A high-resolution comparative map of porcine chromosome 4 (SSC4).

We used the IMNpRH2(12,000-rad) RH and IMpRH(7,000-rad) panels to integrate 2019 transcriptome (RNA-seq)-generated contigs with markers from the porcine genetic and radiation hybrid (RH) maps and bacterial artificial chromosome finger-printed contigs, into 1) parallel framework maps (LOD ≥ 10) on both panels for swine chromosome (SSC) 4, and 2) a high-resolution comparative map of SSC4, thus and human chromosomes (HSA) 1 and 8. A total of 573 loci were anchored and ordered on SSC4 closing gaps identified in the porcine sequence assembly Sscrofa9. Alignment of the SSC4 RH with the genetic map identified five microsatellites incorrectly mapped around the centromeric region in the genetic map. Further alignment of the RH and comparative maps with the genome sequence identified four additional regions of discrepancy that are also suggestive of errors in assembly, three of which were resolved through conserved synteny with blocks on HSA1 and HSA8.

The MetaFam Server: a comprehensive protein family resource.

MetaFam is a comprehensive relational database of protein family information. This web-accessible resource integrates data from several primary sequence and secondary protein family databases. By pooling together the information from these disparate sources, MetaFam is able to provide the most complete protein family sets available. Users are able to explore the interrelationships among these primary and secondary databases using a powerful graphical visualization tool, MetaFamView. Additionally, users can identify corresponding sequence entries among the sequence databases, obtain a quick summary of corresponding families (and their sequence members) among the family databases, and even attempt to classify their own unassigned sequences. Hypertext links to the appropriate source databases are provided at every level of navigation. Global family database statistics and information are also provided. Public access to the data is available at http://metafam.ahc.umn.edu/.

Mammalian mRNAs encoding protein closely related to ubiquitin-conjugating enzyme encoded by yeast DNA repair gene RAD6.

A clone of about 1 kb has been isolated from a human brain cDNA library. The clone possesses a 151 amino acid open reading frame that exhibits 72% amino acid identity with the E2 ubiquitin-conjugating enzyme encoded by the RAD6 gene of Saccharomyces cerevisiae. A 90% amino acid identity was observed in a central sequence surrounding a cysteine, which most likely contributes the sulfhydryl group involved in the formation of the ubiquitin-E2 thiolester linkage. Northern hybridization analyses have identified a poly(A)-containing mRNA of about 1 kb encoding the E2-like sequence in human CEM lymphoblastoid and HeLa cells, Novikoff rat hepatoma cells and S49 mouse leukemia cells. Southern hybridization analyses indicate the presence of a single gene encoding this sequence in both human cell lines, but of two or more related genes in the rodent cell lines.

Common structural features among monoclonal antibodies binding the same antigenic region of cytochrome c.

To examine if there are common physicochemical features among antibodies binding the same antigenic region of a protein, B cell hybridomas were prepared against the two major antigenic regions on mammalian cytochromes c, and the nucleotide sequences encoding the monoclonal antibody (mAb) heavy (H) and light (L) chains were determined and compared. Although the genetic elements used were somewhat diverse, similarities among mAbs to a given antigenic region were observed. In particular, mAbs binding in a region situated at a bend in the antigen around residues 44 and 47 had longer complementarity-determining regions (4-5 additional amino acid residues in L1 and 1-2 in H3) than mAbs binding the other region around residues 60 and 62 located on a relatively flat surface. These observations indicate that the topography of an antigenic site and the lengths of certain complementarity-determining regions are important physicochemical properties determining, at least in part, which antibodies (B cells) will participate in an immune response to a particular site on a protein antigen.

Enzymatic synthesis of deoxyribonucleic acid by the avian retrovirus reverse transcriptase in vitro: optimum conditions required for transcription of large ribonucleic acid templates.

