RESUMO
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.
Assuntos
RNA/isolamento & purificação , Ovinos/genética , Animais , Centrifugação com Gradiente de Concentração , DNA/isolamento & purificação , DNA/farmacologia , Dioxanos , Compostos de MetilmercúrioRESUMO
Total human DNA was fractionated from the three types of Cs2SO4 gradient used to prepare satellites I, II and III. Three satellite DNAs were found: satellite I with a mean buoyant density of 1.6888 g/ml comprising about 1.3% of the total, satellite II with a mean buoyant of 1.696 g/ml, comprising about 1% of the total an satellite III with a mean buoyant density of 1.699 g/ml comprising about 2.2% of the total. The buoyant densities of these satellites after purification were 1.686, 1.694 and 1.697 m/gl, respectively. A preparation with the attributes of satellite IV was isolated from the shoulder region of a satellite III preparative gradient. In situ hybridization using complementary RNA showed that the three satellites were located predominantly on chromosomes 9, Y, 15 and 1. Satellite II also showed marked hybridization to chromosome 16. Satellites I and II and III cross-hybridized to each other but satellites I and II did not. On the basis of our hybridization data, we suggest that some of the same sequences which comprise satellite III also comprise satellite I an II.
Assuntos
DNA Satélite/análise , Animais , Sequência de Bases , Centrifugação com Gradiente de Concentração , Feminino , Humanos , Cariotipagem , Masculino , Hibridização de Ácido Nucleico , Placenta/análise , GravidezAssuntos
Reações Antígeno-Anticorpo , Antígenos , Paramecium/imunologia , Imobilização , Soros ImunesAssuntos
Antígenos , Substâncias Macromoleculares , Paramecium , Peptídeos , Proteínas , Acrilatos , Alquilação , Animais , Fenômenos Químicos , Química , Cromatografia em Gel , Eletroforese , Géis , Concentração de Íons de Hidrogênio , Peso Molecular , Solubilidade , Amido , Sulfetos , Ultracentrifugação , Viscosidade , ÁguaAssuntos
Paramecium/análise , RNA/análise , Ribossomos/análise , Animais , Densitometria , Deutério , Eletroforese , Eletroforese Descontínua , Técnicas In Vitro , Magnésio , Métodos , Peso Molecular , Paramecium/citologia , Proteínas/análise , Ribonucleases/metabolismo , Sacarose , UltracentrifugaçãoRESUMO
An analysis of the 1,217-amino acid residue sequence of the precursor of mouse epidermal growth factor (mEGF) revealed regions of considerable similarity with bovine factor X, a blood coagulation factor. Similarities of mEGF itself with factor X, pancreatic secretory trypsin inhibitor and, most strikingly, transforming growth factor I (TGF-I) have been observed. On the basis of the comparisons described here, it seems that the presumptive 140-residue 19K early protein (relative molecular mass (Mr) 19,000) of vaccinia virus from residues 40-91 shows an overall identity of 36% (19/53 residues) with both mEGF and urogastrone (human epidermal growth factor, hEGF); a single deletion is assumed for vaccinia virus 19K protein which allows the six Cys residues (positions 45-80) to be aligned with those of mEGF or hEGF. This protein is encoded in the 10.3-kilobase (kb) inverted terminal repeat. Because it is an early protein with an EGF-like central portion, the 19K vaccinia virus protein may have an autocrine function and may be required for DNA synthesis.
Assuntos
Fator de Crescimento Epidérmico , Substâncias de Crescimento , Vaccinia virus/fisiologia , Proteínas Virais , Sequência de Aminoácidos , Peso Molecular , Conformação ProteicaRESUMO
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.
Assuntos
Sequência de Aminoácidos , Software , Algoritmos , Cor , Homologia de Sequência do Ácido NucleicoRESUMO
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.
Assuntos
Algoritmos , Proteínas , Aminoácidos , Fenômenos Químicos , Físico-Química , Códon , Conformação Proteica , Proteínas/genéticaRESUMO
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.
Assuntos
Sequência de Aminoácidos , Sequência de Bases , Computadores , SoftwareRESUMO
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.
