Identification of the human melanoma-associated chondroitin sulfate proteoglycan antigen epitope recognized by the antitumor monoclonal antibody 763.74 from a peptide phage library.
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To identify the epitope of the melanoma-associated chondroitin sulfate proteoglycan (MCSP) recognized by the monoclonal antibody (mAb) 763.74, we first expressed random DNA fragments obtained from the complete coding sequence of the MCSP core glycoproteins in phages and selected without success for binders to the murine mAb 763.74. We then used a library of random heptapeptides displayed at the surface of the filamentous M13 phage as fusion protein to the NH2-terminal portion of the minor coat protein III. After three rounds of selection on the bound mAb, several phages displaying related binding peptides were identified, yielding the consensus sequence Val-His-Leu-Asn-Tyr-Glu-His. Competitive ELISA experiments showed that this peptide can be specifically prevented from binding to mAb 763.74 by an anti-idiotypic MK2-23 mouse:human chimeric mAb and by A375 melanoma cells expressing the antigen MCSP. We screened the amino acid sequence of the MCSP molecule for a region of homology to the consensus sequence and found that the amino acid sequence Val-His-Ile-Asn-Ala-His spanning positions 289 and 294 has high homology. Synthetic linear peptides corresponding to the consensus sequence as well as to the MCSP-derived epitope inhibit the binding of mAb 763.74 to the phages displaying the consensus amino acid sequence. Finally, the biotinylated consensus peptide absorbed to streptavidin-microtiter plates can be used for the detection of mAb 763.74 in human serum. These results show clearly that the MCSP epitope defined by mAb 763.74 has been identified. (+info)
Cell-specific peptide binding by human neutrophils.
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Analysis of peptide binding to human neutrophils (PMN) using phage display techniques has revealed cell-specific motifs reactive with the PMN surface. Phage libraries displaying either linear 9-mer or cyclic 10-mer and 6-mer peptides were incubated with normal human neutrophils followed by elution of bound phage with low pH (pH 2.2) and non-ionic detergent. Three rounds of selection generated several related peptide sequences that bound with high avidity to PMN. Using the linear 9-mer library, PMN-binding phage expressed peptides with the motif (G/A)PNLTGRW. The binding of phage bearing this motif was highly specific since no binding was observed on lymphocytes, fibroblasts, epithelial, or endothelial cells. Functional assays revealed that phage bearing the sequence FGPNLTGRW induced a pertussis toxin-sensitive increase in PMN cytosolic calcium analogous to that observed with Galphai coupled receptors. Other prominent motifs identified included phage bearing the consensus DLXTSK(M/L)X(V/I/L), where X represents a non-conserved position. Phage with this motif bound exclusively to a sub population of human PMN that comprised approximately 50% of the total and did not elicit a calcium response. The binding of such phage to PMN was prevented by co-incubation with competing peptides displaying identical or similar sequences (IC50 range from 0.6 micromol/L to 50 micromol/L for DLXTSK and GPNLTG, respectively). We speculate that these techniques will be useful in identifying functional cell-specific binding motifs and contribute to the development of new therapeutic and diagnostic strategies in human disease. (+info)
Synthesis of bacteriophage phi6 double-stranded ribonucleic acid.
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Uracil was incorporated into all three bacteriophage phi6 dsRNA segments throughout the infection cycle; the rates of incorporation into each of the three segments were approx. constant for the first 15 to 20 min and then increased rapidly until 50 min after infection. The medium and small dsRNA segments were produced in greater amounts than the large dsRNA segment at all times in the infection cycle. Inhibition of host RNA and protein synthesis with rifampin and chloramphenicol revealed that virus dsRNA synthesis immediately after infection was independent of either host function. (+info)
Comparison of synonymous codon distribution patterns of bacteriophage and host genomes.
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Synonymous codon usage patterns of bacteriophage and host genomes were compared. Two indexes, G + C base composition of a gene (fgc) and fraction of translationally optimal codons of the gene (fop), were used in the comparison. Synonymous codon usage data of all the coding sequences on a genome are represented as a cloud of points in the plane of fop vs. fgc. The Escherichia coli coding sequences appear to exhibit two phases, "rising" and "flat" phases. Genes that are essential for survival and are thought to be native are located in the flat phase, while foreign-type genes from prophages and transposons are found in the rising phase with a slope of nearly unity in the fgc vs. fop plot. Synonymous codon distribution patterns of genes from temperate phages P4, P2, N15 and lambda are similar to the pattern of E. coli rising phase genes. In contrast, genes from the virulent phage T7 or T4, for which a phage-encoded DNA polymerase is identified, fall in a linear curve with a slope of nearly zero in the fop vs. fgc plane. These results may suggest that the G + C contents for T7, T4 and E. coli flat phase genes are subject to the directional mutation pressure and are determined by the DNA polymerase used in the replication. There is significant variation in the fop values of the phage genes, suggesting an adjustment to gene expression level. Similar analyses of codon distribution patterns were carried out for Haemophilus influenzae, Bacillus subtilis, Mycobacterium tuberculosis and their phages with complete genomic sequences available. (+info)
Analysis of the integration functions of phi304L: an integrase module among corynephages.
