Actinomycin D Binding Research Articles

Spermine Attenuates the Action of the DNA Intercalator, Actinomycin D, on DNA Binding and the Inhibition of Transcription and DNA Replication

The anticancer activity of DNA intercalators is related to their ability to intercalate into the DNA duplex with high affinity, thereby interfering with DNA replication and transcription. Polyamines (spermine in particular) are almost exclusively bound to nucleic acids and are involved in many cellular processes that require nucleic acids. Until now, the effects of polyamines on DNA intercalator activities have remained unclear because intercalation is the most important mechanism employed by DNA-binding drugs. Herein, using actinomycin D (ACTD) as a model, we have attempted to elucidate the effects of spermine on the action of ACTD, including its DNA-binding ability, RNA and DNA polymerase interference, and its role in the transcription and replication inhibition of ACTD within cells. We found that spermine interfered with the binding and stabilization of ACTD to DNA. The presence of increasing concentrations of spermine enhanced the transcriptional and replication activities of RNA and DNA polymerases, respectively, in vitro treated with ActD. Moreover, a decrease in intracellular polyamine concentrations stimulated by methylglyoxal-bis(guanylhydrazone) (MGBG) enhanced the ACTD-induced inhibition of c-myc transcription and DNA replication in several cancer cell lines. The results indicated that spermine attenuates ACTD binding to DNA and its inhibition of transcription and DNA replication both in vitro and within cells. Finally, a synergistic antiproliferative effect of MGBG and ACTD was observed in a cell viability assay. Our findings will be of significant relevance to future developments in combination with cancer therapy by enhancing the anticancer activity of DNA interactors through polyamine depletion.

The Influence of Solutes on the Enthalpy/Entropy Change of the Actinomycin D Binding to DNA: Hydration, Energy Compensation and Long-Range Deformation on DNA

The effects of the changes in the temperature and in the water chemical potential on the energetic of the actinomycin D (ACTD) interaction with natural DNA are studied. At reduced water chemical potential, induced by the addition of neutral solute (sucrose), the ACTD-to-DNA binding isotherms show that the drug accesses two types of binding sites: strong and weak. The binding constants to the stronger sites are sensitive to changes in the temperature and in the water chemical potential, while the weak sites are practically insensitive to these changes. The van't Hoff analyses of the binding in different water chemical potential shows that the binding process to the more specific sites is endothermic in phosphate buffer (ΔH(vH) ∼ 1 kcal/mol) and becomes exothermic when the water chemical potential decreases (ΔH(vH) = -11 kcal/mol in sucrose 30%). The number of water molecules released on the binding to the stronger sites, obtained from the slopes of linkage plots in different temperatures, increases with the decrease in the temperature. Ring closure reactions in the presence of neutral solutes have shown that the reduction in the water activity induces DNA unwinding. It was observed that both reduced water chemical potential and small ratios of daunomycin bound per base pairs have the same effects on the ACTD binding isotherms and consequently on the binding thermodynamic parameters. The results presented indicate that the ACTD binding to the recognition site is enthalpycally unfavorable, which should be compensated by the deformation in the DNA. This compensation would probably be the origin of the synergism observed for these two drugs.

Kinetic Discrimination of Sequence-Specific DNA−Drug Binding Measured by Surface Plasmon Resonance Imaging and Comparison to Solution-Phase Measurements

We demonstrate the use of surface plasmon resonance (SPR) imaging for direct detection of small-molecule binding to surface-bound DNA probes. Using a carefully designed array surface, we quantitatively discriminate between the interactions of a model drug with different immobilized DNA binding sites. Specifically, we measure the association and dissociation intercalation rates of actinomycin-D (ACTD) to and from double-stranded 5'-TGCT-3' and 5'-GGCA-3' binding sites. The rates measured provide mechanistic information about the DNA-ACTD interaction; ACTD initially binds nonspecifically to DNA but exerts its activity by dissociating slowly from strong affinity sites. We observe a slow dissociation time of kd-1 = 3300 +/- 100 s for ACTD bound to the strong affinity site 5'-TGCT-3' and a much faster dissociation time (210 +/- 15 s) for ACTD bound weakly to the site 5'-GGCA-3'. These dissociation rates, which differ by an order of magnitude, determine the binding affinity for each site (8.8 x 10(6) and 1.0 x 10(6) M(-1), respectively). We assess the effect the surface environment has on these biosensor measurements by determining kinetic and thermodynamic constants for the same DNA-ACTD interactions in solution. The surface suppresses binding affinities approximately 4-fold for both binding sites. This suppression suggests a barrier to DNA-drug association; ACTD binding to duplex DNA is approximately 100 times slower on the surface than in solution.