Assuntos
Sequência de Aminoácidos , Sequência de Bases , Computadores , Software , Sistemas de Informação , Ácidos Nucleicos/análise , Proteínas/análiseRESUMO
When Paramecium tetraurelia in log phase growth is treated with 4% dimethyl sulphoxide (DMSO) for five minutes the amount of polyribosomes is reduced 3- to 4-fold while there is a corresponding increase in 80s ribosomal material. Reducing the concentration of DMSO to 1% allows immediate reversal of the condition. Paramecium polyribosomes subjected to 4% DMSO either in whole cell homogenates or during purification through sucrose density gradients appear unaffected while cycloheximide at concentrations up to 100 mug/ml did not prevent DMSO from exerting its effect in vivo. Analyses of 14C amino acid incorporation experiments indicated a strict correspondence between the effect of DMSO on polyribosomes and overall protein synthesis. The reduction of acid precipitable radioactivity in the polyribosomal region after DMSO treatment was associated with a corresponding increase in radioactivity in the 80s region. There was no comparable increase in the acid precipitable radioactivity in the soluble fraction. The overall results of the study suggest that DMSO acts on polyribosommes indirectly through some unknown primary reaction with cell constitutents, and that the mode of action is such as to cause the release of ribosomes from messenger RNA (mRNA) rather than to prevent initiation of the ribosome-mRNA complex. Our data suggest that the effect may be selective. Finally, it is of interest that high concentrations of DMSO (above 8%) appear to have the opposite effect of lower concentrations of DMSO, i.e., they appear to "freeze" the ribosomes to mRNA.
Assuntos
Dimetil Sulfóxido/farmacologia , Paramecium/efeitos dos fármacos , Polirribossomos/efeitos dos fármacos , Biossíntese de Proteínas , Animais , Cicloeximida/farmacologia , Concentração de Íons de Hidrogênio , Paramecium/metabolismo , Polirribossomos/metabolismo , TemperaturaRESUMO
The soluble surface proteins isolated by Cross (1975) from Trypanosoma brucei are compared to those which occur on Paramecium aurelia. Calculations based on the amino acid residue data and the molecular weights of these surface antigens indicate that there is a considerable degree of similarity between the T. brucei and the P. aurelia proteins, and that in neither case can simple mutation be responsible for the differences of molecules within each family of antigens. The analyses are consistent with the suggestion that the P. aurelia system can serve as a model for the phenomenon of phase transformation (relapse syndrome) which occurs in trypanosomiasis.
Assuntos
Antígenos/análise , Paramecium/imunologia , Proteínas/análise , Trypanosoma brucei brucei/imunologia , Aminoácidos/análise , Animais , Membrana Celular/imunologia , Conformação Proteica , SolubilidadeRESUMO
Using as a primary standard a representative set of 208 proteins whose amino-acid-residue mole frequencies have been accurately established, a set of standard distributions of mole frequencies is defined for each amino acids, in terms of which percentile values for the observed mole frequencies of the amino-acid residues in any other protein can be determined. Data so transformed have a distribution much closer to Gaussian than untransformed values, and allow meaningful determinations of correlations between the amino-acid-residue compositions of two proteins as well as between pairs of amino-acid-residues within groups of proteins. Of the 153 possible pairs of amino acids (Asx and Glx are used) 39 are significantly correlated at p less than or equal to 0.01 and 22 at p less than or equal to 0.001. A percentile table is included for those wishing to use the method with programmable calculators. The transformed data for amino-acid compositions have been used to perform principal components analyses on groups of proteins in order to determine if meaningful sub-groupings (observable clusters in scatter diagrams) were detectable. Such analyses are shown for the representative set of proteins and for a group of 184 globins. With regard to the globin chains, a correlation is observed for alpha chains in the first principal component projection (PCP), (accounting for 22% of the variance) with respect to the evolutionary time-scale while beta chains show such a correlation in the first and second PCPs (22% and 18% of the variance respectively). Thus, alpha and beta chains appear to diverge from a common progenitor, similar in position to globin chains from "primitive" forms. Furthermore, globins from "primitive" forms are nearer to one another than they are to globins from the vertebrates, a finding without a priori reason, suggesting perhaps that once a chain has reached a stable relationship with its environment, strong constrains are placed on the co-existing globin chains so that they maintain appropriate interaction with one another. In addition, positions of the epsilon, gamma and delta chains are in the order: epsilon (embryonal) more primitive than gamma (foetal) more primitive than delta equal to beta (adult).
