We examined the requirement of recombination functions for marker rescue of cryptic prophage genes within the Escherichia coli chromosome. We infected lysogenic host cells with imm434 phages and selected for recombinant imm phages that had exchanged the imm434 region of the infecting phage for the heterologous 2.6-kb imm region from the prophage. Phage-encoded activity, provided by either Red or NinR functions, was required for the substitution. Red Ϫ phages with ⌬NinR, internal NinR deletions of rap-ninH, or orf-ninC were 117-, 12-, and 5-fold reduced for imm rescue in a Rec ϩ host, suggesting the participation of several NinR activities. RecA was essential for NinR-dependent imm rescue, but had slight influence on Red-dependent rescue. The host recombination activities RecBCD, RecJ, and RecQ participated in NinR-dependent recombination while they served to inhibit Red-mediated imm rescue. The opposite effects of several host functions toward NinR-and Red-dependent imm rescue explains why the independent pathways were not additive in a Rec ϩ host and why the NinR-dependent pathway appeared dominant. We measured the influence of the host recombination functions and DnaB on the appearance of ori -dependent replication initiation and whether ori replication initiation was required for imm marker rescue.

The bacteriophage lambda replication initiation protein P exhibits a toxic effect on its Escherichia coli (E. coli) host, likely due to the formation of a dead-end P-DnaB complex, sequestering the replicative DnaB helicase from further activity. Intracellular expression of P triggers SOS-independent cellular filamentation and rapidly cures resident ColE1 plasmids. The toxicity of P is suppressed by alleles of P or dnaB. We asked whether P buildup within a cell can influence E. coli replication fidelity. The influence of P expression from a defective prophage, or when cloned and expressed from a plasmid was examined by screening for auxotrophic mutants, or by selection for rifampicin resistant (RifR) cells acquiring mutations within the rpoB gene encoding the β-subunit of RNA polymerase (RNAP), nine of which proved unique. Using fluctuation assays, we show that the intracellular expression of P evokes a mutator effect. Most of the RifR mutants remained PS and localized to the Rif binding pocket in RNAP, but a subset acquired a PR phenotype, lost sensitivity to ColE1 plasmid curing, and localized outside of the pocket. One PR mutation was identical to rpo*Q148P, which alleviates the UV-sensitivity of ruv strains defective in the migration and resolution of Holliday junctions and destabilizes stalled RNAP elongation complexes. The results suggest that P-DnaB sequestration is mutagenic and supports an earlier observation that P can interact with RNAP.

PURPOSE. There are several aspects of the visual system that may be regulated by Ca 2ϩ-and calmodulin (CaM)-stimulated protein phosphatase. In the present study, the distribution and characterization of calcineurin (CaN) in bovine eye was determined. METHODS. Whole bovine eyes were either homogenized for purification or regionally dissected to determine CaN localization and activity. Dissected tissues were homogenized and Western blot analysis performed, using polyclonal anti-CaN antibodies, and assayed using p-nitrophenyl phosphate (PNPP) as a substrate to determine the dephosphorylation activity of CaN. Fresh samples were then prepared for immunohistochemistry and probed with polyclonal anti-CaN antibodies. RESULTS. CaN was found to be present in all eye tissues, although activity and protein expression varied. The highest levels of CaN activity and protein expression were found in the optic nerve, retina, and cornea. Immunohistochemical methods displayed similar results with additional staining of the optic nerve vasculature. Assays of purified CaN demonstrated that bovine eye CaN had regulatory properties similar to CaN isolated from other tissues. Probing eye tissues with CaN A isoform-specific antibodies demonstrated that eye tissues displayed variable distributions of the CaN A␣ and CaN A␤ isoforms. CONCLUSIONS. The presence of CaN in the bovine eye provides a physiological pathway by which the phosphorylated state of proteins and intracellular Ca 2ϩ concentrations can be coordinated. The authors propose that CaN is involved in the immunologic privilege of the cornea, retinal signal transduction, and the toxic effects of immunosuppressants on the eye. Further in vivo studies of CaN function are necessary to understand the contributions of CaN to ocular physiology. (Invest Ophthalmol Vis Sci. 2002;43:15-21) C alcineurin (CaN) is an 80-kDa protein phosphatase that is regulated by intracellular Ca 2ϩ concentrations and is capable of regulating a variety of protein substrates by dephosphorylation. The inositol 1,4,5-trisphosphate (IP3) receptor 1 and N-methyl-D-aspartate receptors 2 are deactivated on dephos-From the