Showing papers by "Torrey Pines Institute for Molecular Studies published in 1990"

Fingerprinting genomes using PCR with arbitrary primers

Abstract: Simple and reproducible fingerprints of complex genomes can be generated using single arbitrarily chosen primers and the polymerase chain reaction (PCR). No prior sequence information is required. The method, arbitrarily primed PCR (AP-PCR), involves two cycles of low stringency amplification followed by PCR at higher stringency. We show that strains can be distinguished by comparing polymorphisms in genomic fingerprints. The generality of the method is demonstrated by application to twenty four strains from five species of Staphylococcus, eleven strains of Streptococcus pyogenes and three varieties of Oryza sativa (rice).

Development of a short-term, in vivo mutagenesis assay: the effects of methylation on the recovery of a lambda phage shuttle vector from transgenic mice

Abstract: Transgenic mice suitable for the in vivo assay of suspected mutagens at the chromosome level have been constructed by stable integration of a lambda phage shuttle vector. The shuttle vector, which contains a beta-galactosidase (beta-gal) target gene, can be rescued from genomic DNA with in vitro packaging extracts. Mutations in the target gene are detected by a change in lambda phage plaque color on indicator agar plates. Initial rescue efficiencies of less than 1 plaque forming unit (pfu)/100 micrograms of genomic DNA were too low for mutation analysis. We determined the cause of the low rescue efficiencies by examining primary fibroblast cultures prepared from fetuses of lambda transgenic animals. The rescue efficiency of 5-azacytidine-treated cells increased 50-fold over non-treated controls indicating that methylation was inhibiting rescue. The inhibitory role of methylation was supported by the observation that mcr deficient E. coli plating strains and mcr deficient lambda packaging extracts further improved lambda rescue efficiency. Present rescue efficiencies of greater than 2000 pfu/copy/micrograms of genomic DNA represent a 100,000-fold improvement over initial rescue efficiencies, permitting quantitative mutational analysis. The background mutagenesis rate was estimated at 1 x 10(-5) in two separate lineages. Following treatment with the mutagen N-ethyl-N-nitrosourea (EtNU), a dose dependent increase in the mutation rate was observed in DNA isolated from mouse spleen, with significant induction also observed in mouse testes DNA.

Elucidation of discontinuous linear determinants in peptides

Abstract: Synthetic peptides, made by the method of simultaneous multiple peptide synthesis, were coupled to the protein carrier keyhole limpet hemocyanin and used to raise mAb. Omission and substitution analogs of the original peptides were tested by ELISA to characterize their reactivity with the respective mAb. Linear antigenic determinants were located for 18 different peptides by using omission analogs. The length of the antigenic determinants ranged from 2 to 8 residues, with an average of 6 residues. The three aromatic amino acids, phenylalanine, tryptophan, and tyrosine, the charged hydrophilic amino acids, aspartic acid and lysine, and the neutral amino acid alanine were found to occur most often in the determinant region of the peptides tested, whereas asparagine, cysteine, and histidine occurred the least often. Alanine substitution analogs provided more information than omission analogs by enabling the determination of which side chain groups of the antigenic determinant residues were not critical for binding to the mAb. Detailed, "fingerprint" information about the interaction of the peptide, GASPYPNLSNQQT, and its mAb was obtained by synthesizing a complete series of analogs with individual substitutions for each position of the antigenic determinant, PYPNLS, with the 19 other amino acids. These results suggest that, at the amino acid level, all antigenic determinants of synthetic peptides defined by mAb can be considered discontinuous linear determinants.

An epitope in human immunodeficiency virus 1 reverse transcriptase recognized by both mouse and human cytotoxic T lymphocytes

Abstract: T-cell-mediated cytotoxicity may play an important role in control of infection by the human immunodeficiency virus (HIV). In this study, we have identified and characterized a relatively conserved epitope in the HIV-1 reverse transcriptase recognized by murine and human cytotoxic T cells. This epitope was identified using a murine antigen-specific CD8+ class I major histocompatibility complex-restricted cytotoxic T-cell (CTL) line, a transfected fibroblast cell line expressing the HIV-1 pol gene, recombinant vaccinia viruses containing different truncated versions of the pol gene, and overlapping synthetic peptides. The optimal antigenic site was identified as residues 203-219 by synthesizing extended or truncated peptide analogs of the antigenic fragment. The optimal peptide was then tested for sensitization of autologous Epstein-Barr virus-transformed B-cell targets for killing by fresh human peripheral blood mononuclear cells. It was recognized by CTLs from several HIV-seropositive patients but not from any seronegative donor. Therefore, this peptide is a good candidate for inclusion in an AIDS vaccine. This study demonstrates that the same CTL epitope can be seen by murine and human CD8+ CTLs, as previously demonstrated for epitopes recognized by CD4+ helper T cells, and suggests the utility of screening for immunodominant CTL epitopes in mice prior to carrying out studies in humans.

