Mutations in bacteriophage lambda repressor that prevent RecA-mediated cleavage.

In this paper, we report on the isolation and sequence analysis of mutations that confer an induction-deficient phenotype to lambda repressor. A total of 16 different mutations, which occur at 13 different sites in the repressor gene, have been characterized. For most of the mutant lysogens, frequencies of spontaneous induction in a recA+ strain were reduced dramatically in comparison with those for a wild-type phage, and these mutant lysogens showed little or no prophage induction after UV irradiation. The immunity properties of cells containing the mutant repressors show that all of the mutants but one exhibit operator-binding properties indistinguishable from wild-type repressor.     

Lambda repressor inactivation: properties of purified ind- proteins in the autodigestion and RecA-mediated cleavage reactions

Under physiological conditions, lambda repressor can be inactivated in vivo or in vitro by RecA-mediated cleavage of the polypeptide chain. The repressor protein is thought to cleave itself, with RecA acting to stimulate autodigestion. ind- repressor mutants are resistant to RecA-mediated inactivation in vivo. In this paper, we report the purification of 15 ind- repressor proteins and the behaviors of these proteins in the RecA-mediated and autodigestion cleavage reactions. None of these proteins undergoes substantial RecA-dependent cleavage. However, eight mutant proteins autodigest at the same rate as wild-type repressor, six mutants do not autodigest or autodigest slower, and one mutant autodigests faster than wild-type. We discuss these results with respect to repressor structure and RecA-binding, and suggest possible roles for the RecA protein in the cleavage reaction.     

Lambda phage mutants insensitive to temperature-sensitive repressor

Summary The effect of the virL, virR or virC mutation (which is related to the virulency of lambda phage) on the gene expression has been studied. The results indicate that the virL is a mutation of operator gene controlling the left-hand operon and virR or virC is that controlling the right-hand operon.     

Lambda phage mutants insensitive to temperature-sensitive repressor

Summary The isolation of recombinant carrying virC mutation from newly isolated virulent carrying virL virC virR, (Horiuchi et al., 1969) was succeeded and the genetic character of virC mutation producing clear plaque was studied. virL virC shows weak-virulent character and produces clear plaque on CIts lysogen but not on wild type lysogen. virC shows avirulent character and no plaque is produced on these lysogen. The virC mutation is located very closely to and on the left side of the virR region (Fig. 1) which is presumed to be the operator of the right-side operon including O and P cistrons. The genetic characters of virL, virR and virC, were compared with v ₁, v ₂, v ₃ mutations of classical vir (Jacob and Wollman, 1954) and c ₁₇ mutation of another type of virulent (Da Silva and Jacob, 1968). The results indicate that virL, virC or virR mutation is similar to v ₂, v ₁ or v ₃ mutation, respectively, and an effect of virC mutation on producing virulent character was somewhat similar to that of c ₁₇ mutation and was stronger than that of virR mutation. The length of virR regions was suggested to be smaller than one tenth of that of the CI cistron.     

Lambda phage mutants insensitive to temperature-sensitive repressor

Summary Weak-virulent mutants of temperate coli-phage were isolated which can grow on the CIts lysogen producing a temperature-sensitive repressor but which cannot grow on the wild type lysogen producing a normal repressor.Genetic analysis on the mutants shows that their weak-virulence is attributable to two mutations, one (virL) in the region between sus N₂₁₃ and c ₄₇ and the other (virR) in the region between c ₁ and sus O₈. Both mutations are located within the region of non-homology between and imm ⁴³⁴ phages.True virulent mutants which can grow on the wild type lysogen can be obtained easily from the weak-virulent mutant by an additional mutation, virC in a region very close to virR. The virulent mutants obtained are similar to the classical vir mutant (Jacob and Wollman, 1954). The virL and virR mutations are probably operator mutations which render the genome insensitive to the repressor.This work was reported at the XII th International Congress of Genetics, held in Tokyo, on August 23, 1968.     

Stimulation of RecA-mediated cleavage of phage phi 80 cI repressor by deoxydinucleotides

The presence of either deoxyguanylyl-(3'----5')-deoxyguanosine (d(G-G] or deoxyadenylyl-(3'----5')-deoxyguanosine (d(A-G] greatly stimulates cleavage of the phage phi 80 cI repressor mediated by the Escherichia coli RecA protein in vitro. No other deoxydinucleoside monophosphate or riboguanylyl-(3'----5')-guanosine (r(G-G] affects the cleavage reaction. Neither the cleavage site of the phi 80 cI repressor nor the requirement for single-stranded DNA and ATP for cleavage is altered by d(G-G). Photoaffinity labeling experiments with 32P-labeled 5'-phosphoryl deoxyguanylyl deoxyguanosine (pd(G-G], which also stimulates cleavage, show that pd(G-G) bound to the repressor under the conditions in which the repressor is cleaved by RecA protein. The binding increases the affinity of the repressor for RecA protein and thus greatly stimulates repressor cleavage. The cleavage reactions of LexA and lambda cI repressors by RecA protein are not affected by d(G-G).     

