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 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 cro repressor: Non-specific DNA binding

The interaction of lambda phage cro repressor with double-stranded non-specific DNA has been investigated by monitoring the quenching of its intrinsic tyrosyl fluorescence. The McGhee & von Hippel (1974) analysis of the binding of cro repressor to DNA showed that cro repressor undergoes structural variations in the ionic strength range from 0.04 to 0.18m-KCl. Under these salt conditions, the excluded binding site size of cro repressor on the DNA lattice changes from three to four base-pairs (6 to 8 nucleotides) at the lower ionic strengths, to seven to eight base-pairs (14 to 16 nucleotides) at the higher ionic strength. Quaternary structure variation, which does not cause the excluded site size variation, was also noted at low ionic strengths. Evidence is presented to indicate that cro repressor binds only one side of the DNA helix, such that cro repressor covers a stretch of 14 to 16 nucleotides along one side of the helix in the presence of 0.2 m-salt. Under conditions where the cro repressor structure is constant, approximately nine ion-pairs are formed in the cro repressor-non-specific DNA complex. These results are in agreement with the model proposed by Anderson et al. (1981).     

Lambda repressor mutants that are better substrates for RecA-mediated cleavage

RecA-mediated cleavage of the bacteriophage lambda repressor results in inactivation of the protein and leads to induction of the lambda prophage. Here, we report the identification of three mutations in lambda repressor that significantly increase the rate of RecA-mediated cleavage. These mutations were isolated as intragenic second-site suppressors of a mutation (ind-) which prevents cleavage. Purified repressor proteins that contain both the ind- mutation and one of the second-site mutations undergo cleavage at near wild-type rates. Purified repressors that contain the second-site mutations in otherwise wild-type backgrounds undergo RecA-mediated cleavage at significantly faster rates than wild-type, and form dimers more poorly than the wild-type protein. In related experiments, we found that other repressor mutants that dimerize poorly are also better substrates for RecA-mediated cleavage. Conversely, we show that a covalent disulfide-bonded repressor dimer is resistant to cleavage. These results support a model in which repressor monomers are the only substrate in the cleavage reaction.     

Lambda phage cro repressor. DNA sequence-dependent interactions seen by tyrosine fluorescence

The thermodynamics of sickle cell hemoglobin gelation in the presence of oxygen has been investigated by measuring the fractional saturation of the solution and polymer phases, and the solubility. The fractional saturation of the solution phase with oxygen and the solubility were measured by near infrared spectrophotometry after sedimentation of the polymers, while the fractional saturation of the polymer phase was determined from linear dichroism measurements on gels formed by nucleation with an argon ion laser. Using the solution binding data of Gill et al. (1979) to calculate the oxygen pressure corresponding to the solution phase saturation, the initial portion of the polymer binding curve was determined. The self-consistency of the data analysis in terms of the two-phase model for the gel was tested by comparing measured and calculated gel (i.e. solution plus polymer) binding curves, and by comparing the observed solubilities with those calculated from the solution and polymer binding curves using Gibbs-Duhem relations.Oxygen binding to the polymer was found to be non-co-operative up to the maximum measured fractional saturation of 0.14. The binding constant was 0.0059 ± 0.0015 torr^?1 (p₅₀ = 170 ± 40 torr), which is about three times smaller than that of hemoglobin in the low-affinity T quaternary structure. Both the non-co-operative binding and the low affinity could be qualitatively explained in terms of an allosteric model and the current information on the polymer structure.     

Lambda phage cro repressor interaction with its operator DNA: 2'-deoxy-5-fluorouracil OR3 analogues

The experiments here show that chemically synthesized DNA containing fluorine at selected sites can be used to test specific predictions of a model for cro repressor--operator interaction. This is done by observation of the perturbation to the fluorine-19 NMR spectra of analogues of OR3 synthesized with 2'-deoxy-5-fluorouracil at specific positions in the DNA helix. Although the three-dimensional structure of the cro repressor from phage lambda has been determined by Matthews and co-workers [Anderson, W., Ohlendorf, D., Takeda, Y., & Matthews, B. (1981) Nature (London) 290, 754-758], direct structural observations on the complex of the protein with its specific DNA recognition sequence, OR3, are limited. From that structure of the protein, alone, a model of its complex to DNA was built by fitting B-form DNA, with some distortion [Ohlendorf, D., Anderson, W., Fisher, R., Takeda, Y., & Matthews, B. (1982) Nature (London) 298, 718-723]. That model proposes that the cro repressor contacts only one side of this DNA double helix and a number of specific protein--DNA contacts. To test the model, 2'-deoxy-5-fluorouracil was used to place the fluorine-19 nuclear spin-label on the side of the DNA contacting the cro repressor and on the opposite side facing away from the cro repressor. The results presented here are consistent with the prediction that lambda phage cro repressor contacts only one side of the DNA double helix.     

Structural and functional analysis of temperature-sensitive mutants of the phage phi 29 DNA polymerase.

The cloning and complete sequencing of gene 2 from four independently isolated temperature-sensitive mutants in the phage phi 29 DNA polymerase (ts2 mutants) is reported. The results obtained indicate that, in vivo, the mutations only affect the initial steps of the replication process. Interestingly, three of these mutations consist in the single amino acid change Ala to Val at position 492 of the protein. The ts2(24) and ts2(98) mutant phi 29 DNA polymerases were expressed, purified and their thermosensitivity was studied at two different steps of DNA replication: 1) protein-primed initiation and 2) elongation of the DNA chain. Whereas the ts2(24) mutation gave rise to a temperature-sensitive phenotype in both reactions, the ts2(98) mutant protein was rather insensitive to the temperature increase. In addition, the ts2(98) mutant protein showed clear differences in the activation by divalent cations. The relationship of these results with structural and functional domains in the phi 29 DNA polymerase are discussed.     