Major population differences in T cell response to a malaria sporozoite vaccine candidate.

Abstract: Using a complete series of overlapping peptides, we have identified the T cell epitopes of a malaria vaccine candidate, the circumsporozoite (CS) protein, that are recognized by sporozoiteexposed residents of a non-endemic country. Thls protein and subunb from tt are belng considered as malarla sporozoite vaccine candidates, as CS-specific antibodies and cytotoxic T lymphocytes have been shown to have a role in protection. The rationale for deveioplng an antibody-based vaccine is that in Plasmodium falclpmmthe lmmunodominant B cell epttope of the protein, (Asn-AieAsn-Ala-Asn-Pro), [(NANP)], is invariant. However, the Ideal vaccine must contain CS protein-derived T cell antigenlc epttopes to allow natural boosting of the antibody response following sporozoite exposure. Here, we show that major differences occur between the CS spectific T cell responses of non-endemic Caucasians and an endemic African population. HLA differencaa between the populations are, in part, responsible. Subunit malaria vacclnes for one population may be ineffective In a different population.

Methylase-limited partial NotI cleavage for physical mapping of genomic DNA.

Abstract: Partial cleavage of DNA with the restriction endonuclease NotI (5'...GC/GGCCGC...3') is an important technique for genomic mapping. However, partial genomic cleavage with this enzyme is impaired by the agarose matrix in which the DNA must be suspended. To solve this problem we have purified the blocking methylase M. BspRI (5'...GGmCC...3') for competition digests with NotI. The resulting methylase-limited partial DNA cleavage is shown to be superior to standard techniques on bacterial genomic DNA. Abbreviations: bp, base-pair; kb, one thousand base-pairs; Mb, one million base-pairs; Tris, Tris(hydroxy-methyl)aminomethane; EDTA, (ethylenedinitrilo)tetraacetic; beta-ME, beta-mercaptoethanol; PMSF, phenyl methyl-sulfonyl fluoride; PEG, polyethyleneglycol (MW = 8000); 3H, tritium; SAM, S-adenosylmethionine; KGB, potassium glutamate buffer; DTT, dithiothreitol; IPTG, isopropyl-beta-D-thiogalactopyranoside; BSA, bovine serum albumin.

Inhibition of Antigen Presentation In Vitro and In Vivo by MHC Antagonist Peptides

Abstract: A series of analogue peptides have been generated, using as a template the core region of the OVA 323-339 peptide identified as critical in determining binding to I-Ad. Several of these "core extended" peptides had increased affinities for the I-Ad molecule compared to the native sequence, and were able to inhibit activation of an I-Ad-restricted T cell hybridoma in vitro. The induction of a T cell proliferative response to a peptide antigen could be inhibited by co-administration of core-extended peptide with antigen in the same adjuvant emulsion. Furthermore, inhibition also occurred when the inhibitor molecule was delivered separately one day before immunization. Finally, the induction of the autoimmune disease, experimental allergic encephalomyelitis (EAE), in susceptible mice could be reduced by the administration of a core-extended peptide with high affinity for the appropriate class II molecule. These findings have implications for the use of MHC antagonists in the control and treatment of MHC-associated autoimmune conditions in humans.

An evaluation of the advantages and effectiveness of picric acid monitoring during solid phase peptide synthesis.

