Cytochemical characterization of deoxyribonucleoprotein in individual cell nuclei

Experimental

CYTOCHEMICAL

Cell Research 55 (1969) 215-224

CHARACTERIZATION

OF DEOXYRIBONUCLEOPROTEIN

INDIVIDUAL

CELL

IN

NUCLEI

Techniques for Obtaining Heat Denaturation Curves with the Aid of Acridine Microfluorimetry and Ultraviolet Microspectrophotometry R. RIGLER,

D. KILLANDER,

L. BOLUND

Orange

and N. R. RINGERTZ

Institute for Medical Cell Research and Genetics, Medical Nobel Institute, Karolinska Institute& IO4 01 Stockholm 60, Sweden

SUMMARY Simple methods for obtaining heat denaturation curves on the deoxyribonucleoprotein DNP of individual cell nuclei or intact cells are described. Cells or nuclei adhering to microscopic slides were heated in a salt solution (SSC) containing formaldehyde. After heating the slides were rapidly cooled. Both the formaldehyde and the rapid cooling served to prevent renaturation of denatured DNA. The heated preparations were analyzed for changes in single- and doublestrandedness by UV microspectrophotometry and by microfluorimetry after a&dine orange staining. These techniques were used to obtain melting profiles of DNP from non-stimulated and PHA stimulated lymphocytes, erythrocyte nuclei activated by cell fusion with HeLa cells, normal erythrocyte nuclei and isolated HeLa nuclei. The results obtained show that the activation of a large number of genes in both human lymphocytes and hen erythrocyte nuclei is paralleled by striking changes in the melting profiles of nuclear DNP.

The cytochemical staining properties of nuclear chromatin may vary considerably depending on the physiological state of the cell nucleus. These variations probably reflect differences in the state of chromatin condensation and in the physicochemical properties of the deoxyribonucleoprotein (DNP), which may be important in the modulation of gene activity. Claims have also been made that DNP preparations isolated from cells differing in gene expression show differences in the binding of histones and other proteins to DNA. The isolation of DNP does, however, involve serious risks for artefacts. Differences in the physico-chemical and chemical properties which exist when intact cell nuclei are compared may in fact be destroyed by the isolation procedure. Another drawback of biochemical methods is that they require material from a large number of cells, whereas changes in the pattern of gene activity e.g. during the early stages of cell differ-

entiation normally affects only a very small number of cells. For these reasons we have attempted to provide methods for studying the DNP in intact cells and nuclei. The methods described in this paper demonstrate that the activation of a large number of genes in human lymphocytes and in hen erythrocytes involves major changes in the stability of DNP to heat denaturation. Emphasis is put on the technical aspects of the methods used. The biological systems studied are described in detail in separate publications [2, 11, 16, 171.

MATERIAL

AND

METHODS

Cells and nuclei Human lymphocytes were cultured directly on hemocytometer cover glasses with or without phytohemagglutinin \l?yA) according to a method previously described [lo, Hen erythrocyte nuclei were activated by fusing hen Exptl Cell Res 55

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erythrocytes with human HeLa cells into heterokaryons with the aid of UV-inactivated Sendai virus Bl. The techniques used for growing these heterokaryons on slides have been reported in a previous communication 121. HeLa nuclei and activated hen erythrocyte nuclei were isolated from heterokarvons _ bv_ the method of Fisher and Harris 171. Nucleonrotein changes characteristic of activation were also induced in “ghosts” prepared from hen erythrocytes by lysis with the detergent Nonident P 40 (Shell Co). These “ghosts” consist of erythrocyte nuclei surrounded onlv bv a cell membrane, all cytoplasmic material and hemoglobin being lost during &is.-The method for inducing the DNP changes involves washing and incubation of redcell ghosts in salt solutions devoid of serum proteins. Control preparations were washed and incubated in the same salt solutions containing 7.5 % (v/v) calf serum. Under those conditions the ghost nuclei retain the same cytochemical DNP properties as nuclei in intact, untreated erythrocytes.

