The mechanisms responsible for reciprocal BrdU-Giemsa staining

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Experimental Cell Research 141 (1982) 127-137

THE MECHANISMS

RESPONSIBLE

BrdU-GIEMSA

FOR RECIPROCAL

STAINING

GARY D. BURKHOLDER Department of Anatomy, College of Medicine, University of Saskatchewan, Saskcrtoon. Suskutchex’un S7N OWO, Ctrnudu

SUMMARY Cytological and biochemical experiments were undertaken to elucidate the mechanisms responsible for the reciprocal Giemsa staining of BrdU-substituted and unsubstituted chromosome regions subjected to high or low pH NaH,PO, treatments. These experiments included staining of chromosome preparations with ethidium bromide (EB), acridine orange (AO), or dansyl chloride, digestion of BrdU-substituted and unsubstituted chromatin with pancreatic DNase I, and SDS polyacrylamide gel electrophoresis of the proteins extracted from, and those remaining in isolated, fixed, air-dried nuclei subjected to either NaH2PG4 treatment. The collective evidence from this and previous work clearly indicates that, although the stajning reactions following the different pH treatments are reciprocal, the mechanisms of induction of the staining effects are not. After the high pH treatment, BrdU-substituted and unsubstituted chromosome regions are palely and intensely stained with Giemsa, respectively. This treatment preferentially solubilizes BrdU-substituted DNA, probably as a result of the photolysis or high temperature hydrolysis of BrdU-DNA. Concomitantly, this treatment selectively denatures the BrdU-DNA. The reduction in the amount of DNA in the BrdU regions leads to a quantitative decrease in Giemsa-dye binding, resulting in pale staining relative to unsubstituted regions. The extraction of BrdU-substituted DNA does not appear to simultaneously extract much chromosomal nrotein. After the low DH treatment. BrdUsubstituted and unsubstituted regions appear intensely-and palely stained with Giemsa, resnectively. BrdU substitution greatly increases the binding affinitv of histone HI to DNA. and the low DH treatment preferentially extracts the less tightly bound Hl of the unsubstituted’chromatin. This extraction of Hl is presumably responsible for the preferential dispersion of unsubstituted DNA outside the boundaries of the chromosome onto the surrounding area of the slide. The unsubstituted chromosome regions subsequently stain relatively palely with Giemsa, because the DNA in these regions is more dispersed than that in the BrdU-substituted regions. The low pH treatment concomitantly denatures the unsubstituted DNA.

When the base analog 5bromodeoxyuridine (BrdU) is substituted for thymidine in the DNA of replicating cells, differentially BrdU-substituted chromosome regions can be cytologically distinguished from one another by a variety of techniques. These techniques produce differential staining of unsubstituted and unifilarly BrdU-substituted chromosome regions, and of unifilarly and bifilarly substituted chromatids. They have therefore greatly facilitated the study of chromosomal DNA replication 9421810

patterns and of sister chromatid exchanges [l-3]. In addition, these methods have aroused interest in the mechanisms underlying the production of the differentially stained chromosome segments. Staining with the fluorescent dye Hoechst 33258 is commonly used to distinguish differentially BrdU-substituted chromosome regions [ 1, 21, and the resulting fluorescence appears to be directly related to a reduction in the dye fluorescence quantum yield by incorporated BrdU [4]. Other methExp Cdl Res 141 (1982)

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ods of BrdU differentiation involve specific pretreatments of the chromosome preparations followed by Giemsa staining. Most of the pretreatments involve exposure of the chromosomes to light and/or a hot salt solution [S-9], and the action of these treatments on the chromosomes is now starting to be understood. When chromosomes are exposed to hot NaH,PO, at pH 9.0 and then stained with Giemsa, BrdU regions are palely stained and unsubstituted regions are intensely stained [5, 91. There is evidence that the exposure of chromosome preparations to light causes photolysis of BrdU-substituted DNA [7, 10, 11, 121, and radioisotope studies have indicated that the high pH treatment preferentially solubilizes and extracts BrdU-substituted DNA into the treatment solution, relative to unsubstituted DNA [13]. The decrease in the amount of DNA in the BrdU-substituted chromosome regions results in a quantitative decrease in Giemsa-dye binding, thereby leading to the production of pale staining in these regions. This basic mechanism probably also accounts for the differential Giemsa staining seen by the FPG method [6] and other related techniques [7, lo]. In this regard, Ockey [ 141 has obtained autoradiographic evidence for the selective loss of BrdU-substituted DNA during the FPG method. If chromosomes are exposed to hot NaH,PO, at pH 4.0 instead of pH 9.0, reciprocal staining patterns are produced. The BrdU regions are intensely stained, and unsubstituted regions are palely stained [9]. The mechanism by which these staining patterns are produced is less clear. Radioisotope studies have indicated that the pretreatment does not solubilize either BrdUsubstituted or unsubstituted DNA [13]. Although this indicates that no DNA loss occurs from the slide during the low pH methautoradiographic experiments have 04 Exp Cell Res 141 (19821