In this communication, we present data which describe optimum conditions for reverse transcription of large ribonucleic acid (RNA) templates into deoxyribonucleic acid (DNA) transcripts by the avian retrovirus reverse transcriptase in vitro. In contrast to previous studies, we have optimized all of the reaction components with respect to their influence on the size of DNA transcripts rather than the incorporation of radio-labeled deoxynucleoside triphosphates into acid-insoluble DNA product. The most dramatic effect on uninterrupted reverse transcription is the presence of physiological concentrations (i.e., 148 mM) of monovalent cation in the reaction mixture, although all of the components of the reaction influence the size of the DNA transcripts synthesized to some extent. The enzymatic conditions described herein for the uninterrupted reverse transcription of large RNA templates (greater than 1000--2000 nucleotides) are superior to those described previously because they are reproducible, do not require the presence of ribonuclease inhibitors, and do not result in the precipitation of components of the reaction mixture during incubation.

Genomic differences between strains of lactate dehydrogenase-elevating virus.

The RNAs of two independently isolated strains of lactate dehydrogenase-elevating virus (LDV), which differ antigenically and in neurovirulence for C58 mice, were isolated and T1 RNase fingerprinted. Of about 30 unique T1 oligonucleotides, 27 seemed to be common for both strains of LDV, whereas 2 or 3 oligonucleotides were unique for each strain. In other physical and biological properties, such as virion density, molecular weights of their structural proteins, interaction with mouse anti-LDV IgG, and replication in primary cultures of peritoneal macrophages from various mouse strains, the two strains of LDV were indistinguishable. The T1 RNase patterns and the affinity of LDV RNA for oligo(dT) indicated that it contained poly-A.

Comparisons of the pestivirus bovine viral diarrhoea virus with members of the flaviviridae.

The molecular features of bovine viral diarrhoea virus (BVDV), a member of the Pestivirus genus currently classified in the Togaviridae, were examined for characteristics resembling those of the Flaviviridae family. Like flaviviruses, BVDV possesses a single-stranded RNA genome (approx. 4.3 x 10(6) Mr) deficient in a 3' poly(A) tract. This RNA has a single open reading frame spanning the length of the genome in the viral RNA sense (positive polarity), implying an expression strategy involving the processing of a precursor polyprotein. With the exception of several short but significant stretches of identical amino acids within two non-structural proteins, no extended regions of nucleotide or amino acid sequence homology between BVDV and representatives of three serological subgroups of mosquito-borne flaviviruses were noted. However, comparison of the organization of protein-coding domains along the genomes and the hydropathic profiles of amino acid sequences revealed pronounced similarities. It is proposed that Pestivirus, of which BVDV is the prototype member, should no longer be grouped in the Togaviridae family, but rather be considered a genus of non-arthropod-borne viruses within the Flaviviridae.

Amplification and detection of lentiviral DNA inside cells.

Visna virus and human immunodeficiency virus are prototypes of animal and human lentiviruses, respectively, that persist and are disseminated despite the host immune response because cells in the tissues and the bloodstream harbor viral genomes in a covert state. To facilitate identification of these latently infected cells, the polymerase chain reaction has been adapted to amplify viral DNA in fixed cells for detection by in situ hybridization. By using a multiple primer set that generates DNA segments with overlapping cohesive termini, visna virus DNA can be amplified, retained, and detected in infected cells with sensitivities that exceed those of existing methods by more than 2 orders of magnitude. This advance in single-cell technology should prove useful in diagnosing and gaining insight into the pathogenesis of viral infections and provide new opportunities to look for viruses in chronic diseases of unknown etiology.

MetaFam: a unified classification of protein families. I. Overview and statistics.

MOTIVATION: Protein sequence classification is becoming an increasingly important means of organizing the voluminous data produced by large-scale genome sequencing projects. At present, there are several independent classification methods. To aid the general classification effort, we have created a unified protein family resource, MetaFam. MetaFam is a protein family classification built upon 10 publicly-accessible protein family databases (Blocks + DOMO, Pfam, PIR-ALN, PRINTS, PROSITE, ProDom, PROTOMAP, SBASE, and SYSTERS). MetaFam's family 'supersets', as we call them, are created automatically using set-theory to compare families among the databases. Families of one database are matched to those in another when the intersection of their members exceeds all other possible family pairings between the two databases. Pairwise family matches are drawn together transitively to create a new list of protein family supersets. RESULTS: MetaFam family supersets have several useful features: (1) each superset contains more members than the families from which it is composed, because each of the component family databases only works with a subset of our full non-redundant set of proteins; (2) conflicting assignments can be pinpointed quickly, since our analysis identifies individual members that are in conflict with the majority consensus; (3) family descriptions that are absent from automated databases can frequently be assigned; (4) statistics have been computed comparing domain boundaries, family size distributions, and overall quality of MetaFam supersets; (5) the supersets have been loaded into a relational database to allow for complex queries and visualization of the connections among families in a superset and the consensus of individual domain members; and (6) the quality of individual supersets has been assessed using numerous quantitative measures such as family consistency, connectedness, and size. We anticipate this new resource will be particularly useful to genomic database curators.

