Computer assisted analysis of S-potentials

S-potentials from the channel catfish were analyzed using a high speed biological data processing system developed at this Institute. The analysis has shown that, when moderately dark adapted, the relation between the (maximal) amplitude of S-potential and log intensity of flash was the hyperbolic tangent throughout the whole visible spectrum. This is what one could expect if the S-potential in the fish was generated by signals from a single class of pigment and each signal had discrete action on the S-potential generating mechanism. The maximal absorption of the pigment involved was at around 625 nm.     

Computer assisted floristic similarity analysis

A program for polythetic numerical evaluation of phytosociological material is described. Using Sörensen's coefficient of floristic similarity it computes the homogeneity of subjectively chosen sets of relevés, the affinity of each constituent relevé to the set as well as the similarity between any pair of sets. It is also able to plot a dendrogram of the hierarchic system obtained by agglomerating individual relevés into complex groupings. The program is suitable for processing phytosociological data arranged in any conventional table.     

Quantitative footprinting analysis

This review outlines the steps for obtaining relative constants for drugs from footprinting data. After correcting the autoradiographic spot intensities for differing amounts of radioactive DNA loaded into the lanes of a sequencing gel, footprinting plots, showing individual spot intensities as a function of drug concentration, are constructed. The initial relative slopes of footprinting plots are proportional to the binding constant of the drug for its DNA sites. Slopes of plots outside the drug binding sites can be used to identify locations of altered DNA structure. It illustrates the power of quantitative footprinting analysis by analyzing the binding of the antiviral agent netrospin to a 139-base pair restriction fragment in the presence of the antitumor agent actinomycin D. While two netrospin binding regions are unaffected by actinomycin D a third region experiences enhanced binding in the presence of the antitumor agent.     

Computer assisted karyotype analysis of Pyrrhopappus carolinianus

With the help of Computer Aided Karyotyping procedures, Ag-NOR staining and C-banding techniques, the karyotype of Pyrrhopappus carolinianus (Asteraceae, Lactuceae) has been studied. The species has 2n=12 chromosomes. Silver staining reveals that the two shortest pairs of chromosomes possess NOR's. On the basis of chromosome length and centromere position, only the longest chromosome pair and the satellite chromosomes can be identified. Two types of C-banding can be obtained, dependent on the temperature of the hydrochloric acid hydrolysis of the root tips. Hydrolysis at 60°C results exclusively in centromeric bands, whereas a treatment at room temperature reveals a pattern of intercalary bands. A computer assisted analysis of the intercalary banding pattern resulted in the construction of schematic representation of the average C-banding pattern. This banding pattern allows an easy identification of each of the chromosome pairs.     

Computer assisted management of warfarin treatment.

Maintenance of anticoagulation is laborious and costly, and the results are often indifferent. An automatic system which adjusts the dose of warfarin has been designed using a formula devised after a survey of prescribing habits. Programs running on a microcomputer maintain a file of the necessary information and deal with the bulk of the weekly clinic. Dosage is advised, the date of the next visit determined, and the file updated. A doctor uses the console to advise some 10% of patients reserved for special reasons. The system produces clinic and ambulance lists, copies of the advice sent to patients, and, as a protection against machine failure, a weekly copy of the updated file. The results after 16 months are at least as good as those achieved manually. Medical and secretarial time is saved, and statistics about the clinic and its efficacy are made available.     

Computer assisted shared care in hypertension.

A computer assisted shared care scheme for the long term management and follow up of hypertensive patients has been developed in the Grampian Region. The scheme aims at facilitating the exchange of clinically important information between doctors and at achieving target levels of blood pressure with treatment in patients at highest risk of cardiovascular events. The shared care scheme has been well received by the local practitioners. Two hundred and fifty seven patients (18%) of 1426 patients under current long term follow up are assigned to follow up in the hospital aspect of the scheme. At the most recent visit 32% of patients in the hospital aspect and 10% of 1169 patients in the general practice aspect had blood pressure recordings above the target levels of 160/95 mm Hg. The stratification of patients formerly attending hospital clinics into grades of risk has rationalised our follow up procedures to allow the specialist resources to be freed and concentrated on those patients at highest risk and with the most complex problems. This computer assisted patient records system could be applied to other groups of high risk patients in whom long term follow up and surveillance are necessary--for example, patients with diabetes mellitus--and has implications for optimising and monitoring the delivery and outcome of care without overwhelming limited hospital resources.     

Quantitative footprinting analysis. Binding to a single site

The theory for measuring ligand binding constants from footprinting autoradiographic data associated with a single binding site is derived. If the ligand and DNA cleavage agent compete for a common site, the spot intensities are not proportional to the amount of DNA not blocked by ligand. The analysis of a single site is experimentally illustrated by using results for the anticancer drug actinomycin D interacting with the duplex d(TAGCGCTA)2 as probed with the hydrolytic enzyme DNase I.     