suggested that unsubstituted DNA is preferentially dispersed outside the boundaries of the chromosomes onto the surrounding area of the slide [ 131. The BrdU regions subsequently stain more intensely with Giemsa, because the DNA in these regions is less dispersed than that in the unsubstituted regions. To illuminate further the mechanisms underlying the reciprocal Giemsa staining ofBrdU-substituted and unsubstituted chromosome regions produced by the high and low pH NaH,PO, pretreatments, additional cytological and biochemical experiments have been conducted. MATERIALS

AND METHODS

Cell culture and chromosome preparation Chinese hamster Don cells were maintained in McCoy’s medium Sa+lO% fetal calf serum (FCS). For cytological experiments, BrdU was added to log phase monolayer cultures at a final concentration of 200 pg/ml and the ceils were grown in the presence of this base analog for 4 h. The cultures were maintained in the dark to prevent photolysis of the BrdU-substituted DNA. Colcemid (0.05 pg/ml) was added to the culture medium for the last 2 h of culture. Mitotic cells were selectively detached from the monolayer by gentle shaking, collected by centrifttgation, treated with 0.075 M KC1 for 10 min at 37”C, and fixed for a total of 60 min with three changes of acetic methanol (1:3). Chromosome preparations were made by air-drying the suspension of fixed cells on dry slides in a humidified room. For the biochemical experiments, BrdU (25-50 pg/ml) was added to roller cultures of Don cells 24 h after initiation, and the cells were grown in the dark for an additional 48 h. This length of time allowed all of the DNA to become substituted with BrdU. Control cells were grown under identical conditions, but in the

absenceOfBrdU. NaHpO,

staining

treatments and chromosome

Chromosome preparations were exposed to two different NaH$O, treatments: (1) the high pH method: Slides were treated in 1 M NaH,PO, (adjusted to pH 9.0 with 10 N NaOH) at 80-88°C for 5 min. rinsed, and stained; (2) the low pH method: Slides were placed in 1 M NaH,FQ, (pH 4.0) at 88°C for IO-20 min, rinsed once in distilled water, and then stained. Untreated (control), and high or low pH NaH,PO,treated preparations were stained with a variety of

Mechanisms of BrdU-Giemsa staining fluorescent dyes. Some slides were stained with ethidium bromide (EB) (5 &ml) in 0.01 M Sorensen phosphate buffer, pH 7.0 for 10 min, rinsed and mounted in buffer. The chromosomes were nhotographed with a Zeiss photomicroscope equipped with an epifluorescence system. The filter set consisted of a band pass 546/10 nm exciter filter, 580 nm chromatic beam splitter, and 590 nm longwave pass barrier filter. Prior to acridine orange (AO) staining, the treated slides were directly washed in 4% formaldehyde and then stained for 5 min in 0.0125% A0 in 0.05 M Sorensen phosphate buffer, pH 6.8. The slides were rinsed in buffer, dried, and mounted in buffer containing 0.05 M dithionite (Na,S,O,). The latter inhibits fluorescence fading 1151. For fluorescence microscopy, the filter set consisted of a band pass 450-490 nm exciter filter, 510 nm chromatic beam splitter, and 520 nm longwave pass barrier filter. Other slides were stained with dansyl chloride. The staininn solution was oreoared bv mixing 0.1 a of dansylchloride with 25*miacetone, then d:luted with 25 ml of 0.2 M NaHCO, adiusted to oH 10 1161.The slides were stained for 30 min immediately after prenarina the solution. washed with three chanees of 50% acetone, air-dried, and mounted in 0.01 M Sorensen nhosnhate buffer, oH 6.8, containina 0.1 M dithionite. -Fluorescence’ microscopy was- performed with a filter set comprising a 365 nm exciter filter, 420 nm chromatic beam splitter, and 418 nm longwave pass barrier filter.

