PubMed · 14240399
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A BROWN, W ARBER. 1964. TEMPERATURE-SENSITIVE MUTANTS OF COLIPHAGE LAMBDA.. https://doi.org/10.1016/0042-6822(64)90114-x
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The O(R) regions from several lambdoid bacteriophages contain the three regulatory sites O(R)1, O(R)2 and O(R)3, to which the Cro and CI proteins can bind. These sites show imperfect dyad symmetry, have similar sequences, and generally lie on the same face of the DNA double helix. We have developed a computational method, which analyzes the O(R) regions of additional phages and predicts the location of these three sites. After tuning the method to predict known O(R) sites accurately, we used it to predict unknown sites, and ultimately compiled a database of 32 known and predicted O(R) binding site sets. We then identified sequences of the recognition helices (RH) for the cognate Cro proteins through manual inspection of multiple sequence alignments. Comparison of Cro RH and consensus O(R) half-site sequences revealed strong one-to-one correlations between two amino acids at each of three RH positions and two bases at each of three half-site positions (H1-->2, H3-->5 and H6-->6). In each of these three cases, one of the two amino acid/base-pairings corresponds to a contact observed in the crystal structure of a lambda Cro/consensus operator complex. The alternate amino acid/base combinations were rationalized using structural models. We suggest that the pairs of amino acid residues act as binary switches that efficiently modulate specificity for different consensus half-site variants during evolution. The observation of structurally reasonable amino acid-to-base correlations suggests that Cro proteins share some common rules of recognition despite their functional and structural diversity.
We examined the requirement of lambda recombination functions for marker rescue of cryptic prophage genes within the Escherichia coli chromosome. We infected lysogenic host cells with lambdaimm434 phages and selected for recombinant immlambda phages that had exchanged the imm434 region of the infecting phage for the heterologous 2.6-kb immlambda region from the prophage. Phage-encoded activity, provided by either Red or NinR functions, was required for the substitution. Red(-) phages with DeltaNinR, internal NinR deletions of rap-ninH, or orf-ninC were 117-, 12-, and 5-fold reduced for immlambda rescue in a Rec(+) host, suggesting the participation of several NinR activities. RecA was essential for NinR-dependent immlambda 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 immlambda rescue. The opposite effects of several host functions toward NinR- and Red-dependent immlambda rescue explains why the independent pathways were not additive in a Rec(+) host and why the NinR-dependent pathway appeared dominant. We measured the influence of the host recombination functions and DnaB on the appearance of orilambda-dependent replication initiation and whether orilambda replication initiation was required for immlambda marker rescue.
A laser-induced fluorescence microscopic system based on optical parametric oscillation has been constructed as a tunable detector for microchip analysis. The detection limit of sulforhodamine B (Ex. 520 nm, Em. 570 nm) was 0.2 mumol, which was approximately eight orders of magnitude better than with a conventional fluorophotometer. The system was applied to the determination of fluorescence-labeled DNA (Ex. 494 nm, Em. 519 nm) in a microchannel and the detection limit reached a single molecule. These results showed the feasibility of this system as a highly sensitive and tunable fluorescence detector for microchip analysis.