The nature of actinomycin D binding to d(AACCAXYG) sequence motifs.

Earlier studies by others had indicated that actinomycin D (ACTD) binds well to d(AACCATAG) and the end sequence TAG-3' is essential for its strong binding. In an effort to verify these assertions and to uncover other possible strong ACTD binding sequences as well as to elucidate the nature of their binding, systematic studies have been carried out with oligomers of d(AACCAXYG) sequence motifs, where X and Y can be any DNA base. The results indicate that in addition to TAG-3', oligomers ending with XAG-3' and XCG-3' all provide binding constants > or =1 x 10(7) M(-1) and even sequences ending with XTG-3' and XGG-3' exhibit binding affinities in the range 1-8 x 10(6) M(-1). The nature of the strong ACTD affinity of the sequences d(A1A2C3C4A5X6Y7G8) was delineated via comparative binding studies of d(AACCAAAG), d(AGCCAAAG) and their base substituted derivatives. Two binding modes are proposed to coexist, with the major component consisting of the 3'-terminus G base folding back to base pair with C4 and the ACTD inserting at A2C3C4 by looping out the C3 while both faces of the chromophore are stacked by A and G bases, respectively. The minor mode is for the G to base pair with C3 and to have the same A/chromophore/G stacking but without a looped out base. These assertions are supported by induced circular dichroic and fluorescence spectral measurements.

Binding of Actinomycin D to Single-Stranded DNA of Sequence Motifs d(TGTCT nG) and d(TGT nGTCT)

Our recent binding studies with oligomers derived from base replacements on d(CGTCGTCG) had led to the finding that actinomycin D (ACTD) binds strongly to d(TGTCATTG) of apparent single-stranded conformation without GpC sequence. A fold-back binding model was speculated in which the planar phenoxazone inserts at the GTC site with a loop-out T base whereas the G base at the 3′-terminus folds back to form a basepair with the internal C and stacks on the opposite face of the chromophore. To provide a more concrete support for such a model, ACTD equilibrium binding studies were carried out and the results are reported herein on oligomers of sequence motifs d(TGTCT nG) and d(TGT nGTC). These oligomers are not expected to form dimeric duplexes and contain no canonical GpC sequences. It was found that ACTD binds strongly to d(TGTC TTTTG), d(TG TTTTGTC), and d(TG TTTTTGTC), all exhibiting 1:1 drug/strand binding stoichiometry. The fold-back binding model with displaced T base is further supported by the finding that appending TC and TCA at the 3′-terminus of d(TGTC TTTTG) results in oligomers that exhibit enhanced ACTD affinities, consequence of the added basepairing to facilitate the hairpin formation of d(TGTC TTTTG TC) and d(TGTC TTTTG TCA) in stabilizing the GTC/GTC binding site for juxtaposing the two G bases for easy stacking on both faces of the phenoxazone chromophore. Further support comes from the observation of considerable reduction in ACTD affinity when GTC is replaced by GTTC in an oligomer, in line with the reasoning that displacing two T bases to form a bulge for ACTD binding is more difficult than displacing a single base. Based on the elucidated binding principle of phenoxazone ring requiring its opposite faces to be stacked by the 3′-sides of two G bases for tight ACTD binding, several oligonucleotide sequences have been designed and found to bind well.

Actinomycin D binds to d(TGTCATG) with 2:1 drug to duplex stoichiometry.

