Actinomycin D and 7-aminoactinomycin D binding to single-stranded DNA

The potent RNA polymerase inhibitors actinomycin D and 7-aminoactinomycin D are shown to bind to single-stranded DNAs. The binding occurs with particular DNA sequences containing guanine residues and is characterized by hypochromic UV absorption changes similar to those observed in interactions of the drugs with double-stranded duplex DNAs. The most striking feature of the binding is the dramatic (ca. 37-fold) enhancement in fluorescence that occurs when the 7-aminoactinomycin is bound to certain single-stranded DNAs. This fluorescence of the complex is also characterized by a 40-nm hypsochromic shift in the emission spectrum of the drug and an increase in the emission anisotropy relative to the free drug or the drug bound to calf thymus DNA. The fluorescence lifetimes change in the presence of the single-stranded DNA in a manner compatible with the intensity difference. Thus, there is an increase in the fraction of the emission corresponding to a 2-ns lifetime component compared to the predominant approximately 0.5-ns lifetime of the free drug. The 7-aminoactinomycin D comigrates in polyacrylamide gels with the single-stranded DNAs, and the fluorescence of the bound drug can be visualized by excitation with 540-nm light. The binding interactions are characterized by association constants of 2.0 x 10(6) to 1.1 x 10(7) M-1.     

A rapid procedure for the isolation of yeast mitochondrial DNA suitable for restriction fragment analysis

A method for the rapid isolation of mitochondrial DNA from the yeast Saccharomyces cerevisiae is described. Cells are first disrupted by vortexing with glass beads and the mitochondiral DNA is then extracted directly from the cell lysate by poly-l-lysine-kieselguhr-exchange chromatography. The method is unique from most other published procedures in that there is no requirement for the isolation of either a crude or purified mitochondrial preparation. Mitochondrial DNA isolated by this procedure is shown to yield restriction endonuclease fragment patterns identical to those obtained from DNA isolated by other previously reported procedures.     

A bacteriophage lambda DNA purification procedure suitable for the analysis of DNA from either large or multiple small lysates

A method for the efficient preparation of high quality bacteriophage lambda DNA from cleared lysates is described. Advantages of the method include high DNA yields (typically around 0.8 micrograms of DNA/1 ml of cleared lysate), speed of processing (approximately 2 h from lysate to DNA), economy, and the absence of any requirement for phenol or chloroform extractions. The technique involves the concentration of phage particles by standard polyethylene glycol precipitation followed by enzymatic treatment to remove contaminating RNA and DNA. Phage particles are then lysed with sodium dodecyl sulfate (SDS) at elevated pH and temperature. Contaminating protein/SDS complexes are rendered insoluble by the addition of potassium acetate and removed by centrifugation. The quality of the resultant DNA is comparable to that prepared by cesium chloride banding for all standard molecular biological purposes providing that spermidine is included in all restriction endonucleases digestions.     

7-Azido-Actinomycin D: A Photoaffinity Probe of the Sequence Specificity of DNA Binding by Actinomycin D

Abstract

Actinomycin D (ActD) is a DNA-binding antitumor antibiotic that appears to act in vivo by inhibiting RNA polymerase. The mechanism of DNA binding of ActD has attracted much attention because of its strong preference for 5′-dGpdC-3′ sequences. Binding is thought to involve intercalation of the tricyclic aromatic phenoxazone ring into a GC step, with the two equivalent cyclic pentapeptide lactone substituents lying in the minor groove and making hydrogen bond contacts with the 2-amino groups of the nearest neighbor guanines. Recent studies have indicated, however, that binding is also influenced by next-nearest neighboring bases. We have examined this higher order specificity using 7-azido-actinomycin-D as a photoaffinity probe, and DNA sequencing techniques to quantitatively monitor sites of covalent photoaddition. We found that GC doublets were strongly preferred only if the 5′- flanking base was a pyrimidine and the 3′-flanking base was not cytosine. In addition we observed a previously unreported preference for binding at a GG doublet in the sequence 5′- TGGG-3′.     

South western blot mapping: a procedure for simultaneous characterization of DNA binding proteins and their specific genomic DNA target sites

A method called "South Western blot mapping" for rapid characterization of both DNA binding proteins and their specific sites on genomic DNA is described. Proteins are separated on a sodium dodecyl sulfate (SDS) polyacrylamide gel, renatured by removing SDS in the presence of urea, and blotted onto nitrocellulose by diffusion. The genomic DNA region of interest is digested by restriction enzymes selected to produce fragments of appropriate but different sizes, which are subsequently end-labeled and allowed to bind to the separated proteins. The specifically bound DNA is eluted from each individual protein-DNA complex and analyzed by acrylamide gel electrophoresis. Evidence that tissue-specific DNA binding proteins may be detected by this technique is presented. Moreover, their sequence-specific binding allows the purification of the corresponding selectively bound DNA fragments and may improve protein-mediated cloning of DNA regulatory sequences.     

Survey and analysis of commercial cellulase preparations suitable for biomass conversion to ethanol

Commercial cellulase enzymes have been used in the food, detergent, and textile industries, and are potentially effective for processing biomass feedstocks. A survey was undertaken to identify major manufacturers/distributors of cellulases in the USA and to evaluate 13 representative commercial preparations for enzyme activity, protein concentration, and chemical composition. Samples were subjected to activity measurements using filter paper, carboxymethylcellulose, cellobiose, and p-nitrophenyl-β-d-glucopyranoside as substrates. To ascertain the microbial origin of the commercial preparations, Western blots utilizing monoclonal antibodies specific for Trichoderma reesei CBH I and Aspergillus niger β-d-glucosidase were developed. Eleven of the cellulases tested were of T. reesei or T. viride origin and two were from A. niger.     