Fixation and storage The slides were rinsed in isotonic saline and fixed in ethanol: acetone (1 : 1) for at least 60 min at room temperature and then stored in the fixative at +4”C until heat denaturation and/or staining could be performed.

Heat denaturation of cell nuclei After stepwise hydration of the fixed cells or nuclei, the preparations were heated at different temperatures first for 10 min in 0.15 M NaCl, 0.015 M Na-citrate (SSC) and then for 20 min in SSC containing 4 % formaldehyde (w/v, pH 7.0). In later experiments the 10 min preheating in SSC was omitted. The preparations were then immediately immersed into ice-cold SSC. After step-wise dehydration through a series of increasing ethanol concentrations the preparations were stored in absolute ethanol at +4”C until they could be analyzed. The formaldehyde present in the heating medium serves to prevent renaturation of the DNP. Without formaldehyde extensive renaturation occurs (fig. 1).

Ultraviolet microspectrophotometry The UV absorption of heated erythrocyte nuclei mounted in glycerin was measured at 265 and 315 nm by scanning and integrating microspectrophotometry [3,12]. The total extinction at 315 nm (E,,,)__“,was found to be insignificant ( < 5 % of EZ6J and therefore no corrections w&e made for unspecific light losses. The results of the UV absorption measurements are expressed in “total extinction units” (decadic extinction times area, integrated over the whole cell).

Fluorescence measurements icrofluorimetry on A0 stained cells was performed in microfluorimeter described by Caspersson et al. [4] sing UV light (A,, = 365 nm) for excitation. Fluorescence ‘ntensities r were recorded at 530 and 590 nm. The fluorescence values given in this publication have neither been corrected for wavelength dependent changes of the photomultiplier sensitivity nor for the transmission of the optical system. The measuring device consisted of a Reichert 100 x objective with a numerical aperture of 1.25 together with a Schott GG9 filter to cut out exciting radiation below 500 nm. The fluorescence emitted was analyzed with the aid of a Zeiss M 4 QII quartz prism monochromator and a RCA 1P 28 photomultiplier. To correct for unequal response of the recording system at the wavelengths measured, all ratios F,,,/F,,, (called a) have to be multiplied by a factor of 2.45.

Calculation of the degree of single strandedness A0 binds stoechiometrically and specifically to nucleic acids in the present type of material [ll, 15, 171. The binding of the dye is dependent on the amount of binding sites on the nucleic acid and also on its secondary structure. Nucleic acids with double helical structure bind A0 in a monomer form giving rise to an emission maximum at 530 nm. Single-stranded nucleic acids bind A0 in associated form with an emission maximum at 590 nm (after correction at 650 nm). If the nucleic acid is regarded as being composed of varying fractions of pure double-stranded-helix and pure single-stranded random coil structures it is possible by measuring the fluorescence intensities at these wavelengths to estimate the amount of single- and doublestranded regions. The fluorescence intensity of the AO-nucleic acid complex can be given as the &n of the intensities characteristic for each region: \

Exptl

Cell Res 55

= D f;o

+ S fko

690

= D f i&o ‘- S f&o

(1)

(2)

where Fsso and Fbgo are the total fluorescence intensities in mV at the two wavelengths examined. D and S are the amount of double- and single-stranded regions, respectively in mole phosphate. The coefficients fgO, ffso, Jgo and $a0 are fluorescence coefficients expressed in mV/mole phosphate of nucleic acid at the respective wavelengths for pure double- and pure single-stranded regions respectively. Simplifying eqs (1) and (2), the ratio S/D is given by: S/D=--- c(-

Acridine orange (AO) staining The staining of heat denatured cells or nuclei was uerformed according to the procedure previously descibed 115. 161. After acetvlation with 40 % acetic acid anhvdride in &&line for 15 min the slides were washed in efhanol and then brought through a series of decreasing ethanol concentrations-to distilled water. Following equilibration in a NaHPO,-citrate buffer (pH 4.1. ionic strength 0.6) for 5 min the preparations were stained in 10e4 M acridine orange for 15 min. The slides were then exposed to three 5 min changes of buffer in order to permit diffusion of unbound dye. Preparations were mounted in buffer and sealed with Entellan (Merck Co.).