Abstract: The effectiveness of picric acid for the monitoring of coupling completion during solid phase peptide synthesis was evaluated. Each coupling step was monitored during the synthesis of 124 peptides by the method of simultaneous multiple peptide synthesis. Of the peptides tested (1622 picrate determinations), approximately 10% underwent a single low yield step, another 10% had 2 or 3 consecutive low yield steps and about 20% contained "humps" 4 to 8 steps long. In virtually all instances, the low yield steps occurred only at or after coupling step 7. High picrate values obtained after incorporation of methionine appear to be caused by a sulfonium salt in the side chain of methionine. Differences in average reactivities of amino acids correlated with structural elements of their side chains. Picric acid, which is currently seldom used for monitoring of coupling completion during solid phase peptide synthesis, was found to be straightforward and nondestructive. While picric acid monitoring is not as sensitive as the more commonly used ninhydrin method, it yields information which is not detectable with the use of ninhydrin and is clearly superior for certain applications and investigations.

Effect of phospholipase A2 inhibitors on mouse T lymphocytes. II. Phospholipase A2 inhibitors induce T cell hybridomas and a T cell clone for the formation of glycosylation-inhibiting factor.

Abstract: The mouse T cell hybridoma 12H5 cells constitutively form glycosylation-enhancing factor (GEF) and produce both IgE-potentiating factor and ovalbumin (OVA)-binding GEF upon antigenic stimulation with OVA-pulsed macrophages. Culture of the 12H5 cells either with nonspecific glycosylation inhibiting factor (GIF) or with a phospholipase A2 (PLA2) inhibitor, ONO-RS-082, stopped the formation of GEF and induced the same cells to form GIF. Induction of the GIF formation by a PLA2 inhibitor was observed even when the 12H5 cells had been treated with mitomycin C, indicating that the switching from the GEF formation to the GIF formation was not due to selective proliferation of a GIF-producing subclone. The OVA-binding GIF produced by the PLA2-inhibitor-treated, antigen-stimulated 12H5 cells binds to homologous antigen (ovalbumin), and shares both antigenic determinant recognized by the monoclonal antibody 14-30 and the lipomodulin-determinant with antigen-specific suppressor inducer factor (TsiF). The present experiments also showed that a typical helper T cell clone, D10, G4.1 cells, constitutively formed GEF and that preculture of the T cell clone with IL-2 and the PLA2 inhibitor switched the cells from the formation of GEF to the formation of GIF. Upon stimulation with antigen-pulsed macrophages, the inhibitor-treated D10.G4.1 cells formed GIF having affinity for conalbumin. The results indicated that the same T cells have the capacity to form either GIF or GEF under different conditions, and suggested that the GIF-producing suppressor T cells may be a phenotype of a subset of helper T cells. Switching of the same cells from the GEF formation to the GIF formation by the PLA2 inhibitor and the ability of the inhibitor to enhance GIF formation suggested that PLA2-inhibitory activity or GIF activity of TsiF is involved in the suppressor T cell cascade.

MODSIM II — a modular, object-oriented language (tutorial session)

Abstract: MODSIM I] is an object-oriented, general purpose programming language which was designed to work with both sequential and parallel processors. It is a compiled language which is available for most systems including mainframes, work-stations and PC's. A research version has been developed for use on parallel processors. The syntax and structure of MODSIM II is based on that of Modula-2. It has additional constructs for object types and simulation. The built-in object-oriented constructs of MODSIM II include single and multiple inheritance, dynarnie binding of objects, polymorphism, data abstraction and information hiding. These capabilities are reinforced by the modular program development environment. MODSIM II supports automated separate compilation and importation of code from modules and libraries. This makes it ideal for large projects. Its built-in interactive, dynamic graphics allow convenient display and manipulation of menus, charts, graphs and animated results. The graphics editor allows users to interactively design their own input and control menus, icons and charts without programming. 1. ~ T R O D U C T I O N MODSIM II was specifically designed to support large programming projects. It is a compiled, modular, object-oriented language with multiple inheritance. To protect the user's investment in applications, MODS/M can be moved to new computer systems as they become available. Its syntax is based on that of Modula-2. Programmers trained in Pascal, Modula-2 or Ada learn the language with ease. This saves training costs and time. Modularity in MODSIM II improves reliability and code reusability. Objects and routines performing related functions can be grouped into modules. These can be put into libraries for reuse by other programs. The optional simulation constructs are based on CACFs widely used SIMSCRIPT 11.5 programming language. The portability of MODSIM II derives from the fact that its compiler emits "C" code which is compiled, in turn, by each computer's "C" compiler. Finally, the integrated dynamic graphics of MODSIM U substantially reduces the time and effort needed to display results with animation and presentation graphics. It only takes a few statements to make dynamic icons, histograms, clocks and meters appear and change as the simulation runs. MODSIM II is a complete, general purpose programming language which is ideal for large software engineering projects. Its features reduce design and coding effort, and improve reliability. The MODSIM II compiler, runtime and graphics libraries are all written in MODSIM II.