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Plasmid p12929 was shown to integrate into the chromosome of Corynebacterium glutamicum RM3 and BL15. The minimal integrating fragment was subsequently defined. The arms flanking the integrated plasmid (attL and attR) were identified, allowing for the determination of the attP and the attB attachment sites. The attB site is located at the 3' end of an ORF presenting 62-78% identity with L19 ribosomal proteins. Integration in the attB site does not result in the inactivation of this gene because its end is also present on the attR arm of the integrated plasmid and is reconstituted. The minimal integrating fragment is 1663 bp long and contains two ORFs. The int ORF was identified as phi304L integrase on the basis of the amino acid homologies it shared with the tyrosine recombinases of the lambda integrase family. Moreover this integrase is highly homologous throughout its sequence with the integrase of phi16 corynephage, the percentage of identity reaching 89% at the NH2 end. The identity also extends upstream of the initiation codon, while both phages are elsewhere nonhomologous. An integrase module was proposed to explain this extensive homology. (+info)
Evolutionary relationships among diverse bacteriophages and prophages: all the world's a phage.
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We report DNA and predicted protein sequence similarities, implying homology, among genes of double-stranded DNA (dsDNA) bacteriophages and prophages spanning a broad phylogenetic range of host bacteria. The sequence matches reported here establish genetic connections, not always direct, among the lambdoid phages of Escherichia coli, phage phiC31 of Streptomyces, phages of Mycobacterium, a previously unrecognized cryptic prophage, phiflu, in the Haemophilus influenzae genome, and two small prophage-like elements, phiRv1 and phiRv2, in the genome of Mycobacterium tuberculosis. The results imply that these phage genes, and very possibly all of the dsDNA tailed phages, share common ancestry. We propose a model for the genetic structure and dynamics of the global phage population in which all dsDNA phage genomes are mosaics with access, by horizontal exchange, to a large common genetic pool but in which access to the gene pool is not uniform for all phage. (+info)
Bacteriophage SPO1 development: defects in a gene 31 mutant.
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SPO1 temperature-sensitive mutant ts14-1, located in cistron 31, has a DD (DNA synthesis-delayed) phenotype at 37 degrees C and produces progeny in a stretched program. At 44 degrees C it behaves as a DO (DNA synthesis-defective) mutant and shuts off the viral RNA synthesis about 10 min after infection. The thermal sensitivity of this mutant is due to the inactivity of gp-31 (the product of gene 31) at 44 degrees C. However, gp-31 is synthesized at that temperature and partly recovers its activity at 37 degrees C. Only 5 min at the permissive temperature is enough to trigger the continuation of the phage program and to produce progeny. The partial defect at 37 degrees C and the expansion of the middle program together with the pleiotropic defects at the nonpermissive temperature could be suitable for the study of the controls involved in bacteriophage development. (+info)
Bacillus subtilis bacteriophages SP82, SPO1, and phie: a comparison of DNAs and of peptides synthesized during infection.
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The genomes of Bacillus subtilis phages phie, SPO1, and SP82 were compared by DNA-DNA hybridization, analysis of DNA fragments produced by digestion with restriction endonucleases, comparison of the arrays of peptides synthesized during infection, and phage neutralization. DNA-DNA hybridization experiments indicated that about 78% of the SP82 DNA was homologous with SPO1 DNA, whereas 40% of the phie DNA was homologous to either SPO1 or SP82 DNA. Agarose gel electrophoresis was used to compare the molecular weights of DNA fragments produced by cleavage of SP82, SPO1, and phie DNAs with the restriction endonucleases Hae III, Sal I, Hpa II, and Hha I. Digestion of the DNAs with Hae III and Sal I produced only a few fragments, whereas digestion with Hpa II and Hha I yielded 29 to 40 fragments, depending on the DNA and the enzyme. Comparing the Hpa II fragments, 51% of the SP82 fragments had mobilities which matched those of SPO1 fragments, 32% of the SP82 fragments matched the phie fragments, and 34% of the SPO1 fragments matched the phie fragments. Comparing the Hha I digestion products, 62% of the SP82 fragments had mobilities matching the SPO1 fragments, 24% of the SP82 fragments matched the phie fragments, and 22% of the SPO1 fragments matched the phie fragments. Analysis of peptides by electrophoresis on one-dimensional sodium dodecyl sulfate-polyacrylamide slab gels showed that approximately 70 phage-specific peptides were synthesized in the first 24 min of each infection. With mobility and the intervals of synthesis as criteria, 66% of the different SP82 peptides matched the SPO1 peptides, 34% of the SP82 peptides matched the phie peptides, and 37% of the SPO1 peptides matched the phie peptides. Phage neutralization assays using antiserum to SP82 yielded K values of 510 for SP82, 240 for SPO1, and 120 for phie. (+info)