430

ED

Q3 - 05

where

xsf?30

(3)

f530

f-50 uszs Go and a=* G(~=~, f 530 f 530 630 F

As previously mentioned the fluorescence coefficient fssO is dependent on the structural organization of the stained nucleic acid and varies with cc according to a function determined experimentally [15]. /22.3\ f530 = \n)

l.16

Cytochemical characterizption of DNP Eq. (3) can thus be written: a- aD S/D = -x +-a

1.2

as ‘a i>C(D

1

217

_

(4)

The a-value is thus a measure for the ratio of the singleto double-stranded regions within a nucleic acid. Increasing values indicate an increase in the amount of single-stranded regions. Changes in a may also be due to other changes in the nucleic acid structure (see below). However, such changes in a are small in comparison with those observed in connection with strand separation.

Determination of a, and as A correct estimate of the actual proportion of singlestranded regions within a DNP complex is dependent on exact values for ocnand as. In the calculations an +,-value of 3.313 was used. This value was obtained on isolated and acetylated, A0 stained ribosomes containing RNA which was found to be almost completely single-stranded under the conditions applied [15]. The mean m-value of the unheated control nuclei (not subjected to formaldehyde) of the same cell type obtained from a great number of measurements was used as an in most calculations, the unheated intracellular DNA thus being referred to as completely doublestranded.

Correction of cc,,for the varying A0 binding capacity of DNP Previous studies [15] on cells with varying A0 binding capacity and containing negligible amounts of RNA (spermatozoa representing different stages of maturation, normal and PHA stimulated polymorphnuclear leucocytes) have shown that the a-value of AO-DNP decreases with increasing number of A0 binding sites. This result was interpreted as showing a lengthening and an increased regularity in the double helical structure due to the exposure of charged (dye binding) phosphate groups in DNP. The regression line for the experimentally determined points could be expressed by the function: -0.163

41 50

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90

90

100

Fig. 1. Effect of formaldehyde on A0 melting profiles tested on human lymphocytes. Formaldehyde is necessary in order to prevent reuaturation (means and S&M:, n = 10). Abscissa: temperature, “C; ordinate: a. 0 - 0, with formalin; w - W, without formalin.

erythrocyte nuclei and on HeLa cell nuclei. These cells and nuclei vary greatly with respect to their growth rates and rates of RNA synthesis and have presented a good test material for the techniques described in this publication. For a more detailed description of the biological experiments performed, the reader is referred to separate and earlier publications where the activation of human lymphocytes by PHA treatment [lo, 11, 161, the activation of hen erythrocyte nuclei by cell hybridization [2] and model experiments on the “activation” of nuclei in red cell ghosts [17] have been described.

(5) where DNA-P is the total amount of binding sites expressed in moles of DNA phosphorus and xDNA represents the F5J&, ratio for the DNA-A0 complex. Since F,,, is only slightly dependent on the presence of singlestranded nucleic acids (single-stranded nucleic acids added to an equal amount of double-stranded nucleic acid where all the binding sites are available increase the FgaO only by 3 x). Thus, a good approximation of an ( = aDNA) can be obtained by this relationship also in the presence of RNA. For higher precision a correction for the contribution of RNA to Fs?53,, can be performed [16]. All calculations were performed in an IBM 7044 computer.

