A physical study of the ovine genome.

The ovine genome has been divided into some seventy-five fractions using 3,6-bis(acetatomercurimethyl)dioxane (BAMD) in conjunction with Cs2SO4 density-gradient-equilibrium centrifugation. Distinct macromolecular populations detected have buoyant densities in CsCl of 1.700, 1.707, 1.714, 1.716, 1.717, 1.721, 1.724 and 1.725 g/cm3. The 1.724 g/cm3 material appears in a number of non-contiguous fractions obtained from BAMD-Cs2SO4 centrifugation suggesting its presence at a number of different sites in the genome. Within two regions of buoyant density (1.701 g/cm3 to 1.707 g/cm3 and 1.708 g/cm3 to 1.717 g/cm3) the analyses were unable to resolve discrete populations.

Apparent relatedness of the main component of ovine 1.714 satellite DNA to bovine 1.715 satellite DNA.

The nucleotide sequence of the principal component of ovine 1.714 g/cm3 satellite DNA was determined from a monomeric fragment inserted at the BamHI site of pBR322 and cloned in Escherichia coli strain RR1. The 816-bp tandemly repeated sequence contains a number of small repeated sequences dispersed within it, one group of which forms a pentameric tandem repeat of a 13-bp segment (positions 548-612). A 20-bp region (60-79) shows an 85% homology with the reverse-complement of the sequence from 455 through 474. There are two regions of 67 bp (75-141) and 59 bp (755-813) which show greater than 70% homology with regions of bovine 1.715 g/cm3 satellite DNA (1402 bp; positions 1218-1284 and 1079-1137, respectively) while a 31-bp region (ovine 62-92, bovine 133-163) shows 80% homology. Quasi-correlation coefficients (Qr) were determined using the triplet numbers of the sheep satellite versus all sequences in the National Biomedical Research Foundation and EMBL nucleotide sequence data bases. Qr equals 0.85 for ovine 1.714 g/cm3 satellite versus bovine 1.715 g/cm3 satellite. The next highest Qr for a bovine satellite segment was 0.58. Thus, the ovine 1.714 g/cm3 and bovine 1.715 g/cm3 satellite appear demonstrably related. Taking into account that sheep and cattle diverged 18-20 million years ago, this suggests that the material may be functional and that its function is related to its sequence.

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).

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 system's 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.

The use of various properties of amino acids in color and monochrome dot-matrix analyses for protein homologies.

Software has been developed to allow the use of a number of parameters in the comparative representation of proteins in color and monochrome dot matrices. They include the parameters of partial specific volume, residue bulkiness, the mean area buried of side chains, seven additional hydropathy scales, mutability, polarity, secondary structure propensities, energy/residue, energy/atom, Rf values, the pKs at the N and C terminals, user-defined parameters and, if desired, randomly generated values. Many of these parameters can be combined in n space using an algorithm based on the Euclidian distance relationship in order to derive consensus values. The problem of scoring matched identities is addressed and the user may stipulate that they score 100 on a 0-100 scale or be determined from the Dayhoff MDM78 values with the rest of the matrix scaled appropriately. The PAMs matrix has been incorporated in such a way to allow the user to stipulate various PAM's values or estimated percentage difference between two peptide sequences, and converting to log odds values. In addition, the similarity ring developed by Swanson and the matrix proposed by Bacon and Anderson have been adapted for use in the program. Color indices have been utilized to give a 'third dimension' to the projections, allowing the user to judge the degree of similarity of different regions which are represented. The software also provides for the plotting of nucleotides in which case color is used to code individual nucleotides, purines versus pyrimidines, or similar colors are used to differentiate between A and T bases on the one hand, and G and C on the other.

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.

Physical analysis of a (dG + dC)-rich fraction of DNA obtained from the ovine genome.

Using the organomercuric compound 3,6-bis(acetatomercurimethyl)dioxane in conjunction with Cs2SO4 density gradient equilibrium centrifugation a (dG + dC)-rich DNA fraction constituting 10% of the ovine genome was separated from the remainder. Further fractionation allowed four distinct classes of DNA to be identified with buoyant densities in neutral CsCl of 1.714, 1.717, 1.725 and 1.716 g/cm3. Computerized curve resolving of the data indicated the presence of several additional DNA classes. Data obtained using restriction endonuclease digestion indicated that the 1.714 g/cm3 satellite consists principally of an 820-base pair (bp) tandemly repeating unit. The 820-bp DNA is also present in the 1.717 g/cm3 satellites but as a minor component. The principal components of the 1.717 and 1.725 g/cm3 fractions are 125-, 176- and 235-bp fragments. In addition, these fractions contain 705-bp tandemly repeated material. Two or possibly three species of 22-bp tandem repeats were found only in the 1.725 g/cm3 DNA.

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.