Despite the apparent single-stranded conformation and the absence of a GpC site, d(TGTCATTG) has been found to bind strongly to actinomycin D (ACTD) with 1:1 drug to strand binding stoichiometry. A hairpin binding model was speculated in which the planar phenoxazone chromophore inserts at the GTC site by pushing out the T-base while the terminal G folds back to form a G small middle dotC base pair so that the 3'-sides of both G-bases stack on the opposite faces of the phenoxazone plane. However, it was also suggested that a slipped duplex binding with similar binding principle could also be operative at higher DNA concentrations. To support such a contention, ACTD binding studies were made with d(TGTCATG) and related oligomers. This heptamer differs from the parent octamer d(TGTCATTG) by a mere removal of a T-base which should result in an enhancement of dimeric duplex formation and a concomitant reduction in monomeric hairpin contribution. It was found that ACTD binds well to d(TGTCATG) with 1 drug to 1 strand (or 2 drugs to 1 duplex) binding stoichiometry. These results are consistent with a slipped duplex binding model in which a dimeric duplex is formed at the self-complementary CATG tetranucleotide sequence with extruding TGT ends. Two drug molecules are bound at both ends of the duplex by pushing out the T-bases of GTC's so that the opposite faces of each phenoxazone are stacked by the 3'-sides of the two G-bases on opposite strands. Such a model provides a ready explanation for the observed enhancement in ACTD binding to d(TGTCATGTC) and d(TGTCATGTCA), where additional base pairs at the ends will stabilize GTC/GTC binding sites, and to d(TGTCAATTG) in which two additional base pairs facilitate the slipped-duplex formation. The observed ACTD affinity reductions for oligomers containing GTTC instead of GTC are also consistent with the T-base displacement model. These findings greatly expand the repertoire of ACTD binding to DNA and may have important implications on understanding the transcription inhibitory activities of this drug.

Non stacking binding of 7-amino-actinomycin D and actinomycin D to DNA and model nucleotide systems in solutions

Mechanism of actinomycin D (AMD) and 7-aminoactinomycin D (7AAMD) interaction with DNA and model nucleotide compounds was studied by absorption and fluorescence spectroscopy (steady-state, phase-modulation, and polarization). It was shown that complex formation does not result in energy transfer from photoexcited nucleotides to phenoxazone chromophore of 7AAMD that indicates the absence of stacking-like intercalation. This fact is fundamentally important to explain the biological effect of actinomycin on cells. It was revealed a fundamental difference in the complex-forming properties of AMD and 7AAMD. Thus AMD is capable of binding to guanine micelles to destroy them. 7AAMD forms complexes neither guanine micelles nor polyguanilic acid. 7AAMD binding sites on DNA can differ substantially from AMD binding sites. However, a strong competition is observed between AMD and 7AAMD for binding site in oligonucleotide HP1 used as DNA hairpin model. The efficient diameters of 7AAMD-HP1 complex and free 7AAMD were determined using the Levshin-Perren equation.

Actinomycin D Binds Strongly to d(CGACGACG) and d(CGTCGTCG)

Earlier calorimetric studies had indicated that despite the absence of a GpC sequence, the self-complementary octamer d(CGTCGACG) binds strongly to actinomycin D (ACTD) with high cooperativity and a 2:1 drug/duplex ratio. A subsequent optical spectral study with related oligomers led us to suggest that ACTD may likely stack at the G · C basepairs of the duplex termini. New findings are reported herein to indicate that despite the lack of complete self-complementarity, oligomers of d(CGXCGXCG) [X = A or T] motif exhibit unusually strong ACTD affinities with binding constants of roughly 2 × 10 7 M −1 and binding densities of 1 drug molecule per strand. The ACTD binding affinity for the corresponding heteroduplex obtained by annealing these two oligomers is, however, considerably reduced. Although spectroscopic results with related oligomers obtained by removing, replacing, or appending bases at the termini appear to be consistent with the end-stacking model, capillary electrophoretic (CE) evidence provides additional insights into the binding mode. CE experiments with the self-complementary oligomers d(CGAGCTCG) and d(CGTCGACG) revealed contrasting migration patterns in the presence of ACTD, with mobility retardation and acceleration exhibited by the GpC- and non-GpC-containing octamers, respectively, whereas the X/X-mismatched d(CGXCGXCG) experienced retardation. These results, along with those of related oligomers, suggest that ACTD may in fact stack at the duplex stem end of a monomeric hairpin or at the 3′-end of dG as a single strand. The seemingly cooperative ACTD binding and the curved Scatchard plot for the self-complementary d(CGTCGACG) may thus be attributed to the drug-induced duplex denaturation resulting from strong binding to single strands of d(CGXCGYCG) motif. Detailed structural information on the ACTD-DNA complexes, however, must await further NMR investigations.