MetaFam: a unified classification of protein families. II. Schema and query capabilities.

MOTIVATION: Protein sequence and family data is accumulating at such a rapid rate that state-of-the-art databases and interface tools are required to aid curators with their classifications. We have designed such a system, MetaFam, to facilitate the comparison and integration of public protein sequence and family data. This paper presents the global schema, integration issues, and query capabilities of MetaFam. RESULTS: MetaFam is an integrated data warehouse of information about protein families and their sequences. This data has been collected into a consistent global schema, and stored in an Oracle relational database. The warehouse implementation allows for quick removal of outdated data sets. In addition to the relational implementation of the primary schema, we have developed several derived tables that enable efficient access from data visualization and exploration tools. Through a series of straightforward SQL queries, we demonstrate the usefulness of this data warehouse for comparing protein family classifications and for functional assignment of new sequences.

Proteins encoded by bovine viral diarrhea virus: the genomic organization of a pestivirus.

The genome of bovine viral diarrhea virus (BVDV) contains a single large open reading frame capable of encoding 449 kDa of protein. Short segments from along the length of the molecularly cloned BVDV genome were engineered so as to be expressed as bacterial fusion polypeptides in Escherichia coli. These BVDV analog fusion proteins were used as immunogens to generate a panel of sequence-specific antisera. These antiserum reagents were in turn employed in immunoprecipitation analyses to identify the authentic BVDV protein to which they were directed. The results allowed for the identification and positioning along the genome of BVDV gene products accounting for approximately 83% of the coding capacity of the virus. A preliminary map of the genetic organization of BVDV is presented and discussed.

Terminal deoxynucleotidyl transferase expression in a Thy-l alloantigen variant lymphoma cell line.

Terminal deoxynucleotidyl transferase (TdT) expression was examined in the clones of the radiation induced murine leukemia, RL male 1, which differ in Thy-1.2 alloantigen expression and tumourigenicity in syngeneic mice. Both cell lines displayed predominant cytoplasmic localization of TdT and equal sensitivities to specific TdT inhibitors. The R1 male 1.3 + cell line (Thy-1.2 positive and tumourigenic) demonstrated overall higher levels of TdT activity and different elution patterns on phosphocellulose chromatography compared with the RL male 1.4 - (Thy-1.2 negative and poorly tumourgenic) cell line. These findings suggest an association of TdT expression with tumourigenicity properties in leukemic T lymphocytes.

Single cell transcript analysis of human immunodeficiency virus gene expression in the transition from latent to productive infection.

In the lymph nodes of individuals infected with human immunodeficiency virus (HIV), there is evidence that points to three kinds of virus-cell relationships. Virions may be associated with CD4+ lymphocytes that are actively producing virus or may be bound at the surfaces of follicular dendritic cells like other antigens. HIV is also harbored in CD4+ lymphocytes and monocytes/macrophages in a latent form as transcriptionally silenced provirus. To ultimately investigate in vivo these and other HIV-cell interactions that play such critical roles in the persistence of virus, immune dysregulation, and depletion, we have developed an in situ hybridization method that discriminates multiply spliced from singly or unspliced viral transcripts. In this report we describe the method and the results obtained with it in an analysis of the switch from latent to productive infection of chronically infected T lymphocytes in culture. We found with this single-cell technique that there are two subpopulations in the culture, a minor one of productively infected cells and a major one of latently infected cells in which only low levels of viral transcripts terminated close to the 5' end of the viral genome were detected. Shortly after activation of viral gene expression with phorbol ester, transcripts encoding Tat and Rev increase in abundancy in individual latently infected cells and this is followed by increases in and cytoplasmic export of singly or unspliced mRNAs encoding structural proteins. These studies provide insights into the regulation of HIV gene expression from a single-cell perspective and, from that perspective, transcript profiles of productively infected cells as a frame of reference for defining HIV-cell relationships in individual cells in tissue sections.

