An overview of the BioExtract Server: a distributed, Web-based system for genomic analysis.

Genome research is becoming increasingly dependent on access to multiple, distributed data sources, and bioinformatic tools. The importance of integration across distributed databases and Web services will continue to grow as the number of requisite resources expands. Use of bioinformatic workflows has seen considerable growth in recent years as scientific research becomes increasingly dependent on the analysis of large sets of data and the use of distributed resources. The BioExtract Server (http://bioextract.org) is a Web-based system designed to aid researchers in the analysis of distributed genomic data by providing a platform to facilitate the creation of bioinformatic workflows. Scientific workflows are created within the system by recording the analytic tasks preformed by researchers. These steps may include querying multiple data sources, saving query results as searchable data extracts, and executing local and Web-accessible analytic tools. The series of recorded tasks can be saved as a computational workflow simply by providing a name and description.

Spliceosomal proteins in plants.

The spliceosome is a large nuclear structure consisting of dynamically interacting RNAs and proteins. This chapter briefly reviews some of the known components and their interactions. Large-scale proteomics and gene expression studies may be required to unravel the many intricate mechanisms involved in splice site recognition and selection.

Patchiness and correlations in DNA sequences.

The highly nonrandom character of genomic DNA can confound attempts at modeling DNA sequence variation by standard stochastic processes (including random walk or fractal models). In particular, the mosaic character of DNA consisting of patches of different composition can fully account for apparent long-range correlations in DNA.

Chance and statistical significance in protein and DNA sequence analysis.

Statistical approaches help in the determination of significant configurations in protein and nucleic acid sequence data. Three recent statistical methods are discussed: (i) score-based sequence analysis that provides a means for characterizing anomalies in local sequence text and for evaluating sequence comparisons; (ii) quantile distributions of amino acid usage that reveal general compositional biases in proteins and evolutionary relations; and (iii) r-scan statistics that can be applied to the analysis of spacings of sequence markers.

Immunity to murine breast cancer cells modified to express MUC-1, a human breast cancer antigen, in transgenic mice tolerant to human MUC-1.

The high incidence of breast cancer in women and the severity of the disease have stimulated a need for improved and novel forms of therapy. The product of the MUC-1 gene has been identified as a breast cancer-associated antigen in breast cancer patients. The gene has been cloned and sequenced. Transgenic mice were prepared that express human mucin and are naturally tolerant to the molecule, providing a unique opportunity to investigate immunotherapeutic strategies in experimental animals that might eventually be applied to breast cancer patients. A cell line (410.4) derived from a mouse mammary adenocarcinoma that arose in a BALB/c mouse was transduced with a retroviral vector (R1-MUC1-pEMSVscribe) that encoded MUC-1. After confirmation of the expression of human mucin, the cells (E3) were further modified by transduction with retroviral vectors encoding interleukin (IL)-2, IL-4, IL-12, or IFN-gamma to evaluate the effect of cytokine-secretion on the immunogenic properties of the cells in the MUC-1 transgenic mice. The results indicated that modification of the breast cancer cells to secrete IL-12 reduced and at times eliminated the tumorigenic growth properties of the cells. Under similar circumstances, progressively growing tumors formed in MUC-1 transgenic mice that received injections of unmodified E3 cells or with E3 cells modified to secrete IL-2, IL-4, or IFN-gamma. Immunity to breast cancer developed in MUC-1 transgenic mice that had rejected IL-12-secreting E3 cells because the animals were resistant to challenge with (non-cytokine-secreting) E3 cells. In vitro analyses confirmed the presence of T cell-mediated cytotoxicity toward the breast cancer cells in MUC-1 transgenic mice immunized with the IL-12-secreting cells. Our data obtained in a unique animal model system point toward an analogous form of therapy for breast cancer patients.

Charge configurations in oncogene products and transforming proteins.