N-terminal sequence of phage lambda repressor

Summary Lambda repressor was purified from an E. coli strain which produces 150 times more lambda repressor than a single lysogen. The sequence of the fifty N-terminal residues was determined by automated Edman degradation. It contains 43% of all arginine and lysine residues of the chain and constitutes according to the genetic data of Oppenheim et al. (1975) a substantial part of the operator-DNA-binding site of the repressor.     

Monoclonal antibodies to Rhizobium meliloti and surface mutants insensitive to them.

Monoclonal antibodies were produced to the surface of the symbiotic nitrogen-fixing bacterium Rhizobium meliloti. Bacterial lysis in the presence of complement or cycles of agglutination and growth were used to select mutants no longer recognized by the antibodies. The mutants were used to produce new antibodies with different specificities. Several mutants had altered sensitivity to one or more bacteriophages. R. meliloti strains from different sources had distinct patterns of sensitivity to monoclonal antibodies and phages, which together can be used for discriminative typing.     

Amino acid changes in the repressor of bacteriophage lambda due to temperature-sensitive mutations in its cI gene and the structure of a highly temperature-sensitive mutant repressor

The mutant cIts genes from seven different lambdacIts phages carrying tsU50, tsU9, tsU46, ts1, tsU51, tsI-22 and ts2 mutations were cloned in plasmid. The positions of these mutations and the resulting changes of amino acids in the repressor were determined by DNA sequencing. The first four mutations mapping in the N-terminal domain show the following changes: I21S, G53S, A62T and V73A, respectively. Of the three remaining mutations mapping in the C-terminal domain, cItsI-22 and cIts2 show N207T and K224E substitutions respectively, while the mutant cItsU51 gene carries F141I and P153L substitutions. Among these ts repressors, CIts2 having the charge-reversal change K224E was overexpressed from tac promoter in a plasmid and purified, and its structure and function were studied. Operator-binding studies suggest that the ts2 repressor is somewhat defective in monomer-dimer equilibrium and/or cooperativity even at permissive temperatures and loses its operator-binding ability very rapidly above 25 degrees C. Comparative studies of fluorescence and CD spectra, sulfhydryl group reactivity and elution behaviour in size-exclusion HPLC of both wild-type and ts2-mutant repressors at permissive and non-permissive temperatures suggest that the C-terminal domain of the ts2 repressor carrying a K224E substitution has a structure that does not favor tetramer formation at non-permissive temperatures.     

Aggregation and assembly of phage P22 temperature-sensitive coat protein mutants in vitro mimic the in vivo phenotype

Aggregation is a common side reaction in the folding of proteins which is likely due to inappropriate interactions of folding intermediates. In the in vivo folding of phage P22 coat protein, amino acid substitutions that cause a temperature-sensitive-folding (tsf) phenotype lead to the localization of the mutant coat proteins to inclusion bodies. Investigated here is the aggregation of wild-type (WT) coat protein and 3 tsf mutants of coat protein. The tsf coat proteins aggregated when refolded in vitro at high temperature. If the tsf coat proteins were refolded at 4 degrees C, they were able attain an assembly active state. WT coat protein, on the other hand, did not aggregate significantly even when folded at high temperature. The refolded tsf mutants exhibited altered secondary and tertiary structures and had an increased surface hydrophobicity, which may explain the increased propensity of their folding intermediates to aggregate.     

The N-degron approach to create temperature-sensitive mutants in Schizosaccharomyces pombe

Conditional mutants are a vital tool for analysis of gene function. The use of temperature-sensitive mutants in Schizosaccharomyces pombe has significantly promoted understanding of many cellular processes. A portable heat-inducible amino-terminal degron (N-degron) for conditional degradation of a gene product has been previously described in Saccharomyces cerevisiae. This paper describes the adaptation of the N-degron method to create temperature-sensitive (ts) mutants in S. pombe. A ts derivative of the mouse dihydrofolate reductase with an amino-terminal arginine (Arg-DHFR(ts)) previously described in S. cerevisiae was fused to the N-terminus of Bir1p, a nuclear protein involved in mitotic chromosome segregation in S. pombe. This fusion allele, referred to as bir1-td, conferred a chromosome segregation defect at 36 degrees C, as with previously described alleles of bir1. Deletion of the S. pombe E3 ubiquitin ligase (N-recognin), Ubr11p, reversed the temperature-dependent lethality of bir1-td, providing evidence for N-end rule mediated destruction of Bir1p. The methods we describe should therefore facilitate analysis of essential genes in fission yeast for which conditionally lethal mutants are unavailable.     

Two temperature-sensitive photosystem II mutants of pea

Two nuclear gene mutants of pea, chlorotica-887 and chlorina-5756, are temperature-sensitive in the development of photosystem II activity. Low temperature flourescence emission spectra of leaves show that the peak at 697 nm from the reaction center of photosystem II is present when the mutants have been grown at 18°C, but absent when they have been grown at 30°C. For leaves of chlorina-5756 grown at 18°C the relative size of the peak at 697 nm is reduced compared to that of leaves of the wild type or chlorotica-887 grown at this temperature. Flourescence induction curves of leaves from wild type plants and chlorotica-887 grown at 18°C possess two steps, while those of leaves from chlorina-5756 grown at 18°C or 30°C and chlorotica-887 grown at 30°C show at fast rise to the maximal level of fluorescence. Measurements on chloroplasts isolated from the mutants indicated that the photosystem I activity per g leaf material is comparable for plants grown at 18°C and plants grown at 30°C. In contrast, no photosystem II activity was detected when the mutants had been grown at 30°C. It is suggested that these mutants are affected in a component required for the assembly of functional photosystem II complexes.