chromomycins and stallimycin

Rat liver nucleus histone H1 was fractionated by polyglutamic acid (PG) in the presence of distamycin A (DM) or chromomycin A(3) (CM). In the absence of the antibiotics, PG extracts from the nuclei about half of the nuclear H1. DM or CM added to the nuclei in saturating concentrations weakens the binding potential of most of H1. Titration of nuclei with DM shows that the number of binding sites for DM in the nuclei is less than in isolated DNA by only 20-25%, and this difference disappears after treatment of nuclei with PG. The lower CD value of DM complexes with nuclei compared to that of DM complexes with free DNA is evidence of a change in the DM-DNA binding mode in nuclear chromatin. About 25% of total histone H1 is sensitive only to DM and ~5% is sensitive only to CM. Half of the DM-sensitive H1 fraction seems to have a different binding mode in condensed compared relaxed chromatin. A small part of H1 (~3%) remains tightly bound to the nuclear chromatin independent of the presence of the antibiotics. Subfraction H1A is more DM-sensitive and H1B is more CM-sensitive. UV irradiation of nuclei results in dose-dependent cross-linking of up to 50% of total H1, which is neither acid-extractable nor recovered during SDS electrophoresis. PG with DM extracts only about 3% of H1 from UV-stabilized chromatin. DM treatment of the nuclei before UV irradiation results in extraction of the whole DM-sensitive H1 fraction (~25%), which in this case is not stabilized in the nucleus. A hypothesis on possible roles of the found H1 fractions in chromatin structural organization is discussed.

Topics: Animals; Anti-Bacterial Agents; Cell Nucleus; Chromatin; Chromomycins; Distamycins; DNA; Female; Hepatocytes; Histones; Interphase; Nucleic Acid Conformation; Polyglutamic Acid; Rats; Ultraviolet Rays

Selaginella is the largest genus of heterosporous pteridophytes, but karyologically the genus is known only by the occurrence of a dysploid series of n=7-12, and a low frequency of polyploids. Aiming to contribute to a better understanding of the structural chromosomal variability of this genus, different staining methods were applied in species with different chromosome numbers.. The chromosome complements of seven species of Selaginella were analysed and, in four of them, the distribution of 45S rDNA sites was determined by fluorescent in situ hybridization. Additionally, CMA/DA/DAPI and silver nitrate staining were performed to investigate the correlation between the 45S rDNA sites, the heterochromatic bands and the number of active rDNA sites.. The chromosome numbers observed were 2n=18, 20 and 24. The species with 2n=20 exhibited chromosome complement sizes smaller and less variable than those with 2n=18. The only species with 2n=24, S. convoluta, had relatively large and asymmetrical chromosomes. The interphase nuclei in all species were of the chromocentric type. CMA/DA/DAPI staining showed only a weak chromosomal differentiation of heterochromatic bands. In S. willdenowii and S. convoluta eight and six CMA+ bands were observed, respectively, but no DAPI+ bands. The CMA+ bands corresponded in number, size and location to the rDNA sites. In general, the number of rDNA sites correlated with the maximum number of nucleoli per nucleus. Ten rDNA sites were found in S. plana (2n=20), eight in S. willdenowii (2n=18), six in S. convoluta (2n=24) and two in S. producta (2n=20).. The remarkable variation in chromosome size and number and rDNA sites shows that dramatic karyological changes have occurred during the evolution of the genus at the diploid level. These data further suggest that the two putative basic numbers of the genus, x=9 and x=10, may have arisen two or more times independently.

Topics: Cell Nucleolus; Chromomycins; Chromosomes, Plant; Coloring Agents; Distamycins; DNA, Plant; DNA, Ribosomal; Evolution, Molecular; Fluorescent Dyes; Indoles; Selaginellaceae; Species Specificity

DNA-binding drugs have been reported to be able to interfere with the activity of transcription factors in a sequence-dependent manner, leading to alteration of transcription. This and similar effects could have important practical applications in the experimental therapy of many human pathologies, including neoplastic diseases and viral infections. The analysis of the biological activity of DNA-binding drugs by footprinting, gel retardation, polymerase chain reaction, and in vitro transcription studies does not allow a real time study of binding to DNA and dissociation of the generated drugs/DNA complexes. The recent development of biosensor technologies for biospecific interaction analysis (BIA) enables monitoring of a variety of molecular reactions in real-time by surface plasmon resonance (SPR). In this study, we demonstrate that molecular interactions between DNA-binding drugs (chromomycin, mithramycin, distamycin, and MEN 10567) and biotinylated target DNA probes immobilized on sensor chips is detectable by SPR technology using a commercially available biosensor. The target DNA sequences were synthetic oligonucleotides mimicking the Sp1, NF-kB, and TFIID binding sites of the long terminal repeat of the human immunodeficiency type 1 virus. The results obtained demonstrate that mithramycin/DNA complexes are less stable than chromomycin/DNA complexes; distamycin binds to both NF-kB and TATA box oligonucleotides, but distamycin/(NF-kB)DNA complexes are not stable; the distamycin analog MEN 10567 binds to the NF-kB mer and the generated drug/DNA complexes are stable. The experimental approach described in this study allows fast analysis of molecular interactions between DNA-binding drugs and selected target DNA sequences. Therefore, this method could be used to identify new drugs exhibiting differential binding activities to selected regions of viral and eukaryotic gene promoters.