Binding of actinomycin D to DNA oligomers of CXG trinucleotide repeats.

Actinomycin D (ACTD) binding propensities of DNA with CXG trinucleotide repeats were investigated using oligomers of the form d[AT(CXG)n = 2-4AT] and their corresponding heteroduplexes, where X = A, C, G, or T. These oligonucleotides contain -CXGCXG-, -CXGCXGCXG-, and -CXGCXGCXGCXG- units that can form homoduplexes containing one, two, and three GpC binding sites, respectively, with flanking X/X mismatches. The corresponding heteroduplexes contain these same sites with flanking Watson-Crick base pairs. It was found that oligomers with X = G exhibit weak ACTD affinities whereas those with X not equal to G and n = 3 exhibit unusually strong ACTD binding affinities with binding constants ranging from 2.3 x 10(7) to 3.3 x 10(7) M-1 and binding densities of approximately 1 drug molecule/strand (or 2/duplex). These binding affinities are considerably higher than those of their shorter and longer counterparts and are about 2- and 10-fold stronger than the corresponding CAG.CTG and CGG.CCG heteroduplexes, respectively. The CTG-containing oligomer d[AT(CTG)3AT] stands out as unique in having its ACTD dissociation kinetics being dominated by a strikingly slow process with a characteristic time of 205 min at 20 degrees C, which is 100-fold slower than d[AT(CAG)3AT], nearly 10-fold slower than the corresponding heteroduplex, and considerably slower than d[AT(CTG)2AT] (63 min) and d[AT(CTG)4AT] (16 min). The faster dissociation rate of the n = 4 oligomer compared to its n = 2 counterpart is in apparent contrast with the observed 10-fold stronger ACTD binding affinity of the former. It was also found that d[AT(CCG)3AT] exhibits the slowest dissociation rate of the CGG/CCG series, being more than an order of magnitude slower than that of its heteroduplex (tau slow of 43 vs 2 min). The finding that a homoduplex d[AT-CXG-CXG-CXG-AT]2 can bind two ACTD molecules tightly is significant since it was thought unlikely for two consecutive GpC sites separated by a single T/T mismatch to do so.

Observation of an anomalously slow association kinetics in the binding of actinomycin D to d(CATGGCCATG)

An unusually slow association process which accounts for the bulk of its dichroic changes at 293 nm is observed for d(CAT-GGCC-ATG) when it reacts with actinomycin D (ACTD). This is in contrast to an order of magnitude faster association rates exhibited by oligomers containing a self-complementary tetranucleotide ACTD binding sequence (-TGCA-, -AGCT-, or -CGCG-). The number of drug molecules bound and the melting temperature increase upon ACTD binding are significantly higher for d(CAT-GGCC-ATG) than for other decamers studied. Temperature-dependent spectral measurements of this oligomer in the presence of ACTD suggest additional drug binding prior to denaturation. This particular decamer sequence may be unique, as other decamers containing central -GGCC- sequence and even those differing only by the terminal bases such as d(TAT-GGCC-ATA) and d(GAT-GGCC-ATC) are only weakly binding and do not exhibit such anomalously slow ACTD association kinetics, whereas the dodecamer d(CCAT-GGCC-ATGG) does. CD evidence indicates that, in contrast to the other -GGCC- containing oligomers, both d(CCAT-GGCC-ATGG) and its parent decamer exhibit nonstandard B conformations. The observed slow association kinetics and its interesting D/P dependence are rationalized in terms of a model in which the ACTD molecules initially end-stack and distort the oligomer duplex to a favorable ACTD-binding conformation so that intercalation at the central G-C sequence can occur via DNA breathing.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding specificities of actinomycin D to self-complementary tetranucleotide sequences -XGCY-.

