The use of distamycin A in human lymphocyte cultures

Summary The effect of the oligopeptide antibiotic distamycin A on human lymphocyte cultures was examined. Distamycin A specifically inhibits the condensation of the Y heterochromatin and induces a fragile site in the chromosome 16 (band q22) in some individuals. The optimal culture conditions under which an undercondensation of the Y heterochromatin and an induction of the fragile site in 16q22 can be achieved by in vitro treatment of lymphocytes were determined. This also permits the use of distamycin A in routine diagnostics of human chromosomes. The use of this technique in the analysis of translocations involving the Y chromosome is presented. The distamycin A-DNA interaction and the different possible explanations for the distamycin A-induced undercondensations of the Y heterochromatin and fragile sites 16q22 are discussed.     

Heterochromatin characterization of sex chromosomes in Triturus marmoratus (Urodela, Salamandridae).

The sex chromosomes of the Iberian marbled newt, Triturus marmoratus, were studied using various banding techniques, including restriction enzyme/nick translation (RE/NT) procedures. Four types of heterochromatin on the sex chromosomes could be distinguished: (1) distamycin A/DAPI and chromomycin A3/distamycin A positive, EcoRI/NT negative, and HaeIII/NT and HinfI/NT positive; (2) distamycin A/DAPI and chromomycin A3/distamycin A positive, but RE/NT negative; (3) AT rich, but RE/NT negative; and (4) distamycin A/DAPI and chromomycin A3/distamycin A positive, EcoRI/NT and HinfI/NT negative, but HaeIII/NT positive. These data suggest a common origin for the terminal heterochromatic domains of both the X and Y chromosomes in this species.     

Reassessment of presumed Y/22 and Y/15 translocations in man using a new technique.

A new chromosome banding technique, distamycin A plus DAPI, has been used to reexamine cases of presumed Y/autosome translocations. In contrast with the results obtained with quinacrine fluorescence (Q-banding), the satellites of acrocentric chromosomes do not fluoresce brightly with this new (DA-DAPI) method, making it more specific for the long arm of the Y chromosome. Previous cases with intensely Q-fluorescent and abnormally long short arms on a chromosome 22 were considered as presumptive 22/Y translocations: The new technique clearly shows that, in these cases, the additional material on 22p is not derived from Yq. In contrast, in other cases the Yq nature of additional material on 15p, in conjunction with the presence of an extra Y-body in interphase nuclei and the presence of a male-specific DNA, supports the previous diagnosis of a presumptive 15/Y translocation.     

Distamycin A/DAPI bands and the effects of 5-azacytidine on the chromosomes of the chimpanzee, Pan troglodytes

The chromosomes of the chimpanzee were stained with distamycin A/DAPI, which labels specific C-bands. Bright distamycin A/DAPI fluorescence was found in the heterochromatic regions of chromosomes 6, 11, 14 to 16, 18 to 20, and 23 and the Y. Lymphocyte cultures from chimpanzees were treated with low doses of 5-azacytidine during the last hours of culture. This cytosine analog induces highly distinct undercondensations in 28 heterochromatic regions of 19 chromosomes. These 5-azacytidine-sensitive regions are predominantly located in the terminal C-bands of the chromosomes. In vitro treatment with 5-azacytidine also preserves into the metaphase stage somatic pairings between the 5-azacytidine-sensitive heterochromatic regions in interphase nuclei. The homologies and differences regarding the chromosomal localization of distamycin A/DAPI-bright C-bands, 5-azacytidine-sensitive heterochromatin, 5-methylcytosine-rich DNA sequences, and satellite DNAs in the chimpanzee and man are discussed.     

Modification of DAPI banding on human chromosomes by prestaining with a DNA-binding oligopeptide antibiotic, distamycin A

A DNA-binding AT-specific oligopeptide antibiotic, distamycin A, was used as non-fluorescent counterstain in conjunction with the DNA-binding AT-specific fluorochrome 4′-6-diamidino-2-phenylindole (DAPI) to investigate the effect of the antibiotic on DAPI fluorescent banding of human chromosomes. Distamycin A-pretreated metaphases and interphase nuclei exhibited a significantly lower overall fluorescence intensity than DAPI controls. Chromosome arms were pale and intercalary DAPI bands (Q bands) were obliterated, but some specific regions of constitutive heterochromatin remained brightly fluorescent. These were mainly the constrictions of chromosomes 1, 9 and 16, the short arm of chromosome 15, and the distal part of the Y. The distamycin A/DAPI banding pattern appears to be comparable to that reported for anti-5-methylcytosine binding [11]. The observations are discussed as they relate to the roles of chromosomal DNAs and proteins in chromosome banding.     