RESULTS Heat denaturation experiments have been performed on normal and PHA activated human lymphocytes, on normal and activated hen

Phytohemagglutiizin (PHA) stimulation of lymphocytes Human lymphocytes cultured in vitro are stimulated to proliferate and undergo blastogenesis when PHA is added to the medium. Within 1 h after the addition of PHA an intense RNA synthesis has been observed [6, 141. The early stages of this activation process are characterized by a drastic change in the ability of nuclear chromatin to bind AO. This increase in A0 stainability must be due to an unmasking of dye binding sites in the DNP since no change in the amount of DNA could be observed at this stage [lo]. The altered DNP complex of the PHA stimulated cells is also more sensitive to heat denaExptl Cell Res 55

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I -

IO0

6u

I

20

50

60

70

90

90

100

2b

’ 5’0

io

lb

io

ob

vi0

C

I 20

Fig. 2. Heat denaturation curves (A0 method) obtained on intact human lymphocytes fixed after 1 h in culture. Control cells ( x --- x) and cells stimluated by the addition of PHA ( 0 - 0). Curves in fig. 2 (a) show the increase in a (Fs90/F680) with increasing temperatures. In fig. 2 (b) and 2 (c) the data in fig. 2 (a) have been used to calculate the increase in amount of single-stranded nucleic acid as the temperatures increase (see text). In Fig. 2 (b) an an-value of 0.199 has been used for both nonstimulated and PHA-stimulated cells. In fig. 2 (c) an ar,value of 0.201 for non-stimulated and of 0.169 for PHAstimulated cells were used. (Means and S.E.M., n=lO.) Abscissa: Temperature, “C; ordinate: (a) a; (b, c) % singlestranded regions in DNP. 50

60

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90

1W

b

turation than that of normal lymphocytes as can be seen from fig. 2 (a). The transition from double- to single-stranded structure in the “melting” DNA results in an increase in the ratio between the fluorescence intensities at 590 and 530 nm (a) of A0 stained cells. It is evident that the increase in cc in stimulated cells takes place at lower temperatures than in control cells. Using eq. (4) the cc-values could be recalculated into percentage single-stranded regions in the DNP complex. Using an tc5 of 3.313 and an aD of 0.199 (mean value of tc for 450 stimulated and 450 unstimulated control lymphocytes containing negligible amounts of RNA, see Material and Methods) the result shown in fig. 2 (b) was obtained. Both curves start from approx. the same percentage of single-stranded regions and Exptl Cell Res 55

reach the same single-stranded state (N 90 %) at the highest temperatures. However, the transition took place earlier in the stimulated than in the control cells. The temperature at which 50 % single-stranded regions are obtained is lower in the stimulated cells than in the control cells. Similar transition curves are obtained if instead of a mean value (fig. 2 (b)), a,-values corrected for the number of A0 binding sites in DNP and for the presence of RNA are used. This type of correction has been performed in fig. 2 (c). The PHA stimulated cells then have a higher percentage of single-stranded regions at the starting point but both PHA stimulated and normal cells reach approx. the same degree of single-strandedness at the higher temperatures. The midpoints for transition lie further apart for the corrected than for the uncorrected curves.

Cytochemical

22

50

60

70

80

90

characterization

of DNP

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la0

cl

I Fig. 3. Changes in the melting profile (A0 method) of hen erythrocyte nuclei induced by activation in heterokaryons (fig. 3 (a)) or by washing and incubation in the absence of serum (fig. 3 (b)). Fig. 3 (a) illustrates the melting profiles obtained on activated hen erythrocyte nuclei isolated from HeLa/hen erythrocyte heterokaryons 41 (B.J and 47 h (B,) after cell fusion. The lower curve (C,) shows the melting profile of erythrocyte nuclei isolated by the same isolation method from normal untreated cells. In fig. 3 (b) are shown 2 experiments where erythrocyte ghost nuclei were “activated” by the removal of serum proteins (A, and AJ together with control nuclei (C, and C,). In fig. 3 (c) the x-values of curves Al, B, and C, obtained by microfluorimetry after A0 staining have been used to calculate the degree of singlestrandedness. (Means and s.E.M., n = 10.) Abscissa: Temperature, “C; ordinate: (a, b) cc;(c) % single-stranded regions in DNP.

