A. H. Reisner

The use of Coomassie Brilliant Blue G250 perchloric acid solution for staining in electrophoresis and isoelectric focusing on polyacrylamide gels.

A method has been developed to stain rapidly protein zones not only in standard but also in isoelectric focusing polyacrylamide gels. It requires no destaining in either case. The technique makes use of the fact that the G250 form of Coomassie Brilliant Blue exhibits a color change in dilute perchloric acid which is reversed when the dye becomes bound to the protein. Under the conditions used, Ampholine shows little interference. In addition, the method selectively visualizes the arginine-rich histones because of the solubility of the lysine-rich histones in PCA.

The MTX package of computer programmes for the comparison of sequences of nucleotides and amino acid residues

A suite of some dozen programmes written in FORTRAN77 to run on VAX computers using the VMS operating system, and which utilizes a Digital Command Language (DCL) shell to allow it to be menu driven has been in use at the Division of Molecular Biology for about nine months. The package allows the user to obtain both dot matrix and line matrix plots, find and output specific regions of similarity and compute statistics for randomly generated sequences. In all these cases the user may specify either a maximum number of gaps in the match that will be tolerated or a minimum percentage similarity allowable for a match to be registered. The system allows the user to create a batch job for any of these analyses; so, for example, a number of line matrix plots can be specified from a remote alpha-numeric terminal which can be plotted later at a graphics terminal. In addition, computation of quasi-correlation statistics (Qr) for nucleotide sequences or correlation statistics (r) for amino acid residue sequences may be computed. Help facilities and documentation including examples are provided.

MBIS--an integrated system for the retrieval and analyses of sequence data from nucleic acids and proteins.

A computer-based system termed MBIS (the Molecular Biological Information Service), written in FORTRAN77 and Digital Command Language (DCL) and running on a Digital Equipment Corporation VAX computer under the VMS operating system (V4.1) is in use at the Division of Molecular Biology. MBIS consists of three main sections: 1) The utility section, used by the systems manager to tailor the five commonly available databases so that they are useable by the applications programmes running on the system; 2) The retrieval section, used to find and extract specific sequences or bibliographic information, and 3) The analytical section, used to analyse and compare sequences either extracted from the databases or input by the user. The nucleotide databases maintained are GenBank, EMBL and PIR (Protein Identification Resource, National Biomedical Research Foundation) and the peptide databases are PIR and NEWAT. In addition, users can originate and maintain their own databases. Those programmes which feature graphics output are compatible with most emulators of the Tektronix 4010 terminal.

Two- and three-dimensional image reconstructions from stained and autoradiographed histological sections

A complete system has been developed to utilize histological serial sections for two- and three-dimensional image reconstructions. Eighty to 120 sections are digitized using a personal computing system augmented with a imaging board and CCD camera. The image files are transmitted to a VAX computer for processing and image reconstruction, and the processed images are transmitted back to the personal computer for display and recording using a film recorder or PostScript printer. The software developed for the system allows serial sections to be placed into proper registration in a 256(3) array, 256 grey levels. Autoradiographs of the sections are obtained in the presence of appropriate standards which are used to recalibrate grey levels to represent linearly the radioactivity of each pixel in the sections and scale the values to allow maximum use of the grey scale. Starting from coronally sectioned material the system has been used to analyse and reconstruct rat nasal turbinates. In two dimensions horizontal and sagittal sections have been obtained while in three dimensions back-to-front and surface-rendered images have been constructed. Useful rendering of differential metabolic activity within an organ of complex geometry has been obtained, and there appears to be no reason why the system cannot be used for any material for which serial sectioning is appropriate.