Lambda repressor mutations that increase the affinity and specificity of operator binding

Intragenic, second-site reversion has been used to identify amino acid substitutions that increase the affinity and specificity of the binding of lambda repressor to its operator sites. Purified repressors bearing the second-site substitutions bind operator DNA from 3 to 600 fold more strongly than wild type; these affinity changes result from both increased rates of operator association and decreased rates of operator dissociation. Three of the revertant substitutions occur in the alpha 2 and alpha 3 DNA binding helices of repressor and seem to increase affinity by introducing new salt-bridges or hydrogen bonds with the sugar-phosphate backbone of the operator site. The fourth substitution alters the alpha 5 dimerization helix of repressor and appears to increase operator affinity indirectly.     

Bacillus subtilis gnt repressor mutants that diminish gluconate-binding ability.

The Bacillus subtilis gnt operon is negatively regulated by GntR, which is antagonized by gluconate. Three GntR mutants with diminished gluconate-binding ability were obtained. Two were missense mutants (Met-209 to Ile and Ser-230 to Leu), whereas the third had a deletion of the C-terminal 23 amino acids. The mutant GntR proteins were unable to become properly detached from the gnt operator even in the presence of gluconate.     

Bacteriophage lambda mutants (lambdatp) that overproduce repressor.

Lambda tp mutants, selected for their ability to form turbid plaques on lon hosts, overproduce repressor. The tp1 and tp2 mutations have been located within (or adjacent to) the cIII gene. The tp1 mutation reduced late gene expression, as measured by endolysin synthesis (in the absence of functional cI repressor) and progeny phage yield. The tp4 mutation was mapped in the cY-cII region, and complementation tests indicated that tp4 affects the diffusible product of the cII gene. The tp4 mutation also reduced progeny production, but did not markedly affect endolysin synthesis.     

Females that experience threat are better teachers

Superb fairy-wren (Malurus cyaneus) females use an incubation call to teach their embryos a vocal password to solicit parental feeding care after hatching. We previously showed that high call rate by the female was correlated with high call similarity in fairy-wren chicks, but not in cuckoo chicks, and that parent birds more often fed chicks with high call similarity. Hosts should be selected to increase their defence behaviour when the risk of brood parasitism is highest, such as when cuckoos are present in the area. Therefore, we experimentally test whether hosts increase call rate to embryos in the presence of a singing Horsfield''s bronze-cuckoo (Chalcites basalis). Female fairy-wrens increased incubation call rate when we experimentally broadcast cuckoo song near the nest. Embryos had higher call similarity when females had higher incubation call rate. We interpret the findings of increased call rate as increased teaching effort in response to a signal of threat.     

Two substrates are better than one: dual specificities for Dnmt2 methyltransferases

Dnmt2 enzymes have been widely conserved during evolution and contain all of the signature motifs of DNA (cytosine-5)-methyltransferases; however, the DNA methyltransferase activity of these proteins is comparatively weak and their biochemical and functional properties remain enigmatic. Recent evidence now shows that Dnmt2 has a novel tRNA methyltransferase activity, raising the possibility that the biological roles of these proteins might be broader than previously thought. This finding has important implications for understanding the evolutionary relationships among these enzymes.     

Bacillus subtilis RecO and SsbA are crucial for RecA-mediated recombinational DNA repair

Genetic data have revealed that the absence of Bacillus subtilis RecO and one of the end-processing avenues (AddAB or RecJ) renders cells as sensitive to DNA damaging agents as the null recA, suggesting that both end-resection pathways require RecO for recombination. RecA, in the rATP·Mg²⁺ bound form (RecA·ATP), is inactive to catalyze DNA recombination between linear double-stranded (ds) DNA and naked complementary circular single-stranded (ss) DNA. We showed that RecA·ATP could not nucleate and/or polymerize on SsbA·ssDNA or SsbB·ssDNA complexes. RecA·ATP nucleates and polymerizes on RecO·ssDNA·SsbA complexes more efficiently than on RecO·ssDNA·SsbB complexes. Limiting SsbA concentrations were sufficient to stimulate RecA·ATP assembly on the RecO·ssDNA·SsbB complexes. RecO and SsbA are necessary and sufficient to ‘activate’ RecA·ATP to catalyze DNA strand exchange, whereas the AddAB complex, RecO alone or in concert with SsbB was not sufficient. In presence of AddAB, RecO and SsbA are still necessary for efficient RecA·ATP-mediated three-strand exchange recombination. Based on genetic and biochemical data, we proposed that SsbA and RecO (or SsbA, RecO and RecR in vivo) are crucial for RecA activation for both, AddAB and RecJ–RecQ (RecS) recombinational repair pathways.     

Designing better coumarin-based fluorogenic substrates for PTP1B

As part of a program to develop better PTP1B fluorogenic substrates that more closely mimic the functionality found in the natural substrate, we have prepared and evaluated nine novel analogs of 4-methylumbelliferone phosphate (MUP) with a variety of additional groups occupying the second phosphate binding pocket.     

Evidence that glucose starvation-sensitive mutants are altered in the relB locus.

Genetic mapping studies indicate that the relaxed-control mutants isolated on the basis of sensitivity to glucose starvation contain lesions in the relB locus. These mutants, which are sensitive to protein synthesis inhibitors such as sulfacetamide, exhibit relaxed control of both RNA and phospholipid syntheses.