Resistance to the peptide-like antibiotic negamycin in Escherichia coli

An $\text{Escherichia coli}$ mutant resistant to the peptide-like antibiotic negamycin carries a mutation, NEG40, which maps at minute 65 on the bacterial genome. Termination of protein synthesis, which is normally blocked by negamycin in wild type cellular extracts, continues with cellular extracts from the mutant in the presence of the drug; indeed, release of complete peptides from the polysomes still proceeds over a wide range of drug concentrations. The data suggest that the NEG40 mutation affects one of the components of the termination complex (ribosome or release factor).     

Phosphorylation by cAMP-dependent protein kinase inhibits the degradation of tau by calpain.

The effects of cAMP-dependent protein kinase (cAMP-PK) phosphorylation on the degradation of the microtubule-associated protein tau by calpain were studied. Purified bovine brain tau that had been phosphorylated by cAMP-PK had a slower migration pattern on sodium dodecyl sulfate-polyacrylamide gels and a more acidic, less heterogeneous pattern on two-dimensional, nonequilibrium pH gradient electrophoresis (NEPHGE) gels compared with untreated tau. Phosphorylation of tau by cAMP-PK significantly inhibited its proteolysis by calpain compared with untreated tau. To our knowledge this is the first demonstration that phosphorylation of tau by a specific kinase results in increased resistance to hydrolysis by calpain. Tau dephosphorylated by alkaline phosphatase migrated more rapidly on sodium dodecyl sulfate-polyacrylamide gels and also showed an altered two-dimensional NEPHGE pattern. Dephosphorylation of tau had no effect on its susceptibility to calpain proteolysis, indicating that regulation of the susceptibility to calpain hydrolysis is due to the phosphorylation of a specific site(s). These results suggest a role for phosphorylation in regulating the degradation of tau. Abnormal phosphorylation could result in a protease-resistant tau population which may contribute to the formation of paired helical filaments in Alzheimer's disease.     

Plasmids pMB123 and pMB124--vectors for obtaining subfragments of DNA with random "sticky" ends

For preparing a DNA fragment with unique protruding ends, plasmid vectors pMB123 and pMB124 were constructed by inserting a synthetic polylinker into plasmid pUR222 at the EcoRI-PstI sites. The polylinker contains two FokI and HgaI sites at its ends in opposite orientation flanking a combination of SalGI, AccI, HindII, HindIII (the latter site is absent from pMB124) and BamHI sites. DNA fragment cloned at the SalGI and BamHI sites can be regenerated by either FokI or HgaI treatment, the SalGI and BamHI sites being deleted from the cloned sequence. Fragments coding for parts of human interleukin-2 were cloned in these vectors.     

Immunoenzyme analysis of adenovirus hexons. Indication and characteristics of rabbit and mouse antibodies against hexons

An enzyme-linked immunosorbent assay (ELISA) was developed for analysis of rabbit and mouse IgG antibodies specific to adenoviral hexon. The anti-hexon antibodies were detected by capture with purified hexon coated onto polystyrene microtiter plates and visualizing them by respective anti-IgG horseradish peroxidase conjugates. In the sera from hyperimmunized rabbits and mice as well as in the mouse ascite fluids the ELISA procedure revealed primarily type-specific (epsilon) and genus-specific (alpha) antigenic determinants in hexon but not those of intermediate specificities.