Studies on polyribosomes of Paramecium: I. Effect of Monovalent Ions

Abstract The monovalent cations Li+, Na+, K+, Rb+, Cs+ and NH4+ and the monovalent anions F−, Cl−, Br− and CH3COO− were tested in the presence of 5 mM and 50 mM Mg2+ and Ca2+ for their effect on the polyribosomes of Paramecium aurelia. In the presence of Mg2+, ribosomes tended to dissociate from messenger RNA (mRNA) in the absence of monovalent cations or in the presence of Li+ or Na+. Little, if any, dissociation occurred when K+, Rb+, Cs+ or NH4+ was the cation. On the other hand, in the presence of Ca2+ only NH4+ gave polyribosome profiles comparable to those seen in the Mg2+ systems and it was possible to adduce an order of effectiveness for maintaining the ribosome-mRNA complex, namely, Li+ Monovalent anions were also found to exert differential effects. At concentrations of 100 and 200 mM the P. aurelia ribosome-polyribosome system appeared to be more tolerant of acetate than Cl− followed by Br− and lastly F−. F− at 50 mM caused complete loss of polyribosomal material while 100 mM F− caused dissociation of ribosomes into subunits. In no case were we able to produce the so-called type I ribosome-polyribosome profile (60 to 70 % polyribosomes, few if any monomeric ribosomes, 30 to 40 % ribosomal subunits) reported for E. coli. This finding may be due to the low concentration of Mg2+ required in Paramecium to maintain the ribosome intact.

Studies on the polyribosomes of Paramecium: II. Effect of divalent cations

Abstract The divalent cations Mg 2+ , Ca 2+ , Mn 2+ , Fe 2+ , Co 2+ , Ni 2+ , Cu 2+ , Zn 2+ , Sr 2+ , Cd 2+ and Ba 2+ were tested for their effect on the polyribosomes of Paramecium aurelia . Broadly, these cations could be classified into one of three groups and two subgroups: 1. (A) Mg 2+ , Ca 2+ and Mn 2+ : Relatively high concentrations (50 mM) were required in the homogenization fluid in order to maintain the ribosome-mRNA complex. 2. (B) 2.1. (1) Fe 2+ , Ca 2+ and Cd 2+ : At concentrations of 5 mM or less these cations caused near total loss of 80 S and polyribosomal material from the 3 000 g supernatant. 2.2. (2) Sr 2+ and Ba 2+ : At concentrations of 25 to 50 mM these cations caused near total loss of polyribosomal material and severe reduction in the amount of monoribosomes in the 3 000 g supernatant. 3

Utilization of sequence libraries on a 16-bit mini computer with particular reference to high speed searching

An interactive menu driven system of programmes written in Fortran and designed to utilize the three main nucleotide sequence libraries and one amino acid sequence library was developed to run on a small 16-bit mini computer with limited main memory and mass storage. The software uses a minimum of system function calls and should be transportable with minimal rewriting to micro computers. Software has also been written to create secondary data bases containing the nucleotide triplet values (4(3) classes) derived from the sequence libraries. Using this secondary set, a given sequence and its reversed complement, once reduced to their trinucleotide values, can be compared to all sequences present in the libraries in about forty minutes on a PDP 11/10 mini computer using the correlation statistic. Because the statistic in this case may not be assumed to be normally distributed, we have termed it a quasi correlation coefficient (Qr).

Analyses of DNA by automated logging and processing of data from the analytical ultracentrifuge.

Abstract Methods to log data from buoyant-density and zonal-sedimentation velocity centrifugations of DNA in an analytical instrument are described, as well as computer programs to analyze such data. It is simple and quick to determine modal molecular weights of DNA in neutral or alkaline solutions or buoyant densities in CsCl or Cs2SO4. An interactive program which simulates the Dupont curve resolver has also proved useful. Correction equations to convert scanner output to true absorbance were developed.

Two-dimensional abstract representations (signatures) of proteins

Algorithms have been developed to allow two-dimensional abstract representations of proteins based on: (i) a non-redundant subset of codons, (ii) hydropathy values of amino acids, (iii) the polarities of the amino acid residues and (iv) predicted secondary structures. In addition the suggestion of Gates on nucleic acid representation was implemented. The two-dimensional projections (signatures) that are obtained have the merit that several plots can be represented simultaneously for ready visualization. The approach appears useful in showing relationships among groups of proteins.