based on our results. One might imagine, for example, that only a subset of specific SAR sequences functions as mitotic loop attachment sites. SAR sequences might also be facultative in the sense that only particular copies of SAR sequences would function as loop attachment sites, depending on spacing of nearby SAR sequences, or even that non-SAR sequences could function similarly to SAR sequences when no nearby SARs are present. In fact, biochemical approaches showing enrichment of SAR sequences in scaffold fractions typically express the relative enrichment based on the enrichment of SAR sequence per DNA concentration. This is similar to the normalization we used for our cytological approach in Fig. 4 D. However, if the absolute amount of scaffold DNA is small, even in cases where significant SAR enrichment in the scaffold fraction is present, the absolute amount of SAR sequence might still be higher in the halo fraction. Along these lines, SAR sequences embedded within repetitive vector transgene copies might not function appropriately as SAR sequences, perhaps failing to bind properly to chromosomal proteins required for normal chromosomal positioning and function. In large repetitive arrays lacking SAR sequences, one would have to imagine that other sequences assume the function of SARs.
Helical coil/radial loop models, in which a 200300-nm prophase chromatid, itself organized as a radial loop structure, is coiled into a metaphase chromatid are derived from observations of human chromosomes isolated from special cell lines or after prolonged inhibition of microtubule polymerization (Rattner and Lin, 1985; Boy de la Tour and Laemmli, 1988). Our results suggesting hierarchical folding involving an 250-nm subunit are consistent with either such a helical/radial loop or other hierarchical models such as successive helical folding or folded chromonema models.
However, a recent paper suggested that the small percentage of chromosomes showing an obvious helical organization by light microscopy (1%) in these previous works may be related to overcondensation of chromosomes observed after a prolonged colcemid-induced mitotic block (Maeshima and Laemmli, 2003). Metaphase chromosomes from cells not exposed to colcemid showed a smaller chromosome diameter, the absence of obvious daughter chromatid coiling, and a centralized core of condensin and topoisomerase II staining, roughly 0.20.3 µm in diameter. This observation of an axial condensin core appears to contradict the helical coil/radial loop model, which would instead have predicted a helical coil to the scaffold proteins with a radius equal to one half the chromatid radius and equal to the predicted radial loop size.
Implications for coiling models of mitotic chromosome structure
A presumably helical folding of an 200300-nm structure to form the final metaphase chromosome has been proposed in several models of chromosomes. Support for this model has come from visualization of surface features of metaphase chromosomes by scanning EM, visualization of apparent gyres by light microscopy of isolated metaphase chromosomes after treatment with specific buffers, isolation of chromosomes from cell lines apparently defective in normal chromosome condensation, or by banding patterns seen in TEM thin sections or suggestions of gyres in whole-mount metaphase chromosomes visualized as stereo-pairs (Belmont, 1997). However, experimental support for this model has been inconclusive, either because of the harsh treatments to which the chromosomes had been exposed to in these experiments and/or due to limitations in the visualization methods used. For example, scanning EM of metaphase chromosomes prepared using normal isolation buffers reveals a smooth surface. Instead, evidence for coiling of 200300-nm gyres comes from a segmented chromosomal appearance observed after G-banding procedures, involving trypsinization and methanol/acetic acid fixation. Similarly, light microscopy methods have relied on harsh buffer conditions that alter chromosome structure to produce obvious zigzag or coiled structures (Ohnuki, 1965), or else treatment with polyglutamic acid that swells chromosomes through extraction of histone H1, producing coiled appearances in
1% of isolated chromosomes from cells blocked in mitosis and possibly overcondensed (see above).
Here, we have demonstrated the existence of an 250-nm-diam substructure within metaphase chromosomes visualized directly within mitotic cells exposed for several seconds to a chromosome isolation buffer, chosen for its preservation of chromosome morphology close to that observed within living cells (Belmont et al., 1989), followed by formaldehyde fixation. The diameter of this substructural unit is similar to the diameter of early prophase CHO chromosomes observed previously in serial section reconstructions (unpublished data). These observations are consistent with a coiling model in which early prophase chromatids coil to form thicker prometaphase and metaphase chromosomes.
Reproducibility of daughter chromatid folding
Reproducible chromosome banding patterns indicate folding reproducibility between daughter chromatids, at least at the level of resolution probed by these works. A fundamental question with regard to models of chromosome compaction is to what level of resolution is this reproducibility between daughter chromatid folding maintained. Our ability to use immunogold staining on relatively intact chromosomes to examine specific chromosome regions smaller than chromosome bands sheds some light on this question of daughter chromatid reproducibility. EM immunostaining results specifically showing close longitudinal alignment of band-like staining across both daughter chromatids indicate a high degree of alignment between daughter chromatids. The boundaries between labeled and unlabeled regions are aligned to an estimated 2040 nm between daughter chromatids. Yet, the examples of insertion sites spanning a "half chromosome band" are intriguing, in that some examples show a transverse band extending across both chromatids, whereas other examples show a band-like staining on one chromatid and a spot-like staining on the opposing daughter chromatid, or two spots on opposite sides of the daughter chromatids, away from their region of contact. These results not only show a variation in folding between different chromosomes, but even within daughter chromatids of the same chromosome. The implication of these observations is that either the packing of sequences within daughter chromatids is not reproducible at this sub-band level of resolution, which might be difficult to reconcile with the observed precision in longitudinal alignment of the labeled bands, or the packing is reproducible between daughter chromatids, but that the daughter chromatids are not rotationally fixed. More detailed analysis of the reproducibility of daughter chromatid alignment is now in progress.