High mobility group proteins HMG-1 and HMG-I/Y bind to a positive regulatory region of the pea plastocyanin gene promoter

A 268 bp region (P268) of the pea plastocyanin gene promoter responsible for high-level expression has been shown to interact with the high mobility group proteins HMG-1 and HMG-I/Y isolated from pea shoot chromatin. cDNAs encoding an HMG-1 protein of 154 amino acid residues containing a single HMG-box and a C-terminal acidic tail and an HMG-I/Y-like protein of 197 amino acid residues containing four AT-hooks have been isolated and expressed in Escherichia coli to provide large amounts of full-length proteins. DNase I footprinting identified eight binding sites for HMG-I/Y and six binding sites for HMG-1 in P268. Inhibition of binding by the antibiotic distamycin, which binds in the minor groove of A/T-rich DNA, revealed that HMG-I/Y binding was 400-fold more sensitive than HMG-1 binding. Binding-site selection from a pool of random oligonucleotides indicated that HMG-I/Y binds to oligonucleotides containing stretches of five or more A/T bp and HMG-1 binds preferentially to oligonucleotides enriched in dinucleotides such as TpT and TpG.     

The effects of local DNA sequence on the interaction of ligands with their preferred binding sites

We have examined the effects of local DNA sequence on the interaction of distamycin, Hoechst 33258, echinomycin, actinomycin and mithramycin with their preferred binding sites using a series of DNA fragments that contain every symmetrical hexanucleotide sequence. In several instances we find that the affinity for the ligands' preferred binding sites is affected by the hexanucleotide context in which they are located. The AT-selective minor groove binding ligand Hoechst 33258 shows a 200-fold difference in binding to the 16 different X(A/T)(4)Y sites; the strongest binding is to AAATTT and the weakest is to (G/C)TTAA(C/G). Although TTAA is generally a poor binding site, ATTAAT is better than TTTAAA and they are both much better than GTTAAC and CTTAAG. Similarly, TTATAA and ATATAT are better binding sites than GTATAC and CTATAG. In contrast, distamycin shows less discrimination between the various X(A/T)(4)Y sites, with a 20-fold difference between the best [(A/T)AATT(T/A)] and worst [GATATC and (G/C)TTAA(C/G)] sites. Although actinomycin binds to GpC it shows little or no interaction with any of the GGCC sites, yet shows only a six-fold variation in affinities for the other XYGCXY sites. Echinomycin binds to CpG yet shows no binding to TTCGAA, TGCGCA and AGCGCT, while the best binding is to AACGTT. The tetranucleotides CCGG and ACGT produce consistently good binding sites, irrespective of the surrounding sequences, while the interaction with TCGA and GCGC is sensitive to the hexanucleotide context. Hexanucleotides with a central GCGC, flanked by A and T are weaker echinomycin sites than those flanked by G and C, especially CGCGCG. The best X(G/C)(4)Y binding sites for mithramycin were located at AGCGCT and GGGCCC, and the worst at CCCGGG and TCCGGA. These footprinting fragments are valuable tools for comparing the binding of ligands to all the potential symmetrical hexanucleotides and provide insights into the effects of local DNA sequence on ligand-DNA interactions.     

Distamycin A-DAPI banding of nonfluorescent Y (Ynf) chromosomes in 45,X/46,XYnf mosaicism

Summary The distamycin A-DAPI banding patterns of nonfluorescent, nonheterochromatic Y chromosomes (Y^nf) in two patients with 45,X/46,XY^nf mosaicism were investigated. In both cases moderately fluorescent bands were observed near the centromere and on the distal long arm of the Y^nf. These bands were similar to the centrometric band on normal Y chromosomes and support the hypothesis that the Y^nf is an isodicentric chromosome derived from the proximal portion of the Y chromosome.     

