The evolution of Tn21 and related multiresistance transposons of natural and artificial origin is described. Variations resulting from insertions and deletions in these transposons indicate the presence of specific recombination sites. There is evidence that these 'hot spots' are flanking several resistance genes. We examined these structures on the basis of the aadA gene, mediating resistance to spectinomycin and streptomycin. Comparison of the DNA sequences of Tn21, Tn7 and pSa indicate the wide spread of these recombination sites.
Human monocytes and macrophages synthesize lysosomal enzymes as larger precursors. The polypeptide patterns of several lysosomal-enzyme precursors and their mature forms are similar to those observed in human fibroblasts. Like fibroblasts, the monocytes and macrophages release small amounts of lysosomal-enzyme precursors. The lysosomotropic NH4+ cation enhances this release. In contrast, zymosan, a degranulating agent, causes release of both the mature and the precursor forms of the lysosomal enzymes. Both NH4Cl and zymosan inhibit maturation of the precursors. The fractional amounts of mature cathepsin D and beta-hexosaminidase released in the presence of zymosan are strikingly different. Probably, in the macrophages several lysosomal organelles are packaged with different relative contents of lysosomal enzymes. The transport of the precursors of cathepsin D into lysosomes is inhibited by tunicamycin. Therefore oligosaccharide side chains are likely to function as signals in packaging of lysosomal enzymes in macrophages also.
A total of 807 gram-negative clinical isolates were treated with five different probes: intragenic segments for the transposase gene tnpA; the resolvase gene tnpR; the modu;ator of the resolvase, tnpM; the integraselike factor gene tnpI; and a 20-mer oligonucleotide for the recombinational site of action for the integrase. A total of 8% of the isolates hybridized with all five Tn2l-related probes, and another 11% represented transposons in which one or more of the tested genes were missing. This 11% included groups whose descriptions have been published as well as groups that have not yet been described. The not-yet-described groups include various deletion products and some precursor structures, as is predicted for the evolution of Tn2l-like transposons.The integration system appears to be coupled with Tn2l-like structures and yet independent from these structures, implying an independent evolution of this system from Tn2l-like transposons. The structures were found with similar incidence levels in all species tested except Pseudomonas aeruginosa, for which a novel separate family of class II transposons has been described before.At present, the Tn2l-like transposons are considered the major structures for transferable multiresistance (5). The Tn2l transposons belong to one subgroup of class II transposons (11); the other subgroup is composed of the Tn3 group. In contrast to the Tn3 group, which mainly encodes only TEM enzymes, the Tn2l group encodes resistance against older and newly introduced antibiotics in various combinations (23). This difference is due to the acquisition of a recA-independent site-specific recombination system, which is responsible for the reorganization of the basic backbone in the resistance-gene-encoding segment surrounding the aadA gene (2, 23). The flanking regions of these genes are short, direct repeated identical sequences. These conserved sequences act as the loci for a site-specific recombination system involving specific recombination enzymes (16). These enzymes show homology to a group of other site-specific recombinases found mostly in phages, in which they are responsible for the integration of the phage DNA into the host DNA. Therefore, the enzyme in Tn21-like transposons was called integraselike factor, or integrase. This enzyme is responsible for insertion, substitution, and deletion of resistance genes (a substitution is defined as a coupled insertion and deletion). The short recombinational sequences which are the points of action were thought to be 54 bp (23) MATERIALS AND METHODSBacterial strains and plasmids. A total of 807 unselected gram-negative clinical isolates were used for hybridization. These strains were collected in 1986 during a multicenter study in hospitals in Austria, Switzerland, and Germany. All other bacterial strains and plasmids used are listed in Table 1.Preparation of DNA and plasmid techniques. Plasmid DNA was prepared by the cleared-lysate method of KupersztochPortnoy et al. (13) and purified by cesium chloride-ethidium bromide ultracentrifug...
A procedure is described that allows the characterization of the molecular forms of beta-hexosaminidase and cathepsin D in controls and pathological specimens of human serum and human urine. The following observations were made. (1) In human serum, beta-hexosaminidase (alpha- and beta-chain) and cathepsin D are present predominantly in their high-molecular-weight precursor forms. In human urine, these enzymes exist as both precursor and mature forms. (2) Cathepsin D precursor from serum and urine differs in the number of oligosaccharides that are sensitive to endo-beta-N-acetylglucosaminidase H. Therefore the urine enzyme is not likely to originate from the serum. (3) The presence exclusively of precursors of beta-hexosaminidase and of cathepsin D in the sera of patients with hepatitis suggests that in hepatitis secretion of lysosomal enzymes is elevated, rather than the enzymes leaking from damaged cells. (4) In the urine of patients with nephrotic syndrome, beta-hexosaminidase and cathepsin D are present in grossly elevated amounts, but do not differ in the polypeptide patterns from controls. (5) In urine from a patient with mucolipidosis II, the elevated activity of beta-hexosaminidase is accounted for mainly by the precursor forms. Mature beta-chain of beta-hexosaminidase is lacking, and incompletely processed beta-hexosaminidase polypeptides are present. Both the precursor and the mature forms of cathepsin D are increased. They contain only complex oligosaccharides.
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