Isolation of chromatin and DNase digestion Unsubstituted or BrdU-substituted cells growing in roller flasks were trvnsinized off the flasks, diluted in culture medium, and washed once in medium, centrifuging at 650 g for 5 min. The cells were then washed 2x in 0.1 M hexylene glycol, 1 mM CaCl,, 0.05 mM PIPES buffer, pH 6.5 (HCP), resuspended in about 30 ml of HCP, incubated at 37°Cfor 10min, and homoeenized with a Teflon homogenizer 1171. The nuclei were sedimented at 650 g for-3 mitt, and the cycle of suspension in HCP, homogenization, and centrifugation was repeated until the nuclei were free of cytoplasmic contamination as monitored by phase contrast microscopy (usually three cycles). The final uellet of clean nuclei was susoended in about 20 ml ‘of 0.14 M NaCl, 5 mM Mgdlz, 3 mM CaCl,. 1 mM NaHSO,. 0.1 mM CdSO,. 1 &ml sovbean ?rypsin inhibitor “(SW buffer), containing 0.05 % Triton X-100, and centrifuged as before. The nuclei were then washed 3x in SW buffer alone, susoended in a small volume of 0.34 M sucrose. 0.1 &ml soybean trypsin inhibitor, 0.01 M Tris HCI,‘pH 7.Gfor 15 min, and sonicated with a Branson W-140 sonicator (standard horn; setting no. lo), using I@sec bursts, until phase contrast microscopy indicated that the nuclei were ruptured. This usually took a total of 30-50 sec. Finally, the chromatin suspension was centrifuged at 400 g for 5 min, and the supernatant was recentrifuged under the same conditions. The final supernatant was dialysed overnight against two changes of 0.01 M Tris

129

HCl, pH 7.0. DNA was isolated from the chromatin samples by the method of Marmur [ 181. DNase digestions were performed on the control and BrdU-substituted chromatin and isolated DNA samples as previously described [ 193.In brief, the samples were adjusted to a concentration of 25 pg/ml equivalent of DNA, warmed to 3PC, and MgCl, and pancreatic DNase I (Worthington) were added in sequence to final concentrations of 3 mM and 5 wg/ml, respectively. One ml samples were removed at various times thereafter and the undigested chromatin or DNA was precipitated by the addition of ice-cold perchloric acid (PCA) to a final concentration of 0.25 M. The samules were left on ice for 30 min and then centrifuged at 37000 g for 15 min to pellet undigested chromatin. The amount of digested DNA in each supernatant was quantitated b; measurement of the absorbance at 260 nm.

In situ treatments of isolated nuclei and gel electrophoresis Nuclei were isolated from unsubstituted and BrdU-substituted Don cells, fixed in acetic methanol 1 : 3, and air-dried in the bottom of glass test tubes [20]. The tubes were washed 3x with distilled water, and 2 ml of the high or low pH NaH,PO,, at the appropriate temperature, were then added. Each tube was incubated at the proper temperature until the treatment time had elapsed. The treatment solution was then decanted and saved for protein precipitation and resolubilization. The proteins in this solution represent those extracted from the nuclei during the treatment. The residual nuclear material left in the tube was directly solubilized for electrophoresis. In one experiment, the nuclei were subjected to a trypsin PCA treatment, a procedure which produces the same staining patterns as the low pH NaH,PO, treatment [21]. The nuclei were successively exposed to 0.02% trypsin (Difco 1 : 250, made in distilled water) at 4°C for 4 min, two washes in distilled water, 20% PCA at 55°C for 15 min, and two further washes in water. The proteins extracted into the PCA and the residual nuclear proteins were collected for electrophoresis. The treatment conditions used in all experiments were the same as those which produced optimal differentiation on cytological chromosome preparations. As controls, nuclei were fixed and dried in tubes, and were resolubilized for electrophoresis without having been exposed to any treatment. The methods of protein precipitation, protein solubilization, and SDS polyacrylamide slab gel electrophoresis were identical with those used previously [20]. The gels were stained with silver [22].