Topics: Biosensing Techniques; Chromomycins; Distamycins; DNA; Molecular Weight; Plicamycin

SARs (scaffold attachment regions) are candidate DNA elements for partitioning eukaryotic genomes into independent chromatin loops by attaching DNA to proteins of a nuclear scaffold or matrix. The interaction of SARs with the nuclear scaffold is evolutionarily conserved and appears to be due to specific DNA binding proteins that recognize SARs by a mechanism not yet understood. We describe a novel, evolutionarily conserved protein domain that specifically binds to SARs but is not related to SAR binding motifs of other proteins. This domain was first identified in human scaffold attachment factor A (SAF-A) and was thus designated SAF-Box. The SAF-Box is present in many different proteins ranging from yeast to human in origin and appears to be structurally related to a homeodomain. We show here that SAF-Boxes from four different origins, as well as a synthetic SAF-Box peptide, bind to natural and artificial SARs with high specificity. Specific SAR binding of the novel domain is achieved by an unusual mass binding mode, is sensitive to distamycin but not to chromomycin, and displays a clear preference for long DNA fragments. This is the first characterization of a specific SAR binding domain that is conserved throughout evolution and has DNA binding properties that closely resemble that of the unfractionated nuclear scaffold.

Topics: Amino Acid Motifs; Amino Acid Sequence; Animals; Binding Sites; Cell Line; Chromatin; Chromomycins; Cloning, Molecular; Conserved Sequence; Distamycins; DNA; DNA-Binding Proteins; Humans; Molecular Sequence Data; Mutation; Nuclear Proteins; Oligodeoxyribonucleotides; Peptide Fragments; Protein Binding; Protein Structure, Tertiary; Recombinant Proteins; Sequence Homology, Amino Acid; Substrate Specificity; Transfection

Sequence-selectivity of DNA-binding drugs was recently reported in a number of studies employing footprinting and gel retardation approaches. In this paper we studied sequence-selectivity of the binding of chromomycin and distamycin to DNA by performing DNase I footprinting and analysis of the cleaved fragments by the Pharmacia ALF DNA Sequencing System. As a model system we employed the long terminal repeat of the human immunodeficiency type 1 virus. The main conclusion of our experiments is that automated analysis of DNase I footprinting is a fast and reliable technique to study drugs-DNA interactions. The results obtained suggest that distamycin and chromomycin differentially interact with the long terminal repeat of the human immunodeficiency type 1 virus; this differential binding depends upon the DNA sequences recognized. The data presented are consistent with a preferential binding of distamycin to DNA sequences of the binding sites of nuclear factor kappa B and transcription factor IID. By contrast, distamycin exhibits only weak binding to DNA sequences recognized by the promoter-specific transcription factor Sp1. Unlike distamycin, chromomycin preferentially interacts with the binding sites of the promoter-specific transcription factor Sp1.

Topics: Antibiotics, Antineoplastic; Antiviral Agents; Automation; Base Sequence; Chromomycins; Deoxyribonucleases; Distamycins; DNA Footprinting; DNA, Viral; Fluorescein; Fluoresceins; HIV-1; Humans; Molecular Sequence Data; Polymerase Chain Reaction

Topics: Animals; Chromomycins; Chromosome Banding; Chromosomes; Chromosomes, Human; Dactinomycin; Distamycins; Evaluation Studies as Topic; Fluorescent Dyes; Heterochromatin; Humans; Indoles; Intercalating Agents; Male; Methyl Green; Solutions; Staining and Labeling