PANAL: an integrated resource for Protein sequence ANALysis.

SUMMARY: We present PANAL, an integrated resource for protein sequence analysis. The tool allows the user to simultaneously search a protein sequence for motifs from several databases, and to view the result as an intuitive graphical summary.

Synthesis in cell culture of the gapped linear duplex DNA of the slow virus visna.

Visna virus is a nontransforming retrovirus that causes slow infections in animals and a rapidly progressive-lytic infection in cell culture. The results of an analysis of the synthesis of viral DNA in cell culture are reported. Region- and strand-specific probes cloned in M13 have been used to define the dynamics of DNA synthesis and the major nucleic acid species formed. It is shown that (i) within the first hours of infection, a full-length copy of the viral RNA genome is synthesized by reverse transcription, (ii) early in infection a major species of DNA is formed that extends from a site near the center of the molecule to the 3' end, (iii) somewhat later a second major species of plus-strand DNA is generated that extends from the 5' end to the middle of the genome. As a consequence, most viral DNA molecules consist of a full-length minus strand, and two plus strands separated by a gap or nick in the center of the molecule (J. D. Harris, J. V. Scott, B. Traynor, M. Brahic, L. Stowring, P. Ventura, A. T. Haase, and R. Peluso (1981). Virology 113, 573-583). The implications of this viral DNA structure for one unusual aspect of the lentivirus life cycle, the production of viral RNA, and virions from extrachromosomal DNA are discussed (J. D. Harris, H. Blum, J. Scott, B. Traynor, P. Ventura, and A. T. Haase (1984). Proc. Natl. Acad. Sci. USA 81, 7212-7215).

The molecular pathogenesis of astrogliosis in scrapie and Alzheimer's disease.

In slow infections caused by scrapie and other unconventional agents, and in Alzheimer's disease (AD), the formation of neuritic plaques and the increase in astrocytes and astrocyte-specific protein, glial fibrillary acidic protein (GFAP), are pathological changes common to both conditions. With the rationale that these parallels imply convergent pathogenetic mechanisms, we identified a gene whose expression increases in both. We now report the results of a more extensive analysis of this gene and show that by sequence analysis it is highly homologous and likely identical to GFAP. GFAP mRNA accumulates late in the course of scrapie in subpial and periventricular astrocytes and in cells in foci in the hippocampus. The increased abundance of GFAP mRNA is accompanied by an increase in the corresponding protein. GFAP mRNA is localized by in situ hybridization to the cell body and processes of astrocytes. In AD, the latter pattern predominates, consistent with induction of GFAP mRNA in the sites of synthesis in glial processes in the neuritic plaque.

Hydrodynamic diameters of murine mammary, Rous sarcoma, and feline leukemia RNA tumor viruses: studies by laser beat frequency light-scattering spectroscopy and electron microscopy.