RESULTS In the Chinese hamster cells used in this study, the long arm of the X-chromosome, the entire Y-chromosome, and numerous regions in the autosomes undergo DNA Exp Cd

Res 141 (1982)

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G. D. Burkholder

Fig.

I. Chinese hamster Don cell chromosomes treated with NaH,PO, and stained with EB. ((1) After high pH treatment, the BrdU-substituted regions are dull red in color (dark in this black-and-white micrograph), whereas the unsubstituted regions are bright red. Note the appearance of the long arm of the X-

chromosome and the entire Y-chromosome, which are BrdU-substituted. (b) After low pH treatment, the BrdU regions are relatively more fluorescent than the unsubstituted regions but the color contrast between these regions is reduced in comparison to that seen after the high pH method. (a) X 1450; (b) X 1 840.

replication late in the S phase. The DNA in these regions becomes unifilarly substituted with BrdU during the time that this base analog is present in the culture medium prior to harvesting. The early replicating chromosome regions do not contain BrdU. Staining of chromosomes with EB specifically detects DNA, and BrdU substitution does not affect the number of EB-binding sites in chromatin [23]. The fluorescence intensity of a chromosome region should therefore reflect the quantity of DNA present in that region. After the high pH NaH,PO, treatment and EB staining, the BrdU-substituted chromosome regions were dull red in color (fig. la). This was particularly obvious in the long arm of the X-chromosome and the entire Y-chromosome. Unsubstituted chromosome regions were a bright red. The low pH treatment produced a reciprocal staining effect, i.e. BrdU regions were bright red, whereas unsubstituted regions were a slightly duller

red (fig. lb). In this case, however, the color contrast between the substituted and unsubstituted regions was considerably less than that seen between these regions after the high pH method. The distribution of double-stranded and single-stranded DNA in chromosomes can be qualitatively assessed from the color of A0 fluorescence [24, 251. Double-stranded DNA fluoresces green, whereas denatured single-stranded DNA is red. A0 staining of BrdU-substituted chromosomes without any pretreatment resulted in the production of late replication banding patterns on the chromosomes (fig. 2~). The unsubstituted chromosome regions were yellow-green in color, whereas the BrdU regions fluoresced a slightly duller yellow-green. The same patterns were observed after high pH treatment (fig. 26). The unsubstituted regions still fluoresced yellow-green, but the BrdU regions appeared orange-red in color, resulting in an overall increase in contrast be-

Exp Cell RPS 141 (1982)

Mechanisms of BrdV-Giemsa staining

131

Fig.

2. Chinese hamster Don cell chromosomes stained with AO. (a) BrdU-substituted chromosomes not subjected to any treatment before staining. Late replication banding patterns are apparent. Both BrdUsubstituted and unsubstituted chromosome regions fluoresce yellow-green, but the former regions are slightly duller in color. (b) After high pH treatment, the unsubstituted chromosome regions are yellow-

green but the BrdU regions are orangered (dull in this micrograph), suggesting denaturation of DNA in the latter regions. (c) Low pH treatment results in BrdU regions which are yellow-green and unsubstituted regions which are orange-red. The latter regions presumably contain denatured DNA. (a) x 1625; (b) x 1500; (c) x 1420.

tween the differentially BrdU-substituted regions. A0 staining of low pH-treated chromosomes produced reciprocal fluorescence patterns, i.e. the BrdU regions were yellowgreen, whereas the unsubstituted regions were orange-red (fig. 2~). In these particular experiments, the slides were washed in 4 % formaldehyde im-

mediately after treatment in order to prevent renaturation of any DNA denatured by the treatment. To test whether there might still be some renaturation during the slide transfer, formaldehyde was directly added to the NaH,PO, solution before the slide was removed at the conclusion of the treatment (final concentration of formaldehyde Exp Cell Res 141 (1982)

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G. D. Burkholder

Fig.