Binding of actinomycin D (ACTD) to self-complementary decamers d(ATA-XGCY-TAT), where XGCY = TGCA, AGCT, CGCG, and GGCC, has been investigated by equilibrium, kinetic, and thermal denaturation studies. The results indicate that despite the presence of a GC dinucleotide sequence, -GGCC- exhibits a much weaker binding affinity toward ACTD than the other three tetranucleotide sequences. Binding constants estimated from Scatchard plots indicate that binding to the -GGCC- site is at least an order of magnitude weaker than binding to -CGCG- and -AGCT-, which in turn is only slightly weaker than binding to the -TGCA- sequence. At 18.5 degrees C and 1% SDS, ACTD dissociates from d-(ATA-TGCA-TAT) with a slow characteristic time of 3300 s, roughly 4 times slower than dissociation from those containing -CGCG- and -AGCT- sequences and more than 2 orders of magnitude slower than that from -GGCC-. An 18.2 degrees C increase in the melting temperature is observed for the -TGCA-containing decamer upon binding of the ACTD, whereas increases of 10.3, 6.7, and 2.0 degrees C are observed for the -CGCG-, -AGCT-, and -GGCC-containing decamers, respectively. The effects observed by changing the adjacent base pair (sequence) may occur as a result of differential stacking and/or peptide ring-DNA groove interactions. Base sequence alterations adjacent to the ACTD binding site may result in differences in the minor groove environment and/or subtle conformational alterations at the intercalation site.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutational Extinction Induced by Sequential X Irradiation and Actinomycin D Treatments in Chinese Hamster Ovary Cells

The interactions of sequential X irradiation and actinomycin D (AMD) treatments for mutagenesis to 6-thioguanine resistance were investigated in CHO cells. Cells were exposed to single doses of X rays followed immediately by 1-h treatments with 0.1 or 1 microgram/ml AMD. X Rays alone induced mutagenesis which increased monotonically with dose to at least 8 Gy. AMD-treated control cultures showed slight to moderate cytotoxicity and little induced mutation. X Rays followed by AMD treatment produced bell-shaped mutagenesis dose-response curves with maximal mutation at approximately 5 or 4 Gy for 0.1 or 1.0 microgram/ml AMD, respectively. Induced mutation frequencies then fell to a negligible level at fractional survival levels below 0.10 for either combination treatment. Application of a stochastic Poisson distribution model to these data led to the prediction that two possible components govern induced mutation frequencies. First, X ray +AMD induced mutations may be depleted progressively with dose from the surviving populations by selective lethality, which we term mutational extinction. Second, X ray +AMD treatments were calculated to induce potentially much greater than additive mutagenesis. However, due to the overriding mutational extinction effect, most of these mutations are not recovered as viable colonies. These studies suggest that AMD binding to DNA immediately following irradiation may cause considerably enhanced mutagenic and often lethal DNA damage, and that mutational extinction may occur because these types of damage are statistically correlated in a sensitive subpopulation of exponentially growing CHO cells.

Effect of CCC on the Cell Cycle and on Actinomycin D Binding in Nuclei of Interphase Periods in Antheridial Filaments of Chara vulgaris L.

The influence of CCC on some processes during successive cell cycles in antheridial filaments of Chara vulgaris L. was -studied using [ 3 H]-thymidine in order to establish the cell cycle duration and [ 3 H]-actinomycin D (AMD) binding as an indicator of cellular genetic activity. The retardant at 10 − 3 M concentration caused, after 24 h incubation, an increment in AMD binding in the S phase and a reduction of radioactivity in the G 2 phase, as well as a considerable prolongation of the cell cycle duration. CCC inhibited cell elongation mainly at the end of G 2 phase, thus changing the rate of cell growth. No significant effect of CCC on the sizes of the mitotic cells was detected. It is suggested that CCC causes an unbalanced RNA and protein synthesis leading to the reduction of the rate of cell growth and to the prolongation of cell cycle durations. This influence is probably due to the effect of CCC on the permeability of membranes.