Statistically significant charge clusters are of infrequent occurrence in all kinds of proteins. In the six standard classes of proto-oncogene products, all of the nuclear class contain a significant charge cluster and several, but not all, of the transmembrane class do, whereas significant charge clusters or patterns are not found in protooncogenes of primarily cytoplasmic location, nor in membrane-bound (src-like) proto-oncogenes, nor in those of the ras family. Among nuclear oncogene families, such as myc, jun, fos, myb, or ets-related, and among homologous proteins across species, the significant charge clusters are part of the most conserved region. These gene families generally have similar charge distributions embodying a significant charge cluster, not of an invariant sign, preceded by a substantial uncharged stretch of predominantly polar residues. The nuclear transforming proteins p53 and p68 also contain significant charge clusters together with long uncharged segments, suggestive of a modular structure of these proteins. The transmembrane oncogene c-mas contains a mixed charge cluster and c-fms displays an unusual (0, +)7 pattern, in both cases positioned within their intracellular activating domain. Distinctive charge configurations for excreted proto-oncogenes are of a mixed character. Possible functions, mechanisms, and associated experimental procedures for studying proteins with anomalous charge distributions are discussed.

Gene structure prediction by spliced alignment of genomic DNA with protein sequences: increased accuracy by differential splice site scoring.

Gene identification in genomic DNA from eukaryotes is complicated by the vast combinatorial possibilities of potential exon assemblies. If the gene encodes a protein that is closely related to known proteins, gene identification is aided by matching similarity of potential translation products to those target proteins. The genomic DNA and protein sequences can be aligned directly by scoring the implied residues of in-frame nucleotide triplets against the protein residues in conventional ways, while allowing for long gaps in the alignment corresponding to introns in the genomic DNA. We describe a novel method for such spliced alignment. The method derives an optimal alignment based on scoring for both sequence similarity of the predicted gene product to the protein sequence and intrinsic splice site strength of the predicted introns. Application of the method to a representative set of 50 known genes from Arabidopsis thaliana showed significant improvement in prediction accuracy compared to previous spliced alignment methods. The method is also more accurate than ab initio gene prediction methods, provided sufficiently close target proteins are available. In view of the fast growth of public sequence repositories, we argue that close targets will be available for the majority of novel genes, making spliced alignment an excellent practical tool for high-throughput automated genome annotation.

Kinetics of complementary RNA-RNA interaction involved in plasmid ColE1 copy number control.

Binding of a small antisense RNA (RNA I) to the primer transcript (RNA II) of plasmid ColE1 inhibits formation of primer for DNA polymerase I-mediated plasmid replication. It is thought that RNA I and RNA II transiently interact via their single-stranded loop regions to form an unstable complex that subsequently converts into a more stable complex by hybridization. Rom (or Rop) protein enhances the inhibitory effect of RNA I on replication by enhancing the binding of the two RNAs. In this paper, we develop a model for the kinetics of the RNA I-RNA II binding reaction, estimate the rate constants, and provide a quantitative description of the effects of Rom protein. We show that the reaction kinetics are consistent with a stepwise binding model in which Rom protein binds to RNA I and RNA II, while the RNAs are held together in a transient complex. Mutations that replace C.G pairs by T.A pairs in the RNA loop regions and thus display weaker hydrogen bonding between the loop regions should be associated with an increased rate of dissociation for the unstable complex. Our model predicts that such destabilization of the loop interactions leads to a greater enhancement in the binding rate by Rom protein. The available data support this prediction.

Quantitative model of ColE1 plasmid copy number control.