Reversion of the B to A transition of DNA induced by specific interaction with the oligopeptide distamycin A

The effect of the DNA-binding oligopeptide distamycin A on the B to A transition of DNA in ethanol/water solutions has been studied by means of CD. (The overbars indicate that it does not matter which particular form of the corresponding families is considered.) The results show that increasing the concentration of distamycin A reverses the A conformation (in 82% ethanol) to the B conformation due to its strong binding and stabilization of the latter. In accordance with previous data for pure aqueous solutions, a site size of 3.5 base pairs is obtained from the studies in water/ethanolic solutions. From the data on the B to A transition in the presence of distamycin A, we estimated the length of the cooperativity ν₀ = 10 base pairs. The results demonstrate that the oligopeptide systems of distamycin, as well as those of netropsin, are effective stabilizers of the DNA B-conformation.     

DA/DAPI-Fluorescent heteromorphism of human Y chromosome

Summary Variation of DA/DAPI intensity in the Yq12 band was observed in five amniotic cell specimens and one blood specimen from the father of one fetus. Three distinct classes of Yq heterochromatin were identified by distamycin A (DA) treatment of the cell cultures and various staining techniques. The heterochromatin in the Yq11.23 sub-band does not under-condense when exposed to DA, and shows pale fluorescence with quinacrine staining, positive C-banding, and bright fluorescence with DA/DAPI technique. This class of heterochromatin was consistently observed in all specimens studied. The other two classes of heterochromatin are in the Yq12 band. Both show undercondensation when exposed to DA, quinacrine-bright fluorescence, and positive C-banding; howover, one class of heterochromatin shows DA/DAPI-bright fluorescence and the other shows pale fluorescence. The size and banding intensity of the two classes of heterochromatin in Yq12 are variable. These results provide cytological evidence of heterogeneity within the Y heterochromatin region containing AT-rich DNA.     

Further resolution of the quail karyotype and characterization of microchromosomes by counterstain-enhanced fluorescence

Sequential staining with a counterstain-contrasted fluorescent R-banding technique (chromomycin A3/distamycin A-DAPI) followed by DAPI-actinomycin D-induced quinacrine-fluorescence-Hoechst 33258 (QFH)-type banding allowed the identification of quail chromosomes up to chromosome 19. The chromomycin A3-positive staining behavior of the W chromosome and of the heterochromatic areas of most microchromosomes indicated their GC-rich nature.     

Specific protection of DNA by distamycin A, netropsin and bis-netropsins against the action of DNAse I

Interaction of netropsin, distamycin A and a number of bis-netropsins with DNA fragments of definite nucleotide sequence was studied by footprinting technique. The nuclease protection experiments were made at fixed DNA concentration and varying ligand concentrations. The affinity of ligand for a DNA site was estimated from measurements of ligand concentration that causes 50% protection of the DNA site. Distribution pattern of the protected and unprotected regions along the DNA fragment was compared with the theoretically expected arrangement of the ligand along the same DNA. The comparison led us to the following conclusions: 1. Footprinting experiments show that at high levels of binding the arrangement of netropsin molecules along the DNA corresponds closely to the distribution pattern expected from theoretical calculations based on the known geometry of netropsin--DNA complex. However, the observed differences in the affinity of netropsin for various DNA sequences is markedly greater than that expected from theoretical calculations. 2. Netropsin exhibits a greater selectivity of binding than that expected for a ligand with three specific reaction centers associated with the antibiotic amide groups. It binds preferentially to DNA regions containing four or more successive AT pairs. Among 13 putative binding sites for netropsin with four or more successive AT pairs there are 11 strong binding sites and two weaker sites which are occupied at 2 D/P less than or equal to 1/9 and 2 D/P = 1/4, respectively. 3. The extent of specificity manifested by distamycin A is comparable to that shown by netropsin although the molecule of distamycin A contains four rather than three amide groups. At high levels of binding distamycin A occupies the same binding sites on DNA as netropsin does. 4. The binding specificity of bis-netropsins is greater than that of netropsin. Bis-netropsins can bind to DNA in such a way that the two netropsin-like fragments are implicated in specific interaction with DNA base pairs. However, the apparent affinity of bis-netropsins estimated from footprinting experiments is comparable with that of netropsin for the same DNA region. 5. At high levels of binding bis-netropsins and distamycin A (but not netropsin) can occupy any potential site on DNA irrespectively of the DNA sequence. 6. Complex formation with netropsin increases sensitivity to DNase I at certain DNA sites along with the protection effect observed at neighboring sites.     