3. Chinese hamster Don cell chromosomes stained with dansyl chloride. (a) Completely unsubstituted chromosomes are uniformly stained. (b)BrdUsubstituted chromosomes not subjected to any treatment before staining. The BrdU regions are slightly more fluorescent than the unsubstituted regions. (c)

After high pH treatment, BrdU-substituted regions are brightly fluorescent and unsubstituted regions are relatively dull. (d) Low pH treatment also produces brightly fluorescent BrdU regions and dully fluorescent unsubstituted regions. (a) Xl 220; (b) xl 460; (c) x 1580; (d) x 1760.

was 4%). The slide was then rinsed, stained, washed, and mounted in solutions containing 4 % formaldehyde. The staining results in terms of the A0 fluorescence properties of the differentially BrdU-substituted chromosome regions were essentiallythe same as those obtained with a single post-treatment formaldehyde wash. This indicates that DNA renaturation did not oc-

cur in the usual experimental protocol. To test for the occurrence of DNA renaturation in the absence of any formaldehyde, slides were exposed to either NaH,PO, treatment, then incubated at room temperature in 0.05 M Sorensen phosphate buffer, pH 6.8 for 30 min prior to A0 staining. Chromosome regions which fluoresced orange-red before the buffer incubation reverted to yellow-

Exp Cell Rcs 141 (1982)

Mechunisms of BrdU-Giemsa staining

IOJ

20

40

60

00

100

120

Fig. 4. Rate of digestion of isolated BrdU-substituted and unsubstituted Don cell chromatin by pancreatic DNase I. Chromatin fractions were digested as described in the text. ---, Control, unsutkituted chromatin; - - -, BrdU-substituted chromatin. The DNA in the BrdU-substituted chromatin is digested at a significantly lower rate than that in unsubstituted chromatin.

green fluorescence afterwards. This experiment suggests that partial renaturation of denatured DNA may occur during the buffer incubation. Dansyl chloride specifically reacts with proteins to form a fluorescent complex [16, 261. Staining of completely unsubstituted chromosomes with dansyl chloride produced uniform fluorescence along the chromosome (fig. 3~). Untreated BrdU-substituted chromosomes, on the other hand, exhibited BrdU regions which were rather more fluorescent than the unsubstituted regions (fig. 36). After either high or low pH treatments, the BrdU regions remained brightly fluorescent and the unsubstituted regions were relatively dull. The low pH treatment frequently enhanced the difference in fluorescence intensity between the

133

differentially BrdU-substituted chromosome regions (cf fig. 3b vs d). Biochemical experiments were undertaken to assess possible differences in DNA-protein interactions in differentially BrdU-substituted chromatin. The rate of digestion of chromatin DNA in the presence of pancreatic DNase I is known to be dependent on the degree of binding of proteins to DNA [27]. Chromatin in which the DNA is loosely associated with protein will be digested at a faster rate than chromatin in which the DNA is more tightly associated with protein. It is therefore possible to study relative rates of digestion as a means to distinguish relative differences in chromatin structure. BrdU-substituted and unsubstituted chromatin and their constituent DNAs were isolated and digested with DNase I. The percentage of DNA digested by the enzyme was then plotted as a function of time to give the rate of digestion (fig. 4). The two chromatin samples both exhibited two steps in their digestion rate: a fast initial rate with 35-50% of the DNA being digested in the first 15 min and a slower rate of digestion thereafter. These are typical chromatin digestion curves [27]. Of significance is the finding that the DNA in the BrdU-substituted chromatin is digested at a significantly lower rate than that in the unsubstituted chromatin. Purified BrdU-substituted and unsubstituted DNA samples were very rapidly digested (100 % digestion within 1 min), and, unlike the chromatin samples, did not demonstrate any differences in their rates of digestion (data not shown). The effect of the NaH,PO, treatments on the proteins of BrdU-substituted and unsubstituted chromatin were investigated by gel electrophoresis. In fig. Sa, the electrophoretic banding patterns of the extracted Exp Cd/ Rcs 141 (1982)

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G. D. Burkholder NaH2PO4 CRE