Theoretical analysis of the footprinting experiment

In the footprinting experiment, an end-radiolabeled DNA restriction fragment is subjected to digest by an endonuclease in the presence and absence of a ligand which alters the endonuclease cleavage rate at sites of ligand-DNA contact. The location of these sites, and the strength of the ligand binding, are then deduced from the measured concentrations of the different oligonucleotides produced by the digest. We analyze the experiment in terms of coupled kinetic equations which take into account the cutting rates of endonuclease for sites with ligand present and absent, and the rates of binding and dissociation of the ligand to a site. As long as the ligand concentration remains essentially constant (which occurs, for example, if digest is terminated early enough to assure that all fragments result from single cuts by the endonuclease), the oligonucleotide concentrations reflect only the ligand binding equilibrium constant (ratio of rate constants) and the cutting rates in the presence and absence of ligand. We also show how the measured oligonucleotide concentrations (from, e.g. an autoradiogram) can be used to deduce the ligand equilibrium binding constants for the various sites on the polymer.     

Computer assisted neuron searching

A microprocessor controlled driver for the Kopf hydraulic microdrive is described. Neuron searching parameters can be specified during an experiment to facilitate the search for active neurons. These parameters include: period between movements, numbers of microns of electrode movement/advance, depth of penetration, automatic cessation of advacement when activity is encountered, and slow or rapid rates of movement. In addition, analog voltage outputs are available that are proportional to impulse frequency and to total impulse count.     

Quantitative footprinting analysis of the chromomycin A3--DNA interaction.

Chromomycin A3 (CHR) binding to the duplex d(CAAGTCTGGCCATCAGTC).d(GACTGATGGCCAGACTTG) has been studied using quantitative footprinting methods. Previous NMR studies indicated CHR binds as a dimer in the minor groove. Analysis of autoradiographic spot intensities derived from DNase I cleavage of the 18-mer in the presence of various amounts of CHR revealed that the drug binds as a dimer to the sequence 5'-TGGCCA-3',3'-ACCGGT-5' in the 18-mer with a binding constant of (2.7 +/- 1.4) x 10(7) M-1. Footprinting and fluorescence data indicate that the dimerization constant for the drug in solution is approximately 10(5) M-1. Since it has been suggested that CHR binding alters DNA to the A configuration, quantitative footprinting studies using dimethyl sulfate, which alkylates at N-7 of guanine in the major groove, were also carried out. Apparently, any drug-induced alteration in DNA structure does not affect cleavage by DMS enough to be observed by these experiments.     

SAFA: semi-automated footprinting analysis software for high-throughput quantification of nucleic acid footprinting experiments

Footprinting is a powerful and widely used tool for characterizing the structure, thermodynamics, and kinetics of nucleic acid folding and ligand binding reactions. However, quantitative analysis of the gel images produced by footprinting experiments is tedious and time-consuming, due to the absence of informatics tools specifically designed for footprinting analysis. We have developed SAFA, a semi-automated footprinting analysis software package that achieves accurate gel quantification while reducing the time to analyze a gel from several hours to 15 min or less. The increase in analysis speed is achieved through a graphical user interface that implements a novel methodology for lane and band assignment, called "gel rectification," and an optimized band deconvolution algorithm. The SAFA software yields results that are consistent with published methodologies and reduces the investigator-dependent variability compared to less automated methods. These software developments simplify the analysis procedure for a footprinting gel and can therefore facilitate the use of quantitative footprinting techniques in nucleic acid laboratories that otherwise might not have considered their use. Further, the increased throughput provided by SAFA may allow a more comprehensive understanding of molecular interactions. The software and documentation are freely available for download at http://safa.stanford.edu.     

Quantitative footprinting analysis of the netropsin-DNA interaction

The results of a series of quantitative footprinting experiments of the netropsin-DNA interaction as studied using two different DNA cleaving probes, the enzyme DNase I and a cationic manganese porphyrin complex, are described. Plots of the relative change in oligonucleotide concentration as a function of drug concentration, covering approximately 110 base pairs of a DNA restriction fragment, revealed netropsin induced changes in the cleavage rates of both probes. These appeared as inhibitions for the binding sites, enhancements where no binding took place, and enhancement/inhibitions for the weak binding sites. Determination of the concentration of drug necessary to reduce the amount of a particular oligomer to half of its initial value allowed a ranking of the affinities of the various binding sites on the fragment. In addition to uncovering the location of a number of overlapping netropsin binding sites, the data allowed additional insight on the manner in which both probes alter their DNA cleavage rates in the drug-footprinting experiment.     

Solid phase technology improves coupled gel shift/footprinting analysis.

For the analysis of protein-DNA interactions by coupled gel-shift/footprinting, DNA fragments need to be extracted from polyacrylamide gels and subsequently separated on high resolution gels. Due to impurities in the extracted DNA, single nucleotide resolution is frequently not achieved. We now describe an improved experimental strategy that employs transient coupling of DNA fragments to a solid support in order to extract DNA of high purity quantitatively, rapidly and reliably. As an example, we describe the application of our protocol to the 'in-gel footprinting' by copper phenanthroline. The method should also find application to the chemical interference assays.