Synthesis and some properties and antitumor effects of the actinomycin lactam analog, (di(1-L-alpha, beta-diaminopropionic))actinomycin D1.

A lactam analog of actinomycin D (AMD) has been synthesized as a potential antitumor chemotherapeutic agent. Both L-threonine residues were replaced by L-alpha,beta-diaminopropionic acid. Starting with Nalpha-benzyloxycarbonyl-Nbeta-tert-butyloxycarbonyl-L-alpha,beta-diaminopropionic acid methyl ester hydrochloride the linear intermediate Nalpha-benzyloxycarbonyl-Nbeta-(tert-butyloxycarbonylsarcosyl-L-N-methylvalyl)-L-alpha,beta-diaminopropionyl-D-valyl-L-proline p-nitrophenyl ester was prepared by conventional methods of peptide synthesis in solution. Selective cleavage of the Nbeta-tert-butyloxycarbonyl group and lactam formation afforded the desired cyclic pentapeptide derivative. The chromophore precursor, Nalpha-(2-nitro-3-benzyloxy-4-methylbenzoyl) substituent, was introduced via its symmetric anhydride. Catalytic reduction followed by ferricyanide-mediated phenoxazinone formation provided the lactam analog, [di(1'-L-alpha,beta-diaminopionic acid)]actinomycin D ([Dpr1]2-AMD). Its binding to natural and synthetic DNA and that of an analogous L-threo-alpha,beta-diaminobutyric acid containing lactam ([Dbu1]2-AMD) compared with the binding of AMD (in which the peptides are in lactone form) was studied by circular dichroic (CD) spectroscopy. The visible and uv CD spectra of free AMD differed from those of the free lactam analogs, indicating that the asymmetric environment of the pentapeptide rings in the region of the chromophore differs in free actinomycin lactone and lactams. In the presence of calf thymus DNA, PM2 DNA, and the synthetic d(A-T)-like copolymers containing 2,6-diaminopurine (DAP), poly[d(DAP-T)], and poly[d(DAP-A-T)], the rotational strengths of the optically active transitions in the visible region of the actinomycins increased, and the CD spectra in the presence of the various DNA duplexes were qualitatively similar. The CD spectra of bound actinomycin lactams resembled the spectrum of bound AMD. This suggests that the lactone and lactam actinomycins acquire a similar environment when bound to DNA. [Dpr1]2-AMD was less cytotoxic than AMD in antibacterial assays but exhibited somewhat higher toxicity in mice than AMD. At optimal dose levels the lactam analog had little or no antitumor activity in three murine tumor systems.

Deoxyribonucleoprotein changes in ram and bull spermatids.

The ability of nuclear chromatin to bind actinomycin-D appears to be related to the synthesis of RNA (Berlowitz, Pallotta & Sibley, 1968, 1969; Brachet & Hulin, 1970). It has also been suggested that binding of actinomycin-D is associated with physicochemical changes in the deoxyribonucleoproteins (Bolund, 1969; Darzynkiewicz, Gledhill & Ringertz, 1969; Pederson & Robbins, 1970). During spermatogenesis, RNA synthesis ceases before the structure and composition of the nucleoproteins change (Monesi, 1964; Utakoji, 1966; Darzynkiewicz et al., 1969; Gledhill, 1971; Gledhill, Darzynkiewicz & Ringertz, 1971). Ram germinal cell squashes were fixed in 40% acetic acid and then incubated at 37° C for 15 min in Ringer-phosphate solution containing 0·166 μCi [3H]actinomycin-D/ml (190 mCi/mmol). Unfixed smears of bull germinal cells were incubated in the same solution for 1 hr at 30° C and

Effect of prednisolone on thymus lymphocytes: II. Studies on [formula omitted] binding and RNA polymerase activity of isolated nuclei