We have studied purified preparations of murine mammary tumor virus (MuMTV), Rous sarcoma virus (RSV; Prague strain), and feline leukemia virus (FeLV) by laser beat frequency light-scattering spectroscopy, ultra-centrifugation, and electron microscopy. The laser beat frequency light-scattering spectroscopy measurements yield the light-scattering intensity, weighted diffusion coefficients. The corresponding average hydrodynamic diameters, as calculated from the diffusion coefficients by the Stokes-Einstein equation for MuMTV, RSV, and FeLV, respectively, are: 144 +/- 6 nm, 147 +/- 7 nm, and 168 +/- 6 nm. Portions of the purified RSV and MuMTV preparations, from which light-scattering samples were obtained, and portions of the actual FeLV light-scattering samples were examined by negatively stained, catalase crystal-calibrated electron microscopy. The light-scattering intensity weighted averages of the electron micrograph size distributions were calculated by weighing each size by its theoretical relative scattering intensity, as obtained from published tables computed according to the Mie scattering theory. These averages and the experimentally observed hydrodynamic diameters agreed to within +/- 5%, which is the combined experimental error in the electron microscopic and light-scattering techniques. We conclude that the size distributions of singlet particles observed in the electron micrographs are statistically true representations of the sedimentation-purified solution size distributions. The sedimentation coefficients (S20, w) for MuMTV, RSV, and FeLV, respectively, are: 595 +/- 29S, 689 +/- 35S, and 880 +/- 44S. Virus partial specific volumes were taken as the reciprocals of the buoyant densities, determined in sucrose density gradients. The Svedberg equation was used to calculate particle weights from the measured diffusion and sedimentation coefficients. The particle weights for MuMTV, RSV, and FeLV, respectively, are: (3.17 +/- 0.32) x 10(8), (4.17 +/- 0.42) x 10(8), and (5.50 +/- 0.55) x 10(8) daltons.

In situ amplification of visna virus DNA in tissue sections reveals a reservoir of latently infected cells.

Maedi and visna are, respectively, the pulmonary and neurological manifestations of slowly progressive infections of sheep caused by retroviruses of the lentivirus subfamily. Lentivirus infections are also persistent infections in which host defenses are generally not successful in eliminating the infectious agent because of restricted viral gene expression in many infected cells. In this report, we describe a method for amplifying and detecting viral DNA in tissue sections which has made it possible to verify experimentally the postulated existence of this reservoir of latently infected cells, as well as to estimate the actual number of cells which harbor viral genomes in infected tissues. In the discussion, we present a simple mathematical model that relates this number to the rate at which inflammatory lesions develop. This model can account for both the slow progression of natural infections and for the rapid accumulation of inflammatory foci in the high dosage experimental system analysed in our studies.

Quantitation of RNA tumor viruses by spectroscopy of density gradient gels.

We have developed a system for virus particle quantitation based on the measurement of the optical absorbance of stained viruses which first have been banded at their buoyant density in an equilibrum 24 to 53% (wt/wt) sucrose density gradient, then fixed in position in the gradient by photopolymerizing an acrylamide-riboflavin mixture in the sucrose, and finally stained and destained. Using plasma from mice infected with leukemia virus (Rauscher) or chickens infected with avian myeloblastosis virus (BAI strain) or suitable controls, we have shown that this technique specifically detects RNA tumor viruses. By using virus stock solutions for which the absolute concentrations were determined by laser beat frequency spectroscopy, we have calibrated the absorbance of the viral bands in terms of virus particle concentration. Using 0.8-ml gradients gels (4 by 45 mm) we can detect as low as 2 x 10(7) viral particles with Coomassie blue staining and 6 x 10(6) viral particles with a more sensitive staining procedure using amido black.

Analysis of xylem formation in pine by cDNA sequencing.

Secondary xylem (wood) formation is likely to involve some genes expressed rarely or not at all in herbaceous plants. Moreover, environmental and developmental stimuli influence secondary xylem differentiation, producing morphological and chemical changes in wood. To increase our understanding of xylem formation, and to provide material for comparative analysis of gymnosperm and angiosperm sequences, ESTs were obtained from immature xylem of loblolly pine (Pinus taeda L.). A total of 1,097 single-pass sequences were obtained from 5' ends of cDNAs made from gravistimulated tissue from bent trees. Cluster analysis detected 107 groups of similar sequences, ranging in size from 2 to 20 sequences. A total of 361 sequences fell into these groups, whereas 736 sequences were unique. About 55% of the pine EST sequences show similarity to previously described sequences in public databases. About 10% of the recognized genes encode factors involved in cell wall formation. Sequences similar to cell wall proteins, most known lignin biosynthetic enzymes, and several enzymes of carbohydrate metabolism were found. A number of putative regulatory proteins also are represented. Expression patterns of several of these genes were studied in various tissues and organs of pine. Sequencing novel genes expressed during xylem formation will provide a powerful means of identifying mechanisms controlling this important differentiation pathway.