C4 from C4-high and C4-low mouse strains have identical sequences in the region corresponding to the isotype-specific segment of human C4.

Abstract: The human complement component C4 isotypes, C4A and C4B, show a substantial and biologically important difference in chemical reactivity. Murine C4 from different mouse strains has recently been reported to have a comparable difference in reactivity. In human C4, the difference in reactivity has been attributed to the effect(s) of one or more of only four amino acid residues, within a six-residue-long segment of the alpha subunit, which distinguish the two isotypes. In the present study, we sought to assess the role of the corresponding four amino acids in mouse C4 in the strain-specific modulation of C4 reactivity. In order to compare the sequences of the corresponding region in murine C4 among different mouse strains, we used the polymerase chain reaction method to amplify an approximately 229-bp segment of the murine gene that includes the codons for these four amino acid residues. Because the difference in chemical reactivity of murine C4 has been reported to be between C4 from strains which express different levels of C4, we examined sequences from mouse strains C57BL/6, DBA/2, C3H/He, B10.BR and CBA/J; these represent two C4-high strains and three C4-low strains. The amplified segments were cloned into the pUC19 vector and 20 independent clones from each mouse strain were sequenced. Across the entire amplified segment, our results revealed expected isotypic differences between C4 and its nonfunctional isotype in the mouse, Slp, as well as allelic differences among the C4 and Slp genes. However, all of these differences were quite distant from the amino acid residues corresponding to the human isotype-specific residues and those corresponding residues were identical in all five mouse strains. This result indicates that any strain-specific difference in chemical reactivity of murine C4 must be due to a mechanism distinct from that operating in human C4.

Introduction to SIMFACTORY II.5 (tutorial session)

Abstract: This paper provides a brief introduction to SIMFACTORY 11.5 and explains how models can be developed without programming. The type of users who benefit most from SIMFACTORY 11.5, the types of systems SIMFACTORY 11.5 can model, and the various implementations of SIMFACTORY 11.5 are also described.

Use of c-20 to c-26 aliphatic alcohols for the manufacture of a medicament for the treatment of viral infections.

Abstract: Est presente un traitement contre les infections virales de l'organisme fonde sur l'utilisation d'une classe reduite d'alcools monovalents a chaine saturee lineaire qui ont de 20 a 26 atomes de carbone dans la chaine et preferablement de 22 a 26, sous forme de compositions physiologiquement compatibles administrees par injection ou par introduction dans la membrane trans-mucus chez l'homme et chez des animaux. Disclosed is a treatment against viral infections of the body based on the use of a small class of monohydric alcohols to saturated linear chain which have from 20 to 26 carbon atoms in the chain and preferably 22 to 26, under form of physiologically compatible compositions administered by injection or by introduction in the trans-mucus membrane in humans and in animals.

Novel cloning host organisms

Abstract: Methods and materials for the cloning of DNA, in particular, for the cloning of "unclonable" DNA using genetically engineered host cells are disclosed. Host cell organisms have been discovered that stabilize and inhibit rearrangement of DNA molecules capable of forming non-standard secondary and tertiary structures. Organisms are engineered to contain at least one mutation which inactivates homologous recombination and at least one mutation in a DNA repair pathway. Examples of such DNA pathways include Uv repair pathway, the SOS repair pathway, the mismatch repair pathway, the adaptive response pathway, the heat shock response pathway, the osmotic shock response pathway, the repair pathway of alkylation damage, the repair pathway of uracil incorporation into DNA and pathways involved in maintaining DNA superhelicity. The host organisms of this invention are suitable for cloning DNA capable of forming non-standard and tertiary structures such as found in eukaryotic DNA.

Use of c-22 to c-26 aliphatic alcohols in the treatment of inflammatory and viral skin diseases

Abstract: not available for EP0428642Abstract of corresponding document: US4874794A method treating virus-induced and inflammatory diseases of skin and membranes in humans or animals, comprising application of a composition consisting of one or more of the aliphatic alcohols docosanol, tetracosanol and hexacosanol in a physiologically compatible carrier is disclosed.