Sequence-selectivity of DNA-binding drugs was recently reported in a number of studies employing footprinting and gel retardation approaches. In this paper we performed polymerase-chain reaction (PCR) experiments to study the in vitro effects of distamycin, daunomycin, chromomycin and mithramycin. As model systems we employed the human estrogen receptor (ER) gene and the Harvey-ras (Ha-ras) oncogene, in order to obtain PCR products significantly differing for the A + T/G + C frequency ratio. Distamycin, daunomycin, chromomycin and mithramycin are indeed known to differentially bind to different DNA regions depending upon the DNA sequences recognized. The main conclusion of our experiments is that distamycin, daunomycin, chromomycin and mithramycin inhibit polymerase-chain reaction in a sequence-dependent manner. Distamycin inhibits indeed PCR mediated amplification of AT-rich regions of the human estrogen receptor gene, displaying no inhibitory effects on PCR-mediated amplification of GC-rich sequences of Ha-ras oncogene. By contrast daunomycin, chromomycin and mithramycin were found to inhibit PCR-mediated amplification of the Ha-ras GC-rich oncogene sequences. We propose that polymerase-chain reaction technique could be applied to study the in vivo interactions of DNA-binding drugs to specific genes in intact cells.

Topics: Animals; Base Sequence; Binding Sites; Cells, Cultured; Chromomycins; Cricetinae; Cricetulus; Daunorubicin; Distamycins; DNA; Drug Interactions; Gene Amplification; Genes, ras; Humans; Intercalating Agents; Molecular Sequence Data; Plicamycin; Polymerase Chain Reaction; Receptors, Estrogen; Sensitivity and Specificity

We identify two high-affinity matrix-attachment regions (MAR elements) located in two introns of the human DNA topoisomerase I gene (TOP1). These intronic MAR elements, designated MI and MII, are specifically bound by the nuclear matrix and partition with scaffolds in vitro. One of these MAR elements, MII, is part of a genomic region which is hypersensitive for endogenous nucleases. We have sequenced both DNA elements and have characterized their mode of binding to the nuclear matrix. Experiments with the minor-groove-binding ligands distamycin and chromomycin indicate that the A+T-rich regions, most likely homopolymeric A tracts, are responsible for binding of these DNA elements to the nuclear matrix. MII contains an alu-like element and a segment of curved DNA. Analysis of subfragments of MII show that the curved DNA region itself shows only weak nuclear-matrix binding, and that the high-affinity binding sites are located on subfragments on the 5' side of the curved DNA. In addition, we found that the alu-like sequence does not contribute significantly to the binding of MII and of subfragments of MII to nuclear-matrix proteins. Comparing the distribution of repetitive sequences in the cloned parts of human DNA topoisomerase I gene with the location of high-affinity matrix-binding sites we find no evidence that repetitive DNA may be located close to MAR elements as has been previously suggested.

Topics: Binding Sites; Chromomycins; Deoxyribonuclease BamHI; Deoxyribonuclease I; Deoxyribonucleases, Type II Site-Specific; Distamycins; DNA; DNA Topoisomerases, Type I; Humans; Introns; Molecular Sequence Data; Nuclear Matrix; Nucleic Acid Conformation; Repetitive Sequences, Nucleic Acid

The DNA of malarial parasites is significantly richer in A and T than that of mammalian cells. Antibiotics which bind to the minor groove of B-DNA with a preference for AT-rich sequences, such as distamycin A, netropsin, 4'-6-diamidino-2-phenylindole (DAPI) and bis-benzimide (Hoechst 33258) were found to inhibit the growth and propagation of Plasmodium falciparum in culture. Distamycin A readily inhibited nucleic acid and protein synthesis and was more toxic to the ring stage than to the trophozoite stage in various parasite strains, irrespective of their susceptibility to chloroquine. Distamycin A, netropsin, DAPI and Hoechst 33258 were considerably more toxic to parasites than to mammalian cells, while chromomycin A3 and mithramycin A, which bind preferentially to GC-rich sequences, were either equally toxic or more harmful to mammalian cells. These results suggest that the mere difference in DNA base composition of parasites and host cells may account for the selective toxicity of minor groove ligands. Distamycin A, DAPI and Hoechst 33258 were also found to be more toxic to Saccharomyces cerevisiae grown on glycerol than to yeast cells grown on glucose, consistent with the preferential binding of these ligands to the relatively AT-rich mitochondrial DNA of yeast cell. These results underscore the generality of selective toxicity of minor groove binders endowed by the DNA base composition.

Topics: Animals; Base Composition; Bisbenzimidazole; Cell Division; Cells, Cultured; Chromomycins; Distamycins; DNA, Protozoan; Dose-Response Relationship, Drug; Humans; Indoles; Intercalating Agents; Mice; Netropsin; Nucleic Acid Conformation; Plasmodium falciparum; Plicamycin; Saccharomyces cerevisiae

Topics: Chromomycins; Chromosome Aberrations; Chromosome Banding; Chromosome Disorders; Distamycins; Female; Humans; Male