Future directions
Previous approaches to understanding mitotic chromosome organization have been largely descriptive and correlative, examining the structure of normal wild-type chromosomes. Although models were presented that invoked specific looped domains organized by specific sequences, no direct experimental testing of these models was conducted. Our experiments establish an experimental paradigm in which sequence composition of chromosome regions are directly manipulated to test sequence requirements for chromosome compaction and stability, and in which specific chromosome sequences can be mapped at high spatial resolution within native mitotic chromosomes. More elaborate DNA construction methods exploiting BAC and/or YAC cloning vectors should allow further dissection of folding motifs underlying mitotic chromosome organization.
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Materials and methods |
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Cell culture
CHO cells with a double deletion in the DHFR locus (DG44 CHO cell line; Urlaub et al., 1986) were grown in Ham's F12 medium (GIBCO BRL) supplemented with 10% defined FBS (HyClone). Ham's F12 medium, lacking thymidine and hypoxanthine, with 10% dialyzed FBS (HyClone) was used for selection and passaging of stably transfected cells. Calcium phosphatemediated transfection of cells with plasmid DNA was based on protocols described elsewhere (Chen and Okayama, 1988). For in vivo expression of EGFPlacINLS fusion protein, isolated clones were transformed with p3'SS-EGFPdimer lacINLS (Tumbar et al., 1999) using FuGENETM reagent (Boehringer). Stable transformants were selected with hygromycin. Cell sorting according to DHFR expression levels was done after growing cells for 8 h in regular Ham's F12 medium with 10% FBS and 20 µM fluorescein-labeled MTX (Molecular Probes, Inc.). 100 µM glycine, 30 µM hypoxanthine, and 30 µM thymine were added to relieve toxic effects of fluorescein-labeled MTX (Sherwood et al., 1990). The copy number was determined by quantitative Southern blot analysis of digested genomic DNA, with transfection plasmid as a control.
Southern blot analysis
High mol wt DNA was isolated from a 5 x 105 cells/ml cell suspension in agarose blocks by a conventional method described earlier (Riethman et al., 1997). DNA probe synthesis and detection were done with the Renaissance® random primer fluorescein labeling kit and CDP-Star kit, respectively (NEN Life Science Products).
In situ hybridization
Labeling of probe DNA was done with ChromaTideTM Alexa® 5945-dUTP from Molecular Probes, Inc., using nick translation. In situ hybridization on halos was done as described elsewhere (Robinett et al., 1996) with minor changes. DNA was denatured at 70°C for 5 min. Hybridization was done in 60% formamide, 2x SSC, 5% dextran sulfate, pH 7.0, with 2 ng/µl of probe DNA and 1 µg/µl of salmon sperm DNA (Sigma-Aldrich) overnight at 37°C. Coverslips were washed 30 min in 60% formamide for 30 min in 2x SSC at 37°C, and finally for 5 min in PBS with extra 5 mM MgCl2 and 0.1 mM EDTA.
Immunostaining
Lac repressor staining of fixed cells was performed as described elsewhere (Li et al., 1998). For 1 l of PBS, 8.00 g NaCl, 2.16 g Na2HPO4(7H2O), 0.20 g KCl, and 0.20 g KH2PO4 are dissolved in diionized water, pH 7.4. DNA was stained either with 0.2 µg/ml DAPI or with 0.5 µg/ml propidium iodide in PBS with extra 5 mM MgCl2 and 0.1 mM EDTA.
Chromosome and nuclear halo preparation
Isolation of mitotic chromosomes was performed according to a previously published protocol (Bickmore and Oghene, 1996). Chromosomes were extracted with high salt buffer (10 mM Tris-HCl, 10 mM EDTA, 0.1% NP-40, 1 µg/ml PMSF, 1 µg/ml aprotonin [Sigma-Aldrich], and 2 M NaCl, pH 8.0) for 5 min at RT with one change of extraction buffer. Alternatively, chromosomes and nuclei were extracted with LIS-containing buffer (Craig et al., 1997; 25 mM LIS lithium salt, 5 mM Hepes/NaOH, 2 mM KCl, 2 mM EDTA/KOH, 0.25 mM spermidine, and 0.1% digitonin, pH 7.4) for 10 min at RT. After extraction, halos were fixed in 1.6% formaldehyde (Polyscience) in PBS with extra 5 mM MgCl2 and 0.1 mM EDTA for 10 min at RT for further staining.
Isolation of the EGFPlacINLS fusion protein
GFPlacINLS fusion protein was expressed in BL21 Escherichia coli cells of the BL21 transformed with the pET28b expression vector. Recombinant protein was isolated with TALONTM resin column (CLONTECH Laboratories, Inc.) as described earlier (Tumbar et al., 1999).
Microscopy and processing and analysis of images
3-D optical sections were collected with an inverted fluorescence microscope (IMT-2; Olympus) and deconvolved as described previously (Hiraoka et al., 1991). Analysis of images was done with NewVision (Pixton and Belmont, 1996), and quantitative analysis of images was done with MATLAB® 6 (The MathWorks, Inc.). EM images were normalized for intensity variation, aligned by cross-correlation, and interpolated to compensate for the larger pixel spacing in the z direction. The interpolated image stack was smoothed twice with a 3 x 3 x 3 box filter and reduced 4x in size in the x and y dimensions. 3-D models were built using MATLAB® 6.
Immunostaining for EM
Staining of mitotic cells for electron and correlative microscopy was done as described earlier (Li et al., 1998; Tumbar et al., 1999). Cells were silver enhanced (Burry et al., 1992) and prepared for EM by dehydration, embedding, and sectioning. Electron micrographs were digitized for analysis and storage, or images were collected with a CCD camera (model 2kx2k; Tietz).
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Acknowledgments |
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This work was supported by National Institutes of Health grants GM42516 and GM 58460 to A.S. Belmont.
Submitted: 14 March 2003
Revised: 27 May 2003
Accepted: 27 May 2003
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References |
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