Initiation of replication of the Escherichia coli plasmid ColE1 is inhibited by formation of a complex between a small plasmid RNA (RNA I) and the pre-primer for DNA synthesis (RNA II). Complex formation (and inhibition of replication) is enhanced by the plasmid-encoded Rom protein. The in vitro kinetics of complex formation were previously studied both experimentally and theoretically. The in vivo concentrations and half-lives of RNA I, RNA II and Rom protein have been measured recently. We present a dynamic model for the in vivo replication control mechanism that accounts for the measured concentration values. From the model we deduce a simple formula for the steady-state plasmid concentration. Our results agree with a previous simple steady-state analysis done by Brenner and Tomizawa, in that plasmid copy number is most strongly dependent on the per plasmid rate of RNA I synthesis. However, our model predicts other parameter dependencies that are not evident from or at variance with the previous analysis. Accordingly, we predict that plasmid copy number is greatly influenced by changes in the rate constant describing the formation of an initial unstable RNA I-RNA II complex, but is only slightly influenced by changes in the dissociation rate of this complex. Plasmid copy number per average cell volume is predicted to increase linearly with increases in the RNA II synthesis rate and with increases in the generation time of the host culture. Rom protein, which promotes conversion of the unstable RNA I-RNA II complex to a stable complex, serves to decrease copy number; however, its presence or absence does not seem to qualitatively alter the copy number control mechanism. Our model predicts the quantitative increase of plasmid copy number in rom- mutants. Several experiments are suggested to investigate the predictions of the model.

Prediction of splice sites in plant pre-mRNA from sequence properties.

Heterologous introns are often inaccurately or inefficiently processed in higher plants. The precise features that distinguish the process of pre-mRNA splicing in plants from splicing in yeast and mammals are unclear. One contributing factor is the prominent base compositional contrast between U-rich plant introns and flanking G + C-rich exons. Inclusion of this contrast factor in recently developed statistical methods for splice site prediction from sequence inspection significantly improved prediction accuracy. We applied the prediction tools to re-analyze experimental data on splice site selection and splicing efficiency for native and more than 170 mutated plant introns. In almost all cases, the experimentally determined preferred sites correspond to the highest scoring sites predicted by the model. In native genes, about 90% of splice sites are the locally highest scoring sites within the bounds of the flanking exon and intron. We propose that, in most cases, local context (about 50 bases upstream and downstream from a potential intron end) is sufficient to account for intrinsic splice site strength, and that competition for transacting factors determines splice site selection in vivo. We suggest that computer-aided splice site prediction can be a powerful tool for experimental design and interpretation.

A method to identify distinctive charge configurations in protein sequences, with application to human herpesvirus polypeptides.

Charge interactions are of great importance for protein function and structure, and for a variety of cellular and biochemical processes. We present a systematic approach to the detection of distinctive clusters, runs and periodic patterns of charged residues in a protein sequence. Criteria and formulae are set forth to assess statistical significance of these charge configurations. For the 80-odd proteins potentially encoded by the Epstein-Barr virus, only the major nuclear antigens of the latent state and the transactivator of the lytic cycle contain separated charge clusters of opposite sign as well as periodic charge patterns. From our studies of the polypeptides of the human herpesviruses and of a broad collection of human and other viral protein sequences, distinctive charge configurations appear to be associated with viral capsid and core proteins (positive clusters or runs, mostly at the carboxyl terminus), with many viral glycoproteins and membrane-associated proteins (negative charge clusters), and with transactivators and transforming proteins (multiple charge structures). The statistics developed in this paper apply more generally to other than charge properties of a protein and should aid in the evaluation of a large variety of sequence features.

Sustained expression of high levels of human factor IX from human cells implanted within an immunoisolation device into athymic rodents.

