M V Norgard

Rapid and simple removal of contaminating RNA from plasmid DNA without the use of RNase

Abstract A procedure for the removal of RNA and RNA fragments from large quantities of pBR322 plasmid DNA without the use of RNase is described. Sephacryl S-300 is employed for the separation of low-molecular-weight RNA from plasmid DNA molecules on the basis of gel filtration. The technique thus circumvents many of the dangers associated with treating plasmid DNA preparations with RNase. The procedure should be generally applicable to the purification of virtually any type of plasmid DNA isolated from a bacterial host.

Digoxigenin-ampicillin conjugate for detection of penicillin-binding proteins by chemiluminescence.

This paper describes a highly sensitive new method for the identification of penicillin-binding proteins (PBPs) that is based on the use of an ampicillin-digoxigenin conjugate (DIG-AMP conjugate) which is detected by immunoblotting and chemiluminescence. The sensitivity of chemiluminescence permitted X-ray film exposure times to be decreased to minutes, as opposed to the days or weeks which are requisite when conventionally radiolabeled beta-lactams are used. Coupling of ampicillin to digoxigenin yielded a product containing digoxigenin (detected by chemiluminescence) which also was inhibitory for Staphylococcus aureus and Escherichia coli. Unconjugated digoxigenin at concentrations of up to 100 micrograms/ml was not inhibitory for either organism. For S. aureus the MICs of DIG-AMP (0.7 microgram of conjugated ampicillin per ml) and of free ampicillin (0.5 microgram/ml) were comparable, indicating that ampicillin retained its bioactivity when coupled to digoxigenin. However, for E. coli the MICs of DIG-AMP (70 micrograms of conjugated ampicillin per ml) and of free ampicillin (8 micrograms/ml) were widely disparate, suggesting that the DIG-AMP conjugate was too large and/or hydrophobic to traverse the E. coli outer membrane via porins. DIG-AMP binding assays with E. coli and S. aureus cell envelopes revealed profiles of PBPs similar to those detected with 125I-ampicillin or [3H]penicillin. DIG-AMP binding to PBPs was completely inhibited in competition experiments with free ampicillin or penicillin, supporting the specificity of the DIG-AMP conjugate for PBPs. DIG-AMP thus represents an advantageous alternative to radioactive beta-lactams for the identification and analysis of PBPs. Images

Monoclonal antibodies directed against major histocompatibility complex antigens bind to the surface of Treponema pallidum isolated from infected rabbits or humans

Evidence is presented for the association of class I major histocompatibility complex (MHC) antigens with the surface of Treponema pallidum during infection. A monoclonal antibody (IgG2a) directed against a murine H-2Kb epitope of public specificity reacted with the cell surface of T. pallidum, as assayed by the binding of protein A-colloidal gold in immunoelectron microscopy. Monoclonal antibodies directed against class I rabbit MHC antigens also reacted in immunofluorescence assays with material on the surface of rabbit-cultivated T. pallidum. In addition, impression smears of human syphilitic genital ulcers that were darkfield-positive for the presence of spirochetes were tested in immunofluorescence assays with monoclonal antibodies directed against human MHC antigens; antibody directed against HLA-ABC (class I) was reactive whereas antibody directed against HLA-DR (class II) was nonreactive. Results of the study suggest that the association of host-derived class I MHC antigens or molecular mimicry may play a role in T. pallidum evasion of host immune defenses.

Phenotypic expression of the major 47 kDa surface immunogen of Treponema pallidum in virulent, tissue-cultured treponemes.

Specific monoclonal antibody and Western blot analysis were used to examine the phenotypic expression of the major 47 kDa surface immunogen of Treponema pallidum among organisms cultivated in vitro. Tissue-cultured treponemes synthesized the 47 kDa immunogen as well as, or better than, organisms cultivated in vivo (rabbit testicles).

Examination of amniotic fluid in diagnosing congenital syphilis with fetal death

The diagnosis of congenital syphilis is difficult, particularly in stillborn fetuses, who are often macerated and have undergone autolysis. These changes can obscure both syphilitic histologic findings and special stains for spirochetes in tissue specimens used to confirm the diagnosis of congenital syphilis. Five gravidas with untreated syphilis and fetal deaths underwent sonographic examination and amniocentesis. In all five cases, dark-field microscopic examination of the amniotic fluid showed spirochetes with morphology and motility characteristic of Treponema pallidum. Organisms were infrequent, but easily identified at 400x magnification and confirmed using an oil-immersion objective yielding a 900x magnification. After delivery, fetal-placental examination and autopsy showed clinical findings typical of congenital syphilis in all five cases. Histologic changes compatible with syphilis were found in all four autopsied fetuses. Silver impregnation stains were positive in two of five tissue specimens, and anti-treponemal monoclonal antibody immunofluorescence assays were positive in one of three amniotic fluid specimens examined retrospectively, further strengthening the specificity of the dark-field microscopic identification of spirochetes. This technique, which can make the diagnosis of congenital syphilis, is recommended for women with syphilis and a fetal death, especially if sonographic hydrops and/or edema is present or if an autopsy will not be performed.

Molecular assessment of S1 endonuclease-resistant snapback hairpin loops generated by DNA polymerase I during the in-vitro nick translation reaction

The in-vitro nick translation reaction used to label DNA to high specific activity also produces aberrant DNA structures known as “snapback” hairpin loops. Hairpin structures are precluded from participating in precise DNA-DNA hybridization interactions. Three nick translation systems were all found to yield significant quantities of snapback hairpins, as determined by their resistance to S1 endonuclease digestion following denaturation. The relative quantities of hairpins produced correlated with both the mass average size of the final DNA probe product synthesized as well as the overall rate of the nick translation reaction. Decreases in the amount of exogenous DNase I used in nick translation reactions produced significant decreases in the amount of hairpin loop structures formed. Hairpins could be effectively removed from nick-translated DNAs by employing hydroxylapatite column chromatography. Strategies to reduce hairpin formation during nick translation and the removal of hairpins from nick-translated DNAs are presented.