b

22

:., 50

60

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C

Reactivation of chick erythrocyte nuclei

In birds the mature erythrocytes retain their cell nuclei in an inactive form. The chromatin of these nuclei is condensed into dense heterochromatin-like clumps. The synthesis of RNA and DNA as well as of protein has stopped. Recently Harris [9] demonstrated that hen erythrocyte nuclei can be reactivated by fusing the erythrocytes with HeLa cells to form heterokaryons. The red cell nuclei in these heterokaryons begin to synthesize large quantities of RNA at the same time as they begin to increase in volume and dry mass. At an early stage of this reactivation process the DNP complex showed marked changes in its cytochemical properties. The ability to bind A0 and ethidium increased markedly already long before any in-

crease in DNA quantity took place, and at the same time the Feulgen reactivity of the DNP changed [2]. The sensitivity of hen erythrocyte DNP to heat denaturation also underwent profound changes during this activation process. Erythrocyte nuclei re-isolated from heterokaryons 41 and 47 h after cell fusion showed melting profiles which were strikingly different from those of nuclei isolated from normal erythrocytes. As shown in fig. 3 (a) the a-value begins to increase at a lower temperature and reaches a much higher end value in the reactivated nuclei. DNP changes similar to those found when hen erythrocyte nuclei are reactivated in heterokaryons can also be induced in the nuclei of normal erythrocytes and erythrocyte ghosts by Exptl Cell Res 55

220

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washing and incubation in salt solutions or tissue culture media devoid of serum proteins [17]. In this model chromatin changes can be induced at low temperatures and in the presence of enzyme inhibitors and thus appear to be of a nonenzymatic nature. The chromatin changes induced in this model system are reflected not only in an increased binding of A0 and ethidium bromide and altered Feulgen reactivity but also in changes in the melting profile of the DNP (fig. 3 (b)). The differences recorded between “activated” and control nuclei in this system are similar or identical to those recorded in the cell hybridization experiments (fig. 3 (u)). In both cases the control nuclei start to melt at higher temperatures and they also seem to melt less completely than the “activated” nuclei. In all the calculations of the degree of singlestrandedness performed on erythrocyte DNP and HeLa DNP (see below) an as-value of 3.313 (see Material and Methods) and an a,-value of 0.226 were used. This &,-value is the mean of the a-values for approx. one thousand erythrocyte nuclei of all the categories included in fig. 3 (c). No significant difference in a was found for the 3 types of erythrocyte nuclei in spite of their very different AO-binding capacity. The same n,-value was used also for the HeLa nuclei since the GC found for untreated HeLa nuclei was close to that found for the erythrocyte nuclei (0.25). The importance of the a,-value for the calculation of the degree of single-strandedness will be discussed further below. In all the experiments described so far, the cells have been acetylated prior to A0 staining. The acetylation step, a part of the standard staining procedure, is necessary for the discrimination between DNA-A0 and RNA-A0 complexes [15, 161. To investigate the influence of the acetylation step on the melting profiles erythrocyte nuclei were stained also without acetylation. This comparison between acetylated and non-acetylated erythrocyte nuclei is possible since no RNA is present. The non-acetylated nuclei showed an increased OLat approx. the same temperature as the acetylated ones and showed Exptl Cell Res 5.5