The rate of actinomycin D (AMD) binding to DNA of isolated nuclei is very rapid. Nearly 90% of the total AMD bound binds during the first minute of incubation, irrespective of AMD concentration or temperature. The amount of AMD bound depends on the concentration of AMD in the medium. Two distinct phases of this concentration dependency were detected. During the initial phase (up to 15 μg/ml AMD concentration) the amount of AMD bound increases in a 1:1 proportion to the increase in AMD concentration; during the second phase (15–500 μg/ml), the increase in bound AMD is in a proportion of 1:8 to the increase in AMD concentration. This phenomenon is considered to reflect AMD binding to DNA in derepressed and repressed chromatin, respectively; AMD “titration” of derepressed sites is completed when 14–16 AMD molecules are bound per 10 3 base pairs of DNA. Increase in ionic strength “opens” new sites for AMD binding while divalent cations specifically decrease the number of sites binding AMD. Treatment of isolated thymocyte nuclei with prednisolone does not significantly change AMD binding; this is in contrast with liver cell nuclei, which bind 10–12% more AMD in the presence of prednisolone. The nuclei isolated from prednisolone-treated thymocytes bind 10–18% less AMD than control nuclei. In some experiments this difference was diminished when AMD binding was studied at higher ionic strength. The mechanism of this phenomenon as well as the possible reasons for its irreproducibility are discussed. No change in RNA-polymerase activity was detected when isolated nuclei were treated with prednisolone. However, when nuclei were isolated from prednisolone-treated cells, they exhibited a decreased activity in the presence of both Mg 2+ or Mn 2+ ions. An increase in ionic strength resulted in stimulated RNA-polymerase activity, with a higher stimulation being observed in nuclei from prednisolone-treated cells. As a result, the difference between control nuclei and those of prednisolone-treated cells diminished at higher ionic strength. The mechanism of the action of prednisolone in modulating thymus chromatin is discussed.

Constitutive heterochromatin in Microtus agrestis: binding of actinomycin-D and transcriptional inactivity.

The constitutive heterochromatin of the field vole, Microtus agrestis, fails to incorporate 3H-uridine. It must be considered to be transcriptionally inactive. Nevertheless, in both living and fixed cells constitutive heterochromatin binds labelled actinomycin-D (3H-AMD) at the same rate or at even a higher rate than euchromatin per unit DNA. Extraction of histones by acids leads to an increase of the AMD-binding capacity of the entire chromatin. The number of free AMD-binding sites is found to be largely independent of the degree of chromatin condensation and heteropycnosis.-It is suggested that AMD-binding to constitutive heterochromatin reflects a genetic inactivity which is not necessarily connected with a repressive function of histones and with chromatin condensation. In this respect a distinct difference seems to exist between constitutive and facultative heterochromatin.

Actinomycin D binding to isolated deoxyribonucleoprotein and intact cells

The binding of actinomycin D (AMD) by isolated deoxyribonucleoprotein (DNP) and chromatin of intact cells has been studied by chemical and autoradiographic experiments with radioactive AMD and by optical titrations. The binding capacity of isolated DNP was found to be one to two orders of magnitude higher than that of DNP in intact cells. In the presence of Ca 2+-ions and at higher ionic strengths the binding capacity of isolated DNP was depressed. EDTA has the opposite effect on intact cells. The degree of AMD binding was highly dependent on the concentration of AMD used. This concentration dependence was most pronounced for intact cells although it was also observed with isolated DNP where two types of binding can be detected. No significant differences were found in the AMD-binding to DNP isolated from hen erythrocytes, human leucocytes and HeLa cells. Differences in AMD-binding capacities were, however, eliminated during the isolation since living hen erythrocytes bound approximately twice as much AMD per unit DNA as HeLa cells which in turn bound 3–4 times as much as human leucocytes. Kinetic studies suggested that there are two phases of binding to the living cells tested: one early rapid phase and a second slower one. The differences in binding capacity were diminished or even reversed if the cells were fixed in ethanol-acetic acid (9:1) prior to incubation with AMD. This fixation also dramatically increased the AMD-binding capacity of all cell types tested. The large proportion of non-AMD-binding cells seen in human lymphocyte populations was found to bind AMD after fixation. The penetration of AMD into living cells was studied with “dry” autoradiography. The results show that the varying extent of binding cannot be explained solely on the basis of differences in penetration. The biological significance of the observations is discussed.