PubMed · 4415352
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R W Hendrix, S R Casjens. 1974. Protein cleavage in bacteriophage lambda tail assembly.. https://doi.org/10.1016/0042-6822(74)90250-5
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Nuclease footprinting techniques were initially developed to investigate protein-deoxyribonucleic acid (DNA) interactions but these tools of molecular biology have also become instrumental for probing sequence-selective binding of small molecules to DNA. Here, the method is described and technical details are given for performing deoxyribonuclease (DNase) I footprinting with DNA-binding drugs. An example is presented where DNase I is used (as well as DNase II and micrococcal nuclease) to probe the patterns of sequence-selective recognition of DNA by the anticancer antibiotic actinomycin D. DNase I is a convenient endonuclease for detecting and locating the position of actinomycin-binding sites within GC-rich sequences.
A novel technique was developed to spatially map the phosphorus net influx capacity in intact root systems. The method is based on digital autoradiography and permits the quantification of phosphorus influx at high spatial resolution (2 mm). Roots of 18-d-old common bean plants were exposed to (32)P-labelled orthophosphate, quickly frozen, excised, lyophilized, scanned, and exposed to a storage phosphor screen. Plots of (32)P content versus root length (distance from the root tip or from the base of the root) were obtained for three different root classes: basal, basal laterals, and taproot laterals. Radioactivity detected by filmless autoradiography correlated well (r(2)=0.99) with measurements made by scintillation counting. Basal roots absorbed 2.5 times and 1.9 times more phosphorus than the taproot lateral and basal lateral root classes, respectively, in the first 20 mm from the root apex. External phosphorus markedly affected influx: roots averaged 5, 16, and 34 pmol P min(-1) in the apical 20 mm when exposed to 1, 5, and 10 microM P solutions, respectively. The spatial pattern of phosphorus influx along the root axes of the different root classes was rather homogeneous when measured on a root surface area basis. Phosphorus influx in the older segments of basal roots (those next to the hypocotyl) did not differ from the newer segments close to the root apex. However, a heterogeneous pattern was detected for basal roots when measured on a length basis, indicating that both root class and diameter constitute main factors controlling the spatial pattern of net influx.
Hydroxyl radical footprinting can probe the solvent accessibility of the ribose moiety of the individual nucleotides of DNA and RNA. Semi-automated analytical tools are presented for the quantitative analyses of nucleic acid footprint transitions in which processes such as folding or ligand binding are followed as a function of time or ligand concentration. Efficient quantitation of the intensities of the electrophoretic bands comprising the footprinting reaction products is achieved by fitting a series of Lorentzian curves to line profiles obtained from gels utilizing sequentially relaxed constraints consistent with electrophoretic mobility. An automated process of data 'standardization' has been developed that corrects for differences in the loading amounts in the electrophoresis. This process enhances the accuracy of the derived transitions and makes generating them easier. Together with visualization of the processed footprinting in false-color two-dimensional maps, DNA and RNA footprinting data can be accurately, precisely and efficiently processed allowing transitions to be objectively and comprehensively analyzed. The utility of this new analysis approach is illustrated by its application to the ion-meditated folding of a large RNA molecule.