The chromosomes of Micromys minutus (Rodentia, Murinae). II. Pairing pattern of X and Y chromosomes in meiotic prophase

Both light and electron microscopy were used to study the pairing behavior of the sex chromosomes of the harvest mouse, Micromys minutus, in surface-spread pachytene spermatocytes. The XY pairing pattern is very exceptional in that the site of synaptic initiation is located interstitially in the short arms of the X and the Y, next to their centromeric regions. From this tiny euchromatic site, synapsis proceeds unidirectionally along the homologous heterochromatic short arms of the X and the Y toward the ends of the chromosomes. After pairing of the short arm is concluded, synapsis begins between the nonhomologous long arms of the X and the Y in the immediate vicinity of the centromeres and progresses unidirectionally toward the end of the long arm of the Y. A synaptic complex develops between the constitutive heterochromatin of the long arm of the Y and the euchromatin of the long arm of the X. Analysis of C-banded and distamycin A/DAPI-stained diakineses revealed a trefoil-like XY bivalent, which was interpreted to be the result of an interstitial chiasma occurring in the paired short arms of the X and the Y. A conspicuous, electron-dense body, about 1 micron in diameter, was found closely associated with the centromeres of the X and the Y in numerous pachytene spermatocytes. A review of the literature showed that comparable XY-associated bodies have been found in only eight other mammals to date.     

Synergistic induction of the senescence-associated genes by 5-bromodeoxyuridine and AT-binding ligands in HeLa cells

5-Bromodeoxyuridine induces a senescence-like phenomenon in mammalian cells. This effect was dramatically potentiated by AT-binding ligands such as distamycin A, netropsin, and Hoechst 33258. The genes most remarkably affected by these ligands include the widely used senescence-associated genes and were located on or nearby Giemsa-dark bands of human chromosomes. We hypothesize that AT-rich scaffold/nuclear matrix attachment region sequences are involved in this phenomenon. In fact, upon substitution of thymine with 5-bromouracil, a rat S/MAR sequence reduced its degree of bending and became insensitive to cancellation of the bending by distamycin A. The S/MAR sequence containing 5-bromouracil also bound more tightly to nuclear scaffold proteins in vitro and this binding was not inhibited by distamycin A. Under the same conditions, the S/MAR sequence containing thymine easily dissociated from the nuclear scaffold proteins. Taken together, the synergistic induction of the genes may be explained not only by opening of condensed chromatin by distamycin A but also by increase in the binding of 5-bromouracil-containing S/MAR sequences to the nuclear scaffolds.     

Interaction of lambda cro repressor with synthetic operator OR3 studied by competition binding with minor groove binders.

In the present work, we employ a combination of CD spectroscopy and gel retardation technique to characterize thermodynamically the binding of lambda phage cro repressor to a 17 base pair operator OR3. We have found that three minor groove-binding antibiotics, distamycin A, netropsin and sibiromycin, compete effectively with the cro for binding to the operator OR3. Among these antibiotics, sibiromycin binds covalently to DNA in the minor groove at the NH2 of guanine, whereas distamycin A and netropsin interact preferentially with runs of AT base pairs and avoid DNA regions containing guanine bases in the two polynucleotide strands. Only subtle DNA conformation changes are known to take place upon binding of these antibiotics. Both the CD spectral profiles and the results of the gel retardation experiments indicate that distamycin A and netropsin can displace cro repressor from the operator OR3. The binding of cro repressor to the OR3 is accompanied by considerable changes in CD in the far-UV region which appear to be attributed to a DNA-dependent structural transition in the protein. Spectral changes are also induced in the wavelength region of 270-290 nm. The CD spectral profile of the cro-OR3 mixture in the presence of distamycin A can be represented as a sum of the CD spectrum of the repressor-operator complex and spectrum of distamycin-DNA complex at the appropriate molar ratio of the bound antibiotic to the operator DNA (r). When r tends to the saturation level of binding the CD spectrum in the region of 270-360 nm approaches a CD pattern typical of complexes of the antibiotic with the free DNA oligomer. This suggests that simultaneous binding of cro repressor and distamycin A to the same DNA oligomer is not possible and that distamycin A and netropsin can be used to determine the equilibrium affinity constant of cro repressor to the synthetic operator from competition-type experiments. The binding constant of cro repressor to the OR3 is found to be (6 +/- 1).10(6)M-1 at 20 degrees C in 10 mM sodium cacodylate buffer (pH 7.0) in the presence of 0.1 M NH4F.     