Immunoisolation of allogeneic cells within a membrane-bound device is a unique approach for gene therapy. We employed an immunoisolation device that protects allograft, but not xenograft, cells from destruction, to implant a human fibroblast line (MSU 1.2) in athymic rodents. Cells, transduced with the MFG-human factor IX retroviral vector, and expressing 0.9 microg/10(6) cells/day in vitro, were implanted in rats (four 40-microl devices, each containing 2 x 10(7) cells, two subcutaneously, two in epididymal fat) and in mice (two 20-microl devices, each containing 2 x 10(6) cells, subcutaneously). Plasma factor IX levels increased for 50 days, reaching maxima of 203 ng/ml (rat) and 597 ng/ml (mouse), and both continued at greater than 100 ng/ml for more than 140 days. A clone derived from the transduced cells, making 5 microg of factor IX/10(6) cells/day, was implanted within a device (one 20-microl device containing 2.5 x 10(6) cells), or without a device (1 x 10(7) cells implanted freely), either subcutaneously or in epididymal fat. The freely implanted cells expressed transiently, reaching more than 100 ng/ml in each site by day 4, but dropped to zero by day 20 (subcutaneous) or day 90 (epididymal fat). In devices, levels gradually increased to 100 ng/ml (subcutaneous) or 300 ng/ml (epididymal fat), remaining high for more than 100 days. These results show long-term, high-level expression of a human protein: (1) when cells are implanted within a cell transplantation device, but not when the cells are freely implanted, and (2) from a transgene driven by a viral promoter. An alloprotective device will enable the use of cloned cell lines that can be subjected to stringent quality control assessment that is impossible to achieve with autologous approaches.

Immunization with interleukin-2-secreting allogeneic cells transfected with DNA from mouse melanoma cells induces immune responses that prolong the lives of mice with melanoma.

Altered genes in tumor cells specify tumor-associated antigens. Because genetic instability is a characteristic of the malignant cell phenotype, a large number of different, altered genes may be present in a population of neoplastic cells, specifying an array of undefined tumor-associated determinants. We hypothesized that immunogenic cells transfected with DNA from malignant cells will include cells that specify tumor-associated antigens. To test this question, we deliberately mutagenized a population of B16 melanoma cells (H-2b) by ultraviolet-B irradiation. DNA from the surviving cells was used to transfect LM cells (H-2k), a mouse fibroblast cell line modified previously to secrete interleukin-2. The transfected allogeneic cells were then tested for their immunogenic properties in C57BL/6J mice (H-2b) syngeneic with the melanoma. Mice injected with a mixture of the mutagenized B16 cells and the transfected cells survived significantly longer than untreated mice injected with the mutagenized B16 cells alone. Mice injected with a mixture of mutagenized B16 cells and cells transfected with DNA from unirradiated B16 cells died in shorter intervals. Based on the results of cytotoxicity assays performed in vitro, the cellular immune responses of greatest magnitude were directed toward the type of cell from which the DNA was obtained.

The missense errors in protein can be controlled by selective synonymous codon usage at the level of transcription.

In the cases of the 6-fold degenerate residues and the stop signal, selective codon usage at the level of transcription can account for a 10-20% variation in their mistranslation rate. For all other residues, the mistranslation rate is dependent upon the degree of degeneracy only, but not upon the pattern of synonymous codon usage.

Intervening sequences exhibit distinct vocabulary.

Little is known about the origin and function of eukaryotic introns. Application of a novel linguistic approach to the analysis of intervening sequences reveals, however, that they exhibit a specific non-random vocabulary whose major feature is the utilization of mirror-symmetrical words ("mirrorrim"). Introns also manifest a significant tendency to avoid local complementarities. Possible biological implications of the corresponding loop regions in the RNA transcripts are discussed.

Terminators of transcription with RNA polymerase from Escherichia coli: what they look like and how to find them.

We present here a compilation of prokaryotic transcription terminator sequences (ref. 1-152). The compilation includes 49 independent terminators, 52 speculated independent terminators, 27 sites shown to function in vivo, and some 20 proven or speculated rho-dependent terminators. In addition to the well-known features of independent terminators (dyad symmetry and T-run), two consensus are found: CGGG(C/G) upstream and TCTG downstream of the termination point. A subset of the collection of sequence has been used to construct a computer algorithm to locate independent terminators by sequence analysis.

Linguistics of nucleotide sequences: morphology and comparison of vocabularies.

The concept of "words" in continuous languages devoid of blanks is introduced and an operational definition of words given. With this novel concept nucleotide sequences become object for linguistic analysis. The typical word size of the nucleotide language is found to be 3 to 5 (tri- to pentamers). Different genomes have distinct vocabularies. Comparison of these vocabularies can serve as a basis for revealing functional and evolutionary relatedness of sequences.