140

130

120

110

T

22

,i,

50

60

70

80

90

loo

Fig. 4. Melting profiles obtained by UV microspectro-

photometry on control A-.-A and “activated” erythrocyte nuclei A-A . “Activation” was achieved by washing and incubation in the absence of serum proteins. (Means and s.E.M., n=20.) Abscissa: Temperature, “C; ordinate: Etot 265 nm. the same differences between activated and control nuclei as shown in fig. 3 (b). There may have been a slight tendency, however, for the curves representing the non-acetylated nuclei to be skewed towards higher temperatures. Finally the degree of melting was measured by means of UV absorption measurements on erythrocyte nuclei. This was done in order to check that the altered A0 staining properties of heated nuclei are in fact due to a change in the degree of single-strandedness. In the present experiments the erythrocyte nuclei were made to adhere to quartz slides which were then subjected to heat denaturation by the same heating technique as was used in A0 experiments. Ghost nuclei “activated” by washing and incubation in the absence of serum were found to melt at a lower temperature than did the control nuclei (fig. 4). Both melting curves start at the same absorbancy values and end at approx. the same values. The midpoints of transition from the single to the doublestranded state were approx. lO-15°C apart. In both the experiment in fig. 4 and in a duplicate experiment the same result was obtained. In both cases it was possible to “melt” the “activated” and control DNP to a similar extent. The increase in UV absorbancy recorded in these cytochemical experiments (approx. 33 %) was the same as that observed in parallel bio-

Cytochemical

characterization

of D NP

221

both from measurements of small areas of nucleoplasm free from visible nucleoli and from measurements of whole HeLa nuclei. As can be seen from fig. 5 (a, b) the same melting profile is obtained by both types of measurements. This suggests that the presence of considerable quantities of RNA in the nucleoli does not noticeably affect the measurement of cc. The melting profile of HeLa DNP is similar to that of “activated” erythrocyte DNP.

60

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-

60 -

60-

22

SO

60

70

60

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b

Fig. 5. Melting profiles obtained by A0 microfluorimetry on HeLa nuclei isolated from heterokaryons. Measurements were performed on points in the nucleoplasm where no nucleoli could be seen ( 0 - - - 0 ) as well as on whole nuclei ( 0 - 0). Fig. 5 (a) shows the temperature dependence of the a-value and in fig. 5 (b) these data have been used for calculations of the degree of single-strandedness. (Means and s.E.M., n = 10.) Abscissa: (a, b) Temperature, “C; ordinate: (a) a, (b) % single-stranded regions in DNP.

chemical experiments [17] for hen erythrocyte DNA (3 l-33 %) and erythrocyte DNP (3 l-34 %) isolated by Zubay & Doty’s [18] method. HeLa cell nuclei

For comparison, melting profiles were also recorded on HeLa nuclei isolated from heterokaryons. These nuclei are very active with respect to nucleic acid synthesis and contain one or more big nucleoli. The a-values were obtained

DISCUSSION In comparison with current biochemical methods used in studying the melting profile of isolated DNP the present methods offer major advantages. The physicochemical state of the DNP is very sensitive to alterations in the ionic environment and to dilution [13]. Since the isolation of DNP involves extensive washing in salt solutions containing EDTA [18] and the conventional optical methods for studying changes in the UV absorption of DNP make it necessary to work with very dilute solutions, the properties recorded for isolated DNP may be only remotely relevant with respect to the situation which prevails in the cell nucleus. The cytochemical methods described here make it possible to study the state of DNP in individual cell nuclei at the biological concentration levels and also avoids the risks involved in forcibly removing metal ions bound to DNP. The present work has provided two simple methods for obtaining “melting profiles” on the deoxyribonucleoprotein of intact cells or cell nuclei. In both cases the cells/nuclei are heated in the presence of formaldehyde which prevents the renaturation of the DNP complex as the temperature is lowered. After cooling the individual nuclei are analyzed for the degree of single-strandedness induced by the heat treatment, either by A0 microfluorimetry or by measuring the increase in UV absorption by microspectrophotometry. Previously Chamberlain & Walker [5] have performed heat denaturation experiments on spermatozoa using UV microspectrophotometry Exptl Cell Res 55

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Table 1. Sensitivity of A0 and UV methods in detecting an increase in singlestrandednessof DNA Increase in the degree of singlestrandedness (%) o- 10 o- 20 o- 50 O-100

i

d.5

lb

Fig. 6. Theoretical between Fss,,/F6J0(u) age single-stranded dbscissa: a; ordinate:

