chromomycin-a3 and stallimycin

The interactions of self-complementary oligonucleotides with a group of metal-mediated DNA-binding drugs, including chromomycin A(3), mithramycin and the novel compound UK-1, were examined via electrospray ionization quadrupole ion trap mass spectrometry. Both chromomycin and mithramycin were shown to bind preferentially to GC-rich oligonucleotide duplexes in a 2:1 drug:metal ratio, while UK-1 was shown to bind in a 1:1 drug:metal stoichiometric ratio without a strong sequence preference. These trends were observed in the presence of Co(2+), Ni(2+) and Zn(2+), with the exception that chromomycin-Zn(2+) complexes were not readily observed. The binding stoichiometries as well as the sequence specificities are in agreement with literature reports for solution studies. Binding selectivities and stabilities of the complexes were also probed using electrospray ionization mass spectrometry. Both of the GC-rich oligomers 5'-GCGCGC-3' and 5'-GCGCATGCGC-3' exhibited a binding preference for chromomycin over mithramycin in the presence of Co(2+) and Ni(2+). Energy-variable collisionally activated dissociation of the complexes was employed to determine the stabilities of the complexes. The relative metal-dependent binding energies were Ni(2+) > Zn(2+) > Co(2+) for UK-1-oligomer complexes and Ni(2+) > Co(2+) for both mithramycin and chromomycin complexes.

Topics: Antineoplastic Agents; Base Sequence; Benzoxazoles; Binding Sites; Chromomycin A3; Distamycins; DNA; Dose-Response Relationship, Drug; Hydrogen-Ion Concentration; Metals; Molecular Weight; Nucleic Acid Synthesis Inhibitors; Oligodeoxyribonucleotides; Plicamycin; Spectrometry, Mass, Electrospray Ionization; Streptomyces; Substrate Specificity; Temperature; Thermodynamics

Using nucleotide-specific agents Hoechst 33258, actinomycin D, chromomycin A3, and distamycin A, the Paris quadrifolia L. karyotype, and the location and nucleotide composition of heterochromatic bands were studied. The chromosome ideogram of H33258/AMD and CMA/DA heterochromatic bands was created by an image analysis system with the Videotest-Kario software. By fluorescence in situ hybridization, the 18S and 26S rRNA genes were mapped.

Topics: Bisbenzimidazole; Chromomycin A3; Chromosome Mapping; Dactinomycin; Distamycins; Heterochromatin; In Situ Hybridization, Fluorescence; Plants; RNA Probes; RNA, Ribosomal; RNA, Ribosomal, 18S

Nucleoporins are the main components of nuclear pore complexes (NPCs) involved in nucleo-cytoplasmic transport. Starting with an expressed DNA fragment retrieved by exon trapping from pooled human BAC clones mapped to the short arm of chromosome 5, we identified a human nucleoporin cDNA sequence by PCR from a human testis cDNA library. The coding sequence showed high homology to that of the rat nucleoporin 155 (Nup155) cDNA. FISH analysis with the human BAC clone as probe localized the human NUP155 gene to chromosome band 5p13. Northern analysis showed that the human NUP155 gene was expressed at different levels in all tissues tested. Two species of transcripts were observed with estimated lengths of 5.4 and 4.7 kb, respectively, in concordance with the finding of two alternative polyadenylation sites in the cDNA. The genomic location of the human NUP155 gene suggests a possible role in the mental and developmental retardation associated with hemizygous deletions of the 5p13 region.

Topics: Amino Acid Sequence; Animals; Base Sequence; Blotting, Southern; Chromomycin A3; Chromosomes, Human, Pair 5; Distamycins; Exons; Expressed Sequence Tags; Humans; In Situ Hybridization, Fluorescence; Membrane Proteins; Models, Genetic; Molecular Sequence Data; Nuclear Pore Complex Proteins; Nuclear Proteins; Rats; Sequence Analysis, DNA; Sequence Homology, Nucleic Acid; Tissue Distribution

Adjacent binding sites for early growth response factor-1 (EGR1) and TATA box-binding protein (TBP) were identified on the herpes simplex virus latency promoter in previous work. The binding of EGR1 to the GC-rich region prevented TBP binding to the AT-rich region. With the simultaneous addition of both EGR1 and TBP, the intercalator nogalamycin prevented EGR1 complex formation, resulting in a dose-dependent increase of the TBP.DNA complex. The minor groove binder chromomycin A3 inhibited EGR1 complex formation but resulted in a smaller increase of the TBP complex. In contrast, an alkylating intercalator hedamycin strongly inhibited binding of both proteins. The ability of these GC-binding drugs to prevent EGR1.DNA complex formation was in the following order: hedamycin > nogalamycin > chromomycin A3, and the specificity was nogalamycin > chromomycin A3 > hedamycin. With transcription factor IIA (TFIIA) in the assay, TBP was able to bind the promoter whereas formation of the EGR1.DNA complex was reduced. An AT minor groove-binding drug, distamycin A, disrupted the TBP.TFIIA.DNA complex and restored the EGR1.DNA complex. We conclude that the binding motif and sequence preference of DNA-interactive drugs are manifested in their ability to inhibit the transcription factor-DNA complexes.