Ewing's tumor X mouse hybrids expressing the MIC2 antigen: analyses using fluorescence CDD-banding and non-isotopic ISH

Summary We present a highly sensitive method that has been applied to map the chromosomal origin of the prominent cell surface antigen of Ewing's tumor cells recognized by monoclonal antibody HBA-71. The technique allows an unambiguous identification of human chromosomal material in interspecific cell hybrids. This is achieved by fluorescent in situ hybridization of biotinylated total human DNA, followed by high resolution fluorescence banding with the chromomycin/distamycin/ DAPI triple stain. An advantage of this method is that all signals can be visualized in one single operation by simply switching the appropriate filter blocks. The protocol has proved extremely useful in gene mapping by means of interspecific cell hybrids, a technique that depends on the accurate and unambiguous recognition of the relevant (e.g., human) genetic material in the clonal genome. Our studies confirm that this antigen is the product of the MIC2 gene, which is so far the only well-studied pseudoautosomal gene in man, and which is located on the short arms of chromosomes X and Y. Furthermore, no influence of the Ewing's tumor-specific translocation t(11;22) on MIC2 expression could be discerned.     

Enhancement and alteration of bleomycin-catalyzed site-specific DNA cleavage by distamycin A and some minor groove binders.

The effects of compounds which bind in the DNA minor groove of A.T rich sequences, on bleomycin-catalyzed site-specific DNA cleavage were investigated by a DNA sequencing technique. Distamycin A enhanced bleomycin-catalyzed DNA cleavage in G.C rich sequences such as 5'-GGGGC-3' (under scoring; the cleaved nucleotide). The cleavage in such a sequence in the presence of distamycin A was greater than that in the absence of distamycin A by as much as about 100 times. Neither Hoechst 33258, 4',6-diamidino-2-phenylindole (DAPI) nor berenil caused extensive enhancement. The results suggest that the distamycin-induced conformational changes of DNA through interactions other than the DNA minor groove binding in A.T-rich sequences are specifically suitable for the bleomycin action.     

Synthesis and DNA binding properties of saturated distamycin analogues

A series of saturated heterocyclic analogues of distamycin were prepared and examined. A fluorescent intercalator displacement (FID) assay conducted on p[dA]-p[dT] DNA to obtain C(50) values and a hairpin deoxyoligonucleotide containing an A/T-rich binding site was used to evaluate DNA binding affinity. It is observed that saturated heterocycles greatly reduce the DNA binding relative to distamycin.     

Molecular orbital calculations on the oligopeptides netropsin, distamycin and related compounds

The electronic structure of the antibiotics netropsin, distamycin A, and related compounds has been examined by various theoretical models. Calculations of both the Pariser-Parr-Pople (PPP) and the semiempirical CNDO/S types can account for the absorption spectrum of netropsin and distamycin A. The CD spectrum has been calculated for the conformation of netropsin found in the crystal structure of a netropsin/DNA dodecamer. Not all the CD spectral features can be attributed entirely to the chiral conformation of netropsin, indicating that there are significant interactions between netropsin and DNA. A CD calculation was also performed for distamycin A in a similar conformation. An examination of the charge-density maps of the excited states suggests that there is substantial charge transfer from the pyrrole ring to either of the adjacent peptide linkages in these systems. At higher energies, even longer distance charge transfer can be observed. Similar behavior was seen in the monomers pyrrole-2-carboxamide and 3-(formylamino)pyrrole.     

Counterstained enhancement of TaqI resistant sites after distamycin A/diamidinophenylindole treatment

Numerous selective and differential staining techniques have been used to investigate the hierarchical organisation of the human genome. This investigation demonstrates the unique characteristics that are produced on fixed human chromosomes when sequential procedures involving restriction endonuclease TaqI, distamycin A (DA) and 4,6-diamidino-2-phenylindole (DAPI) are employed. TaqI produces extensive gaps in the heterochromatic regions associated with satellite II and III DNAs of human chromosomes 1, 9, 15, 16 and Y. DA/DAPI selectively highlights, as brightly fluorescent C-bands, the heterochromatin associated with the alpha, beta, satellite II and III DNAs of these chromosomes. When DA and DAPI are used on chromosomes before TaqI digestion, and then stained with Giemsa, the centromeric regions appear to be more resistant, producing a distinct C-banding pattern and gaps in the heterochromatin regions. Sequential use of the DA/DAPI technique after TaqI treatment produces a bright fluorescence on the remaining pericentromeric regions of chromosomes 1, 9, 16 and Y, which also displayed a cytochemically unique banding pattern. This approach has produced specific enhanced chromosomal bands, which may serve as tools to characterize genomic heterochromatin at a fundamental level.