1:5

i0

is

i.0

curve describing the relationship in the A0 method and the percent-

regions in deoxyribonucleoprotein. % single-stranded

regions in DNP.

with a microscope stage which could be heated at the same time as the measurements were performed. The techniques described here offer the advantage that no special microscope stage for the heating of the cells is required. Furthermore detection of single-stranded nucleic acid by A0 microfluorimetry is in several respects a more sensitive method than UV microspectrophotometry. Analysis of the degree of single-strandedness by AO-microfluorimetry is based on measurements of the green fluorescence (F5;,,,) of the double-stranded AO-nucleic acid complexes relative to the red fluorescence (FSno) of singlestranded AO-nucleic acid complexes. The ratio F590/F530(CY)increases as the temperature is increased and the double-stranded nucleic acid undergoes the transition to single-strandedness. The theoretical relationship between the increase in a and the increase in single-strandedness is illustrated by fig. 6. From this curve and from table 1 it is obvious that the A0 method is more sensitive than the UV method in detecting an increase in singlestranded regions particularly at the end of the melting process, The sensitivity of both depends, however, on the intercellular variation of the measured values for c( and Ezss which were found to vary with the cell material used in this study. In Exptl Cell Res 55

Increase in a (%I 5 15 15::

Increase ina Em (%I 3 7 ::

a Assuming a linear relationship between the increment in UV absorption and the increment in single-strandedness.

lymphocytes with a coefficient of variation of 20% for CI and of 10% for EzG5both methods are equally sensitive to detect an increase in single-stranded regions from O-l 5 %. In erythrocytes with coefficients of variation of 20% (OZ) and 3 % (E2& respectively, the sensitivity of the UV method is greater in detecting the same increase in single-strandedness (from O-l 5 %). For both erythrocytes and lymphocytes the A0 method soon becomes the most sensitive technique for detecting further melting. Furthermore, the A0 method offers a great advantage being less dependent on intercellular variation in the amount of DNA, since the degree of single-strandedness is determined from the ratio of the fluorescence intensities at two wavelengths. As demonstrated by the melting profiles obtained on HeLa nuclei it is even possible to obtain melting profiles on parts of cell nuclei by this method. In both the cell hybridization experiments [2] and in those experiments where erythrocyte nuclei are “activated” by the removal of serum proteins [17] it was very difficult to obtain a complete denaturation of control nuclei as evidenced by A0 microfluorimetry. The “activated” nuclei were, however, easily denaturated to the same extent as HeLa nuclei. At the same time UV melting profiles (fig. 4) showed that the control nuclei were denaturated to the same extent as the activated erythrocyte nuclei. This observation makes it necessary to point out that

Cytochemical

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J

Fig. 7. Calculation of a series of curves describing the temperature dependent increase in single-strandedness for normal untreated human lymphocytes. The calculations were based on a series of different a,-values (0.140.30) in order to illustrate how errors in the determitiation of a, may affect the appearance of melting profiles derived from fluorescence measurements. Abscissa: Temperature, “C; ordinate: % single-stranded regions in DNP.

the two techniques for determining the degree of single-strandedness do not necessarily measure precisely the same physical phenomena. In making the calculations of the degree of single-strandedness from measurements of A0 fluorescence it is necessary to determine experimentally the value for CQ,. As pointed out (in methods) a, varies with the number of binding sites in DNP accessible to AO. (Extreme values: 0.14-0.29.) Corrections for @,-variations can be made with the aid of eq. (5). The need for such a correction is not very great, however, since, as shown in fig. 7 a decrease in CI~ from 0.24 to 0.18 only lowers the temperature at which 50 % single-strandedness is achieved by approx. 5°C. If the correction is made, however, a higher initial level of single-strandedness is found in activated, non-heated lymphocytes. This may be due to the true presence of singlestranded regions in DNA which, in turn, may be due to an increased sensitivity of the activated DNP complex to the formaldehyde treatment. It can also be due to the presence of RNA. Though it is difficult to distinguish which of these interpretations is the more accurate, the