PHO ERC

NI H2PO4

pH4

CREERC

Trypsin-HC104 CREERC

Hl

30

-21 @

BrdU

No BrdU

BrdU

No BrdU

a Fig. 5. SDS polyacrylamide gels demonstrating the ef-

b

C

fect of(u) the high pH NaH*PO,; (b) low pH NaH,PO,; (c) trypsin-PCA treatments on the proteins of BrdUsubstituted and unsubstituted nuclei. In each case, the proteins in control untreated nuclei, C, are compared with those extracted into the treatment solution, E, and the residual proteins left in the nuclei after treat-

ment, R. The extract E, in (c) represents the PCAextracted proteins. The treatment conditions are described in the text. In ((I) the histone bands are identitied. Numbers along the ordinate in (d) refer to MWx IO-“. Equivalent amounts of protein (5 pg) were applied to the C and R wells; the E wells received 0.01-2.0 /Lg.

and residual proteins from the fixed nuclei treated with high or low pH NaH2P0, are compared with the banding patterns of untreated nuclei. The high pH treatment extracts very little protein from either the BrdU-substituted or unsubstituted nuclei. Only a few faint protein bands are apparent in the lanes in which the extracted proteins were electrophoresed. The residual protein patterns in each case are virtually identical to those of the control, further indicating the absence of a marked effect of this treatment on the chromosomal proteins. The low pH treatment, on the other hand, extracted a significant quantity of

histone H 1, a trace amount of histone H26, and some of each of a small number of nonhistones from unsubstituted nuclei (fig. 56). Hl formed the most conspicuous band in the low pH extract. The same treatment extracted only trace amounts of histone H 1, and small amounts of histone H26 and the same non-histones from the BrdU-substituted nuclei (fig. 56). The trypsin-PCA treatment produced an effect identical with that of the low pH NaH,PO, treatment, but the amount of histone Hl extracted from the unsubstituted nuclei into the PCA was more pronounced (fig. 5~). In comparison, much less Hl was extracted from the BrdU-

Exp Cell Res 141 (1982)

Mechanisms

of BrdU-Giemsa

staining

135

substituted nuclei by this treatment, a result also identical to that obtained by the low pH treatment.

ference is much smaller than that observed after high pH treatment. Again, these observations are consistent with the previous autoradiographic data suggesting a preDISCUSSION ferential dispersion of unsubstituted DNA The results of this cytological and bio- outside of the chromosome onto the surchemical investigation provide further in- rounding area of the slide [ 131.A side effect sight into the nature of the mechanisms of low pH treatment, indicated by A0 stainresponsible for the reciprocal Giemsa stain- ing (fig. 2c), is the denaturation of the unsubstituted DNA, leaving the BrdU-DNA ing of BrdU-substituted and unsubstituted chromosome regions subjected to high or in a double-stranded state. This denaturation of DNA could also contribute to the low pH NaH,PO, treatments. Based on the EB fluorescence prop- reduced EB fluorescence of the unsuberties of BrdU-substituted and unsub- stituted DNA. BrdU substitution results in an increase in stituted chromosome regions, there appeared to be much less DNA in the BrdU the thermal stability of chromatin [30], and regions than in the unsubstituted regions af- several studies have shown that the hister high pH treatment (fig. la). This is con- tones bind more tightly to BrdU-substituted sistent with previous radioisotope studies than to unsubstituted DNA [31-331. There which shdwed that considerably more BrdU- is also some evidence of an increased stabilsubstituted DNA is extracted into the high ity of non-histone proteins bound to BrdUpH treatment solution than unsubstituted DNA [34, 351.Collectively, these data demonstrate that BrdU substitution in DNA DNA [ 131. This preferential solubilization of BrdU-substituted DNA is probably a re- alters DNA-protein interactions. To further substantiate this conclusion, sult of previous photolysis of the BrdUDNA during exposure to light [7, 10-12, the rate of digestion of chromatin DNA by 281, or of BrdU-DNA fragmentation during pancreatic DNase I was used to assay high temperature hydrolysis at alkaline pH DNA-protein interactions in isolated BrdU[29]. The quantitative reduction of DNA in substituted and unsubstituted chromatin. the BrdU regions would lead to a decrease BrdU-substituted chromatin was digested at in the amount of Giemsa-dye binding, re- a much slower rate than unsubstituted chrosulting in pale staining relative to the un- matin (fig. 4). This differential sensitivity to substituted chromosome regions. The re- DNase digestion did not appear to be insults of A0 staining indicate that high pH herent in the DNA that each chromatin treatment also preferentially denatures the sample contained, but minor differences in BrdU-substituted DNA. The fluorescence of DNase digestibility of purified DNA resultEB is reduced by denatured DNA; thus the ing from BrdU substitution would probably denaturation of BrdU-DNA by high pH escape detection under the experimental treatment may partially contribute to the conditions used. Nevertheless, these results indicate that the difference in the rates observed reduction in EB fluorescence. The EB fluorescence patterns seen after of digestion of the chromatin samples is prelow pH treatment suggest that there is less dominantly related to a difference in the asDNA in the unsubstituted than in the BrdU sociation of the chromosomal proteins with regions (fig. lb), but quantitatively the dif- the DNA, such that the chromosomal proExp Cell Res 141 (1982)