Topics: Alkylating Agents; Anthraquinones; Base Composition; Chromomycin A3; Deoxyribonucleoproteins; Distamycins; DNA-Binding Proteins; Early Growth Response Protein 1; Gene Expression Regulation, Viral; Humans; Immediate-Early Proteins; Intercalating Agents; Macromolecular Substances; Nogalamycin; Promoter Regions, Genetic; Recombinant Fusion Proteins; Simplexvirus; TATA-Box Binding Protein; Transcription Factor TFIIA; Transcription Factors; Virus Latency

This report deals with the cleavage reaction of calf thymus (CT) topoisomerase II with oligonucleotides containing one main cleavage site and adjacent binding sites for minor groove binders. The sequences of the oligonucleotides were derived from a pBR 322 sequence, which contains one main topoisomerase II cleavage site. The cleavage reaction was performed under increasing concentrations of minor groove binders and it showed characteristic inhibition dependences of topoisomerase II to the binding sites and to the binding length of the minor groove binders. The extension of the minor groove binder length on DNA from 4 to 10 base pairs (bp) by netropsin and bis-netropsin, respectively, causes a strong increase of the topoisomerase II cleavage inhibition. The same is observed by the introduction of a second minor groove binder sequence symmetrically positioned around the topoisomerase II main cleavage site. The combination of two different minor groove binders can lead to an increased topoisomerase II inhibition but also to a prevention of total inhibition as shown with chromomycin A3 and distamycin A at concentrations of 0.1 and 0.25 microM, respectively.

Topics: Animals; Autoradiography; Base Sequence; Binding Sites; Cattle; Chromomycin A3; Distamycins; DNA; DNA Topoisomerases, Type II; Enzyme Inhibitors; Indoles; Kinetics; Netropsin; Oligonucleotides; Topoisomerase II Inhibitors

The influence of netropsin (Nt), distamycin A (Dst-3) and chromomycin A3 (CHR) on the binding of gyrase from Streptomyces noursei to an 162 bp-fragment of pBR 322 containing a strong gyrase cleavage site and on the gyrase mediated cleavage of this fragment was analyzed. Binding of the enzyme to the fragment is effectively inhibited by the GC-specific drug CHR, but poorly influenced by Dst-3, while Nt is ineffective. Cleavage of the fragment catalysed by the enzyme is inhibited by all three ligands but to different extent. Dst-3 and Nt inhibit the enzyme cleavage reaction at 20- or 250-fold higher concentration than that required for CHR. The inhibitory mechanism of CHR on gyrase-DNA binding and cleavage may be related to a competitive interaction of the ligand to GC sequences located at and around the gyrase cleavage site. The fact that AT-specific minor groove binders Dst-3 and Nt poorly inhibit the binding of gyrase to the fragment due to the low amount of the AT basepair sequences contained in the fragment and their inhibitory influence on the cleavage step underlines the role of the DNA minor groove during enzyme action.

Topics: Base Sequence; Chromomycin A3; Distamycins; DNA Topoisomerases, Type II; DNA-Binding Proteins; DNA, Bacterial; Hydrolysis; Molecular Sequence Data; Netropsin; Protein Binding; Streptomyces

Upstream Binding Factor (UBF) is important for activation of ribosomal RNA transcription and belongs to a family of proteins containing nucleic acid binding domains, termed HMG-boxes, with similarity to High Mobility Group (HMG) chromosomal proteins. Proteins in this family can be sequence-specific or highly sequence-tolerant binding proteins. We show that Xenopus UBF can be classified among the sequence-tolerant class. Methylation interference assays using enhancer DNA probes failed to reveal any critical nucleotides required for UBF binding. Selection by UBF of optimal binding sites among a population of enhancer oligonucleotides with randomized sequences also failed to reveal any consensus sequence. The minor groove specific drugs chromomycin A3, distamycin A and actinomycin D competed against UBF for enhancer binding, suggesting that UBF, like other HMG-box proteins, probably interacts with the minor groove. UBF also shares with other HMG box proteins the ability to bind synthetic cruciform DNA. However, UBF appears different from other HMG-box proteins in that it can bind both RNA (tRNA) and DNA. The sequence-tolerant nature of UBF-nucleic acid interactions may accommodate the rapid evolution of ribosomal RNA gene sequences.