Survey and analysis of commercial cellulase preparations suitable for biomass conversion to ethanol

Commercial cellulase enzymes have been used in the food, detergent, and textile industries, and are potentially effective for processing biomass feedstocks. A survey was undertaken to identify major manufacturers/distributors of cellulases in the USA and to evaluate 13 representative commercial preparations for enzyme activity, protein concentration, and chemical composition. Samples were subjected to activity measurements using filter paper, carboxymethylcellulose, cellobiose, and p-nitrophenyl--d-glucopyranoside as substrates. To ascertain the microbial origin of the commercial preparations, Western blots utilizing monoclonal antibodies specific for Trichoderma reesei CBH I and Aspergillus niger -d-glucosidase were developed. Eleven of the cellulases tested were of T. reesei or T. viride origin and two were from A. niger.     

Buccal DNA samples for DNA typing: new collection and processing methods

With the recent expansion of DNA database laws in many states, there is a critical need for the rapid and simple collection of DNA samples and streamlined processing for downstream applications. The Buccal DNA Collector was developed to address the need for a reliable, practical alternative to blood collection that is compatible with high-throughput operations. The collection area consists of filter paper that is placed against the inside of the cheek, and the sample is taken by swiping the cheek several times while pulling the device out of the mouth. Using this method, DNA profiles have been obtained from samples stored for 2 years at room temperature. Cells are collected on all regions of the filter paper with the maximum DNA recovery from the tip. The processing of DNA for DNA typing is accomplished with BodeElute, a new product that prepares DNA for amplification in a single 30-min heating step. Extracted DNA samples were successfully amplified with four commonly used multiplex short tandem repeat (STR) amplification kits. These products provide simplified approaches for collecting and processing buccal cell samples.     

Triostin A derived hybrid for simultaneous DNA binding and metal coordination

The natural product triostin A is known as an antibiotic based on specific DNA recognition. Structurally, a bicyclic depsipeptide backbone provides a well-defined scaffold preorganizing the recognition motifs for bisintercalation. Replacing the intercalating quinoxaline moieties of triostin A by nucleobases results in a potential major groove binder. The functionalization of this DNA binding triostin A analog with a metal binding ligand system is reported, thereby generating a hybrid molecule with DNA binding and metal coordinating capability. Transition metal ions can be placed in close proximity to dsDNA by means of non-covalent interactions. The synthesis of the nucleobase-modified triostin A analog is described containing a propargylglycine for later attachment of the ligand by click-chemistry. As ligand, two [1,4,7]triazacyclononane rings were bridged by a phenol. Formation of the proposed binuclear zinc complex was confirmed for the ligand and the triostin A analog/ligand construct by high-resolution mass spectrometry. The complex as well as the respective hybrid led to stabilization of dsDNA, thus implying that metal complexation and DNA binding are independent processes.     

A procedure for the small-scale isolation of plant RNA suitable for RNA blot analysis

A small-scale method for the isolation of total RNA from plant tissue is described. The method provides RNA of suitable quantity and quality from 0.2 g fresh tissue for the detection of mRNA species by RNA blot analysis. The entire procedure is adapted to 1.5-ml microfuge tubes and takes less than 5 h. This method is well suited for the isolation of RNA from large numbers of samples or from samples of limited quantity.     

Actinomycin D complexes with oligonucleotides as models for the binding of the drug to DNA. Paramagnetic induced relaxation experiments on drug-nucleic acid complexes.

Mn(II) ions have been used as a paramagnetic probe to investigate the geometry of drug-oligonucleotide complexes. Nuclear magnetic resonance and electron spin resonance experiments show that Mn(II) ions bind approximately two orders of magnitude stronger to the 5'-terminal phosphate group than to the 3'-5' phosphodiester linkage of deoxydinucleotides. By using mixtures of nucleotides in which only one nucleotide contains a terminal phosphate group, the location of the Mn(II) ion in the drug-nucleotide-Mn(II) complexes may be preselected. The paramagnetic induced relaxation of the nuclear spin systems in these complexes has been used to investigate the geometry of these complexes. These data confirm that actinomycin D is able to recognize and preferentially bind guanine (as opposed to adenine) nucleotides in the quinoid portion of the phenoxazone ring, while both adenine and guanine will bind to the benzenoid portion of the phenoxazone ring. These results suggest that stacking forces are primarily responsible for the general requirement of a guanine base when actinomycin D binds to DNA.     

Nonstacking Binding of 7-Aminoactinomycin D and Actinomycin D to DNA and Model Nucleotide Systems in Solutions

The 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 the phenoxazone chromophore of 7AAMD, which indicates the absence of stacking-like intercalation. This fact is fundamentally important to explain the biological effect of actinomycin on cells. A basic difference was revealed in the complex-forming properties of AMD and 7AAMD. Thus AMD is capable of binding to guanine micelles to destroy them; 7AAMD forms no complexes with either guanine micelles or polyguanylic acid. 7AAMD binding sites on DNA can differ substantially from AMD binding sites. However, strong competition is observed between AMD and 7AAMD for the binding site in oligonucleotide HP1 used as a DNA hairpin model. The effective diameters of 7AAMD–HP1 complex and free 7AAMD were determined using the Levshin–Perren equation.