gal4 transcription activator protein of yeast can function as a repressor in Escherichia coli

The chromosomal lac operator of Escherichia coli was replaced by a 22 bp oligonucleotide containing the binding site of the yeast gal4 protein. Induction of gal4 protein synthesis in these bacteria repressed beta-galactosidase synthesis at least 30-fold. These results show that it is possible to detect in bacteria with a simple assay the DNA binding activity of a eukaryotic protein with a defined sequence specificity. This opens new avenues for the isolation in E. coli of mutants of DNA binding proteins unable to bind to their DNA targets, and for direct cloning in bacteria of cDNA coding for DNA binding proteins with defined sequence specificity.     

ABA is required for Leptosphaeria maculans resistance via ABI1- and ABI4-dependent signaling

Abscisic acid (ABA) is a defense hormone with influence on callose-dependent and -independent resistance against Leptosphaeria maculans acting in the RLMcol pathway. ABA-deficient and -insensitive mutants in Ler-0 background (abal-3 and abil-1) displayed susceptibility to L. maculans, along with a significantly decreased level of callose depositions, whereas abi2-1 and abi3-1 remained resistant, together with the abi5-1 mutant of Ws-0 background. Suppressor mutants of abil-1 confirmed that the L. maculans-susceptible response was due to the dominant negative nature of the abil-1 mutant. Highly induced camalexin levels made ABA mutants in Col-0 background (aba2-1, aba3-1, and abi4-1) appear resistant, but displayed enhanced susceptibility as double mutants with pad3-1, impaired in camalexin biosynthesis. beta-Aminobutyric acid (BABA) pretreatment of Ler-0 contributed to an elevated level of endogenous ABA after L. maculans inoculation. Comparisons between (RLM1co1)pad3 and rlmlLerpad3 showed that ABA and BABA enhancement of callose deposition requires induction from RLM1col. ABII, but not ABI2, was found to be involved in a feedback mechanism that modulates RLM1co, expression. Genetic analysis showed further that this feedback occurs upstream of ABI4 and that components downstream of ABI4 modulate ABIJ activity. ABA and BABA treatments of the L. maculans-susceptible callose synthase mutant pmr4 showed that ABA also induces a callose-independent resistance. Similar treatments enhanced callose depositions and induced resistance to L. maculans in oilseed rape, and BABA-induced resistance was found to be independent of salicylic acid.     

Crystallization of the arginine-dependent repressor/activator AhrC from Bacillus subtilis

The arginine-dependent repressor/activator AhrC from Bacillus subtilis has been crystallized in space group C222(1), with unit cell dimensions a = 229.8 A, b = 72.8 A, c = 137.7 A and one aporepressor hexamer per asymmetric unit. Preliminary X-ray photographs show measurable intensities beyond 3.0 A.     

4-Thiofuranoid glycals: versatile synthons for stereoselective synthesis of 4'-thionucleosides

Beta-anomers of 4'-thionucleosides have been synthesized stereoselectively, through PhSeCl- or N-iodosuccimide (NIS)-initiated electrophilic glycosidation to 3,5-O-(di-t-butylsilylene)-4-thiofuranoid glycal (1). This synthetic method has been applied to the synthesis of those analogues branched at the anomeric position using 1-C-carbon-substituted 3,5-O-(tetraisopropyldisiloxane-1,3-diyl)-4-thiofuranoid glycals (11-14) prepared based on lithiation of 10.     

Evidence for activator and repressor functions of the response regulator MtrA from Corynebacterium glutamicum

Previous analysis of a Corynebacterium glutamicum Delta mtrAB mutant showed that the MtrAB two-component signal transduction system influences the expression of genes involved in cell wall metabolism or osmoregulation, but it remained unknown whether this influence is direct or indirect. In order to identify the direct target genes of the response regulator MtrA, chromatin immunoprecipitation as a genome-wide approach and DNA affinity chromatography as a gene-specific approach were used. The results indicate that mepA and nlpC, both encoding putative cell wall peptidases, are directly repressed by MtrA, whereas proP and betP, both encoding carriers for compatible solutes, are directly activated by MtrA.     

ABA-insensitive (ABI) 4 and ABI5 synergistically regulate DGAT1 expression in Arabidopsis seedlings under stress

Triacylglycerol (TAG) accumulation is essential for seed maturation in plants. Diacylglycerol acyltransferase 1 (DGAT1) is the rate-limiting enzyme in TAG biosynthesis. In this study, we show that TAG accumulation in Arabidopsis seedlings is correlated with environmental stress, and both ABI4 and ABI5 play important roles in regulating DGAT1 expression. Tobacco transient assays revealed the synergistic effect of ABI4 with ABI5 in regulating DGAT1 expression. Taken together, our findings indicate ABI5 is an important accessory factor with ABI4 in the activation of DGAT1 in Arabidopsis seedlings under stress.     

Regulation of the Escherichia coli allantoin regulon: coordinated function of the repressor AllR and the activator AllS

The allantoin regulon of Escherichia coli, formed by three operons expressed from promoters allA(P), gcl(P) and allD(P), is involved in the anaerobic utilization of allantoin as nitrogen source. The expression of these operons is under the control of the repressor AllR. The hyperinduction of one of these promoters (allD(P)) by allantoin in an AllR defective mutant suggested the action of another regulator, presumably of activator type. In this work we have identified ybbS (proposed gene name allS), divergently transcribed from allA, as the gene encoding this activator. Analysis of the expression of the three structural operons in DeltaallS mutant showed that the expression from allD(P) was abolished, suggesting that AllS is essential for the expression of the corresponding operon. In a wild-type strain expression of allS takes place mainly anaerobically and is hyperinduced when the nitrogen source limits growth. However, expression of allS is independent of regulators of the Ntr response, NtrC or Nac. Band shift experiments showed that AllR binds to DNA containing the allS-allA intergenic region and the gcl(P) promoter and its binding is abolished by glyoxylate. Both DNA fragments contain a highly conserved inverted repeat, which after site-directed mutagenesis, has been proven to be the AllR-binding site. This site displays similarity with the IclR family recognized consensus. Interaction of AllR with the single operator present in the allS-allA intergenic region prevented binding of RNA polymerase to either of the two divergent promoters. The regulator AllS interacts only with allD(P) even in the absence of allantoin. Analysis of this promoter allowed us to identify an inverted repeat as a motif for AllS binding. We propose a model for the coordinate control of the allantoin regulon by AllR and AllS.