Topics: Animals; Base Sequence; Chromomycin A3; Dactinomycin; Distamycins; DNA; DNA-Binding Proteins; Enhancer Elements, Genetic; High Mobility Group Proteins; Methylation; Molecular Sequence Data; Nucleic Acid Conformation; Pol1 Transcription Initiation Complex Proteins; RNA Polymerase I; RNA, Transfer; Transcription Factors; Xenopus laevis

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.

Topics: Animals; Chromomycin A3; Chromosome Banding; Distamycins; Endodeoxyribonucleases; Ethidium; Female; Heterochromatin; Male; Triturus; X Chromosome; Y Chromosome

The employment of certain DNA-specific fluorescent stains on unbanded and C-banded chromosomes of two species of grasshoppers shows remarkable differences among C-heterochromatic regions supposed to be similar in their base pair composition, according to their response to the standard fluorescence techniques. The possible interspersion of the opposite DNA base pairs in these regions as well as the role played by proteins in chromosome banding are discussed.

Topics: Animals; Bisbenzimidazole; Chromomycin A3; Chromosome Banding; Chromosomes; Distamycins; Fluorescent Dyes; Grasshoppers; Heterochromatin; Staining and Labeling

Three different fluorochrome and specific counterstain combination (DAPI/AMD, DA/DAPI and CMA/DA) treatments were applied to the chromosomes of four Microtidae (Rodentia) species. The results complete the data obtained in our previous paper (Burgos, M., Jiménez, R., & Dìaz de la Guardia, R., Genome 30:540-546, 1988) and prove that the changes in the constitutive heterochromatin in the evolution of the karyotypes of these species are not only due to gain or loss of heterochromatin, but are qualitative with respect to their nucleotide composition, repeated base pair organization or DNA-protein complex modification. These variations lead to the differential response to the fluorescence dye combinations used.

Topics: Animals; Biological Evolution; Chromomycin A3; Chromosome Banding; Distamycins; Fluorescent Dyes; Heterochromatin; Metaphase; Rodentia

The chromosomes of the gorilla were extensively studied with various staining techniques labeling the different classes of heterochromatin. The chromosomal distribution of distamycin A/DAPI-, D287/170-, quinacrine-, and chromomycin A3-positive heterochromatic regions, as well as the nucleolus organizer regions, is described and compared with the karyotypes of other hominoid species. Lymphocyte cultures were treated with low doses of 5-azacytidine during the last hours of culture. This cytidine analog induces distinct undercondensation in 37 heterochromatic regions in the 24 gorilla chromosomes. The 5-azacytidine-induced undercondensations are localized not only in most of the distamycin A/DAPI-bright heterochromatic regions but also in many telomeric C-bands of the chromosomes. Furthermore, 5-azacytidine preserves the somatic pairing between heterochromatic regions from the interphase nuclei into the metaphase stage. The homeologies and differences in the chromosomal localization of the various classes of heterochromatin, 5-azacytidine-sensitive regions, 5-methylcytosine-rich DNA sequences, and satellite DNAs in the gorilla, chimpanzee, orangutan, and man are discussed.

Topics: 5-Methylcytosine; Animals; Azacitidine; Cells, Cultured; Chromomycin A3; Chromosome Banding; Cytosine; Distamycins; DNA; Female; Fluorescent Dyes; Gorilla gorilla; Heterochromatin; Indoles; Lymphocytes; Male; Nucleolus Organizer Region; Primates; Quinacrine; Species Specificity; Staining and Labeling

Metaphase chromosome bands were induced in Allium flavum (Liliaceae) by protecting the chromosomal DNA with DNA-binding compounds of different base specificities against DNase digestion. The bands obtained represented different subsets of C-band heterochromatin.

Topics: Chromomycin A3; Chromosome Banding; Dactinomycin; Deoxyribonucleases; Distamycins; DNA; Fluorescent Dyes; Karyotyping; Plants

DNase I and MPE.Fe (II) footprinting both employ partial cleavage of ligand-protected DNA restriction fragments and Maxam-Gilbert sequencing gel methods of analysis. One method utilizes the enzyme, DNase I, as the DNA cleaving agent while the other employs the synthetic molecule, methidium-propyl-EDTA (MPE). For actinomycin D, chromomycin A3 and distamycin A, DNase I footprinting reports larger binding site sizes than MPE.Fe (II). DNase I footprinting appears more sensitive for weakly bound sites. MPE.Fe (II) footprinting appears more accurate in determining the actual size and location of the binding sites for small molecules on DNA, especially in cases where several small molecules are closely spaced on the DNA. MPE.Fe (II) and DNase I report the same sequence and binding site size for lac repressor protein on operator DNA.

Topics: Base Composition; Base Sequence; Chemical Phenomena; Chemistry; Chromomycin A3; Dactinomycin; Deoxyribonuclease I; Distamycins; DNA; Edetic Acid; Endodeoxyribonucleases; Plasmids; Repressor Proteins; Substrate Specificity