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G. D. Burkholder

teins are more tightly associated with BrdUsubstituted DNA than with unsubstituted DNA. The even distribution of dansyl chloride fluorescence in completely unsubstituted chromosomes (fig. 3~) suggests that under normal circumstances protein is uniformly distributed along the chromosome. This is in agreement with the observations of Utakoji & Matsukuma [ 161.In untreated BrdUsubstituted chromosomes, the BrdU regions were somewhat more fluorescent than the unsubstituted regions (fig. 3b). Assuming that dansyl chloride reaction sites are freely available on the chromosomal proteins, this result suggests that there is more protein associated with BrdU-substituted than with unsubstituted chromosome regions. Presumably, there is either more protein bound to the BrdU regions in the native chromosome or else there may be a greater degree of protein extraction from unsubstituted regions compared to BrdU regions during chromosome harvesting. The difference in the amount of protein associated with BrdU-substituted and unsubstituted regions was further increased by the low pH NaH,PO, treatment, as demonstrated by the increased contrast in fluorescence between these regions relative to untreated chromosomes (cf fig. 36 vs d). Differences in dansyl chloride fluorescence of BrdU-substituted and unsubstituted chromosome regions could also be partially due to factors other than the amount of protein present. For example, cysteine residues are among the protein reaction sites for dansyl chloride [36]. During the various treatments, cysteines may arise from cystines in the chromosomal proteins. This may preferentially occur in BrdU-substituted chromosome regions [8], resulting in an increase in dansyl chloride fluorescence relative to the unsubstituted regions. Exp Cell Res 141 (1982)

The electrophoretic studies revealed that high pH treatment had a relatively minor effect on the chromosomal proteins of either BrdU-substituted or unsubstituted nuclei (fig. 5~). This suggests that protein extraction does not play a major role in the mechanism of the differential staining produced by this treatment. On the other hand, low pH treatment extracted considerably more histone Hl from unsubstituted than from BrdU-substituted nuclei (fig. 5b). A trypsin-PCA treatment, which produces the same cytological effect as the low pH NaH2P0, [21], had an identical effect on histone Hl (fig. 5~). It therefore appears that Hl is preferentially extracted from unsubstituted chromatin during low pH treatment . The preferential extraction of histone H 1 from unsubstituted chromatin during low pH treatment is likely responsible for the preferential dispersion of unsubstituted DNA occurring simultaneously during this treatment. Histone Hl has long been thought to play a role in chromosome condensation [37-411, and BrdU substitution considerably increases the binding affinity of this protein to DNA, relative to unsubstituted DNA [31]. The difference in the binding affinity of histone Hl to BrdU-substituted and unsubstituted chromosome regions can readily explain the differential extraction of Hl from these regions by the low pH treatment and the concomitant differential dispersion of chromatin. The unsubstituted chromosome regions would subsequently stain relatively palely with Giemsa because the DNA in these regions is more dispersed than that in the BrdUsubstituted regions. The results of this study clearly indicate that although the staining reactions after the different pH treatments are reciprocal, the mechanisms of induction of the staining

Mechanisms of BrdU-Giemsa staining effects are not. Substantially different mechanisms are responsible, one involving the selective extraction of BrdU-substituted DNA, and the other involving the selective extraction of histone Hl from unsubstituted DNA followed by the dispersion of the histone-depleted chromatin. This work was supported by grant MT-5125 from the MRC of Canada.

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