Genetic selection of lambda phage clones from a gene clonotheque

Summary A genetic procedure for selection of specific clones, by homologous recombination between clones from a gene clonotheque and sequences cloned into a plasmid, was developed. Resulting clones are isolated in transduction experiments by plating infected Escherichia coli cells under conditions selecting for the antibiotic resistance marker carried by the plasmid. The feasibility of the method was demonstrated in a model test system as well as by isolation of -interferon-specific sequences from the human gene clonotheque.     

Ultraviolet resonance Raman spectroscopy as a probe of protein structure in the fd virus

Resonance Raman spectroscopy can provide details of molecular structure via the enhancement of specific vibrational bands in the spectrum of the scattered light when the laser excitation is tuned to electronic absorption wavelengths of the molecule. The availability of lasers operating in the deep ultraviolet region makes it possible to apply this technique to problems of protein structure. The backbone conformation and the environments of aromatic side chains can be probed via appropriate enhancement of selected vibrational modes. In this article we investigate ultraviolet resonance Raman (UVRR) spectra from the coat protein of the filamentous bacteriophage, fd, in the intact virus and in sodium dodecyl sulfate (SDS) suspension. The results indicate that 1) the protein is completely alpha-helical in the mature virus, but loses a large fraction of its helix content in the SDS micelles. 2) The two tyrosine residues appear to behave as H-bond acceptors in the intact phage but this interaction is lost in the micelles. 3) The tryptophan residue is not solvent-exposed in either protein conformation, although in SDS it is accessible to H/D exchange with the solvent. 4) The three phenylalanine residues are involved in stacking interactions in the intact virus; these are disrupted in the SDS micelles. 5) The single proline residue appears to be in a trans conformation both in the virus and in the micelles.     

Comparison of lipid/gramicidin dispersions and cocrystals by Raman scattering

Gramicidin crystals, dimyristoylphosphatidylcholine (DMPC)/gramicidin dispersions, and DMPC/gramicidin cocrystals were examined by Raman scattering to determine lipid/gramicidin stoichiometries and lipid organization. Calibrations of the choline (716-cm-1) and tryptophan (756-cm-1) peaks indicate that the cocrystals contain two lipids for each gramicidin monomer, a result confirmed by chemical analyses of washed crystals. In dispersions with high lipid/gramicidin ratios (e.g., 25:1), the lipid is ordered but becomes increasingly disordered as the gramicidin content is increased. Paradoxically, the DMPC/gramicidin cocrystals have highly ordered lipids that possibly contain no gauche bonds at all, despite their low lipid/gramicidin ratio. In addition, the polypeptide amide I peak position near 1670 cm-1 is found to be independent of the lipid/gramicidin ratio in the complexes and may indicate a beta-helix-type secondary structure at all ratios. However, the amide I peak broadens significantly at low lipid/gramicidin ratios and broadens still further in the cocrystals, suggesting that protein-protein interactions may induce band-broadening distortions of the polypeptide structure.     

Lipid peroxidation of rabbit small intestinal microvillus membrane vesicles by iron complexes

Fe(II)- and Fe(III)-induced lipid peroxidation of rabbit small intestinal microvillus membrane vesicles was studied. Ferrous ammonium sulphate, ferrous ascorbate at a molar ratio of 10:1, and ferric citrate, at molar ratios of 1:1 and 1:20, did not stimulate lipid peroxidation. Ferrous ascorbate, 1:1, induced low stimulation, while ferrous ascorbate, 1:20 gave higher stimulation of lipid peroxidation. These results show that in our experimental system, ascorbate is a promotor rather than an inhibitor of lipid peroxidation. Ferric nitrilotriacetate (at molar ratios of 1:2 and 1:10), at an iron concentration of 200 microM, was by far the most effective in inducing lipid peroxidation. Superoxide dismutase, mannitol and glutathione had no effect, while catalase, thiourea and vitamin E markedly decreased ferrous ascorbate 1:20-induced lipid peroxidation. Ferric nitrilotriacetate-induced lipid peroxidation was slightly reduced by catalase and mannitol, significantly reduced by superoxide dismutase, and completely inhibited by thiourea. Glutathione caused a 100% increase in the ferric nitrilotriacetate-induced lipid peroxidation. These results suggest that Fe(II) in the presence of trace amounts of Fe(III), or an oxidizing agent and Fe(III) in the presence of Fe(II) or a reducing agent, are potent stimulators of lipid peroxidation of microvillus membrane vesicles. Addition of deferoxamine completely inhibited both ferrous ascorbate, 1:20 and ferric nitrilotriacetate-induced lipid peroxidation, demonstrating the requirement for iron for its stimulation. Iron-induced peroxidation of microvillus membrane may have physiological significance because it could already be demonstrated at 2 microM iron concentration.     

Biological activity of different forms of bacteriophage lambda DNA]

Hershey circles and linear tandem aggregated forms of DNA have been obtained in vitro and treated with polynucleotide ligase to form phosphodiester bond. Using zone centrifugation in glycerol gradient covalently closed circles and linear dimers have been purified and their biological activity investigated. It was found that closed circular molecules lost most, if not all, of their activity in CaCl2-dependent system. In order to investigate the biological activity of tandem dimer molecules, hybrid dimers consisting of DNA's from lambda C1857 and lambda 1434 have been obtained. In plaque assay with the appropriate non-permissive strains of E. coli the efficiency of infectivity of hybrid dimers was measured. Biological activity of dimer molecules sealed with ligase was about 5% of the activity of linear monomers. Ig has been suggested that tandem dimers of lambda DNA joined by phosphodiester bond are able to penetrate into the CaCl2-treated host cells and both components of dimers are active during subsequent multiplication.