Many proteins are organized as a set of compact functional domains connected by flexible, exposed segments of the polypeptide chain. To study one of these connector regions, we isolated a series of functional in-frame deletions in the central portion of a specific DNA-binding protein, the LexA repressor of Escherichia coli. These mutant proteins fell into two main classes: those with small deletions of two to eight amino acids functioned as repressor about as well as did wild type, while those with large deletions of 17-22 amino acids functioned well only at considerably higher concentrations. The mutant proteins were resistant to the specific cleavage reaction that triggers the SOS response. These data suggest that the conformation of the hinge region in LexA protein is important for cleavage. By contrast, the hinge plays a topological role in repressor function, connecting the two functional halves of the protein; in the SOS response, this function of the hinge is inactivated by cleavage, leading to inactivation of the repressor. 2301The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
The influence of light quality on positive phototopotaxis by the gliding, unicellular red alga Porphyridium purpureum was obtained using interference filters. Cells exposed to 3 × 10−7 mol · m2· s−1 of various wavelengths for 72 h showed maximum topotaxis at 420 and 440 nm. The lower threshold for positive, movement was approximately 5 × 10−8 mol · m−2· s−1. Random movement occurred at nonactinic wavelengths, and no movement occurred in the dark. Cell motility appeared to be unaffected by light polarity, suggesting that the photoreceptor(s) for topotaxis and photokinesis are randomly oriented.
We replaced the Escherichia coli lexA gene by a segment of DNA coding for resistance to spectinomycin and streptomycin. The use of this segment expands the range of selectable markers usable for allele replacement. The availability of this null lexA mutation will facilitate genetic analysis of lexA and the SOS regulon.The Escherichia coli LexA protein controls the expression of the SOS regulon (11,17). Many In order to overcome these problems, we have substituted another selectable marker, resistance to spectinomycin (Spcr), for the lexA gene and have introduced it into the chromosome by homologous recombination (18). The new allele of lexA, which we term lexA300(Del) [where "(Del)" extends the nomenclature of reference 11 and signifies a deletion], was made from plasmid pJWL197 (Fig. 1A), in which a DNA segment coding for resistance to spectinomycin and streptomycin was flanked by homology with E. coli sequences lying on both sides of lexA. Plasmid DNA was linearized by digestion with Sacl and PstI and introduced into strain KP520 (a sulA100::TnS derivative [3] of strain JC9387 recB21 recC22 sbcB15 [16]) as described previously (18), and then selection for resistance to 30 jig of spectinomycin per ml was carried out. Since this level of drug gave heterogenous colony morphology, 20 ,ug/ml was used in all subsequent selections. Because strain KP520 and most other strains used in work on the SOS system carry an rpsL allele conferring resistance to streptomycin (Strr), simultaneous selection for Strr was not possible. In a later step, the lexA300 marker was transduced into other recipient strains by P1 transduction and selection for Spcr.Several lines of evidence indicated that lexA300(Del) completely replaces the resident lexA sequences (Fig. 1B). * Corresponding author.First, recipients carrying lexA7J::TnS became sensitive to kanamycin upon transduction to Spcr, indicating tight linkage between this marker and lexA71. Second, Southern analysis of DNA from Spcr transductants of recipients carrying lexA+ or lexA71::TnS (data not shown) showed that the substitution altered the size of restriction fragments in the region spanning lexA in a manner consistent with the maps in Fig. 1B and that flanking regions were unaltered. Analysis of four of the initial Spcr transformants of strain KP520 also showed that the chromosomal lexA sequences were deleted and that the omega fragment was present on fragments of the predicted size. Third, lexA300 inactivated the LexA repressor function, as judged by several tests using sulA::lacZ operon fusions. Levels of P-galactosidase were identical to those observed in strains carrying lexA7J::Tn5.When strain JL1478, a derivative of JL1436 (L. Lin and J. W. Little, manuscript in preparation) carrying lexA3(Ind-) on the chromosome and expressing low levels of f3-galactosidase, was transduced to Spcr, it also displayed a constitutive level of enzyme. In addition, when a lexA7J: :TnS strain and a lexA300 derivative were transformed with a multicopy plasmid, pJWL147, carrying a lacP-lexA+ ...
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