characterization

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experimental data on HeLa cell nuclei suggest that RNA is of relatively little importance for the melting profile obtained from acetylated nuclei. In this case almost identical curves were obtained in the two sets of measurements, one obtained on nucleoplasmic areas only and one obtained on entire nuclei containing prominent nucleoli. Another difficulty which militates against corrections for RNA is that though RNA may be present in the untreated cell at the lower temperatures studied it may be selectively lost at the higher temperatures. That some loss of nucleic acid does occur at the higher temperature is stressed by some of the melting profiles obtained. Thus, at the highest temperatures a decrease in cc-values was observed in activated lymphocytes (fig. 2a) and erythrocyte nuclei (fig. 3 a) as well as in HeLa nuclei (fig. 5 a). In these cases a preferential loss of single-stranded DNA may have taken place. Such losses, are, however of very minor importance for the interpretation of the melting profiles obtained. A number of data suggest that the decrease in thermal stability of cellular DNP complexes observed after growth stimulation is due to a weakened interaction between the DNA and the protein parts of the DNP complex [2, 10, 11, 16,171. The processes leading to this altered DNAprotein binding are still obscure. The results obtained on hen erythrocyte nuclei [17] suggest that the DNP changes which occur during “activation” are due to non-enzymatic, physicochemical changes. In the lymphocyte system more subtle mechanisms have been suggested [l, 141 e.g. enzymatic acetylation of histones within the DNP complex. Whichever explanation will be found valid it seems likely that these DNP changes reflect an early step in the activation of a large number of genes. This work was supported by grants from the Swedish Natural Science Research Council, The Swedish Medical Science Research Council, the Swedish Cancer Society and Reservationsanslaget, Karolinska Institutet. The development of the biophysical instruments used in these investigations was supported by grants from the Swedish Medical Science Research Council and the Wallenberg Foundation to Professor T. Caspersson. The computer work was supported by grants from the Data Processing Committee at Karolinska Institutet. Exptl Cell Res 55

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REFERENCES 1. Allfrey, V G, Faulkner, R & Mirsky, A E, Proc natl acad sci US 51 (1964) 786. 2. Bolund, L, Ringertz, N R & Harris, H, J cell sci 4 (1969)

71.

3. Caspersson, T & Lomakka, G, Instrumentations in biochemistry: Biochem sot symp (ed T W Godwin) vol. 26, p. 25. Academic Press, New York (1966). 4. Caspersson, T, Lomakka, G & Rigler, R, Jr, Acta histochem, suppl. VI (1965) 123. 5. Chamberlain, P J & Walker, P M B, J mol biol 11 (1965) 1. 6. Cooper, H L & Rubin, A D, Blood 25 (1965) 1014. 7. Fischer, H W & Harris, H, Proc roy sot B 156 (1962) 521.

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8. Harris. H. Watkins. J F. Ford. C E & Schoefl, G I. J cell sci 1 (1966) 1.’ . 9. Harris. H. J cell sci 2 (1967) 23. 10. Killander; D & Rigler,‘R, Exptl cell res 39 (1965) 701. 11. - Ibid. 54 (1969) 163. 12. Lomakka, G, A&a histochem, Suppl. VI (1965) 47. 13. Luzzati. V & Nicolaieff. A. J mol biol 7 (1963) 142. 14. Pogo, B G T, Allfrey, V G.& Mirsky, A E, Prdcnatl acad sci US 55 (1966) 805. 15. Rigler, R, Acta physiol Stand 67, suppl. 267 (1966). 16. Rialer. R & Killander. D, Exptl cell res 54 (1969) 171. 17. Ringertz, N R & Bolund, L,-Exptl cell res 55 (1969) 205.

18. Zubay, G & Doty, P, J mol biol 1 (1959) 1. Received August 16, 1968