Assuntos
Aminoácidos/análise , Evolução Biológica , Proteínas/genética , Animais , Computadores , Globinas/genética , Substâncias Macromoleculares , Matemática , Modelos GenéticosRESUMO
Although no major structural or numerical abnormalities were found in the karyotypes of 12 aborted equine fetuses, two unrelated abortuses each carried a large polymorphism for the amount of heterochromatin in chromosome 1. In both karyotypes this chromosome was shown to be larger than its homolog. To determine the nature of the extra DNA in these chromosomes, equine DNA was isolated and characterized by buoyant density analysis. Equine mainband DNA had a buoyant density in neutral CsCl of 1.699 g/cm3, while the highly repetitive (dG+dC)-rich fraction had a buoyant density of 1.715 g/cm3. A radioactive RNA probe complementary to the purified satellite fraction was used for in situ hybridization to chromosomal spreads containing the enlarged chromosome 1. The results indicated that an increase in highly repetitive (dG+dC)-rich DNA was responsible for the increase in the size of the abnormal No. 1 chromosomes. While two of the 12 aborted fetuses exhibited marked heterochromatic dimorphisms, none of the karyotypes obtained from individuals with no family history of abortion exhibited such obvious polymorphisms.
Assuntos
Aborto Animal/genética , DNA/genética , Doenças dos Cavalos/genética , Animais , Aberrações Cromossômicas , Feminino , Feto , Idade Gestacional , Cavalos , Cariotipagem , Polimorfismo Genético , Gravidez , Cromossomos SexuaisRESUMO
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.
Assuntos
DNA Satélite , Animais , Sequência de Bases , Evolução Biológica , Bovinos , Sequências Repetitivas de Ácido Nucleico , Ovinos , Especificidade da EspécieRESUMO
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).
Assuntos
Sequência de Aminoácidos , Sequência de Bases , Computadores , Sistemas de Informação , Software , Genes , Genes Virais , Hemaglutininas Virais/genéticaRESUMO
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.
Assuntos
DNA , Desoxicitidina/análise , Desoxiguanosina/análise , Genes , Animais , Composição de Bases , Enzimas de Restrição do DNA , DNA Satélite , Peso Molecular , Desnaturação de Ácido Nucleico , OvinosRESUMO
In an extensive analysis, using a range of restriction endonucleases, HinfI and TaqI were found to differentiate satellites I, II and III & IV. Satellite I is resistant to digestion by TaqI, but is cleaved by HinfI to yield three major fragments of approximate size 770, 850 and 950bp, associated in a single length of DNA. The 770bp fragment contains recognition sites for a number of other enzymes, whereas the 850 and 950bp fragments are "silent" by restriction enzyme analysis. Satellite II is digested by HinfI into a large number of very small (10-80bp) fragments, many of which also contain TaqI sites. A proportion of the HinfI sites in satellite II have the sequence 5'GA(GC)TC. The HinfI digestion products of satellites III and IV form a complete ladder, stretching from 15bp or less to more than 250bp, with adjacent multimers separated by an increment of 5bp. The ladder fragments do not contain TaqI sites and all HinfI sites have the sequence 5'GA(AT)TC. Three fragments from the HinfI ladder of satellite III have been sequenced, and all consist of a tandemly repeated 5bp sequence, 5'TTCCA, with a non-repeated, G+C rich sequence, 9bp in length, at the 3' end.
Assuntos
DNA Satélite , Composição de Bases , Enzimas de Restrição do DNA , Feminino , Humanos , Leucemia , Masculino , Placenta/análise , Gravidez , Sequências Repetitivas de Ácido NucleicoRESUMO
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.