Danuta Hulanicka

An antisense coat protein gene confers immunity to potato leafroll virus in a genetically engineered potato

The Bzura commercial potato cultivar was transformed by sense or antisense constructs which included the coat protein gene of potato leafroll virus RNA. In the sense construct, the coat protein gene was preceded by a leader sequence shorter than that in the subgenomic RNA formed in infected cells. The antisense construct consisted of a sequence complementary to the first 2020 nucleotides of the subgenomic RNA. Selected transformants expressing viral RNA were resistant to virus challenge by viruliferous aphids. In one line, expression of the antisense RNA prevented virus infection even after grafting with scions from infected plants and therefore this transformant might be regarded as virus immune.

Effect of genomic and subgenomic leader sequences of potato leafroll virus on gene expression

The effect of the genomic and subgenomic leader sequence of potato leafroll polerovirus on the efficiency of translation of the downstream located genes has been studied. The results obtained in vitro and in vivo indicate that neither leader sequence functions as translational enhancer, a generally important feature of leader sequences. Deletion analyses demonstrated that both leader sequences not only decrease translation of the downstream located genes but also alter the ratio of the synthesized proteins. A correlation between the in vitro and in vivo results can be established in the case of the subgenomic leader sequence.

Analysis of merodiploids of the cysB region in Salmonella typhimurium.

SummaryWe have studied the regulation of two cysteine biosynthetic enzymes in S. typhimurium merodiploid strains which are heterozygous at the cysB regulatory locus. This gene codes for an element of positive control which is necessary for the expression of the enzymes of the biosynthetic pathway. Under conditions of sulfur deprivation levels of sulfite reductase (coded for by cysI, cysJ and cysG) and of O-acetylserine sulfhydrylase (coded for by cysK) are derepressed in cysB+ haploid strains, but not in cysB- haploid strains. Growth on a rich sulfur source such as l-cystine results in low levels of both enzyme activities in cysB+ and cysB- haploid strains but not in cysBc haploid strains, where enzyme expression is constitutive, i.e. substantially greater than in a cysB+ strain grown on l-cystine, regardless of the nutrients used for growth.We find that cysB-/F cysB+ merodiploid strains can be derepressed for sulfite reductase and O-acetylserine sulfhydrylase by growth on a poor sulfur source, and therefore cysB+ is dominant to cysB-. Enzyme levels are also derepressed in l-cystine-grown cysBc/F cysB+ strains indicating that cysBc is dominant to cysB+. The cysB484 allele is known to be cysB- in regard to the regulation of sulfite reductase activity, but cysBc with respect to O-acetylserine sulfhydrylase. In a cysB484/F cysB+ strain the cysB- character of cysB484 is recessive to cysB+, while cysBc is dominant to cysB+.Merodiploids of the type cysB-/F cysB+, bearing chromosomal point mutations are derepressed by sulfur deprivation to levels which are either less than, equal to, or greater than those of wild type. These results can be explained by assuming a multimeric structure for the cysB protein and the formation in merodiploids of cysB-/cysB+ hybrid molecules with altered capacities for gene activation. The dominance of cysBc over cysB+ indicates that in contrast to the araC regulatory protein, which acts as both a gene activator and repressor, the cysB protein serves only as an element of positive control.

Method of isolation of cysteine constitutive mutants of the cysteine regulon in Salmonella typhimurium

SummaryA method for selection of constitutive cysB mutation is described which takes advantage of the resistance of cysteine constitutive mutants to 1,2,4-triazole. Since cysM cysK double mutants are cysteine auxotrophs, by selecting for triazole resistance in cysM strains, mutants arising under this condition also should be constitutive for cysteine biosynthesis. Genetic analysis of mutants isolated by this technique showed that their mutational sites are located in the cysB region. Biochemical assays of cysteine enzymes, sulphite reductase and O-acetylserine sulfhydrylase of the mutants showed the derepressed level of these enzymes and the lack or slight repression by l-cysteine.

Integration host factor positively regulates cysJIH transcription

Abstract We report the identification of integration host factor (IHF) as an additional element involved in the regulation of the cysJIH promoter of Salmonella typhimurium and Escherichia coli. An IHF-binding site was located in the regulatory region of the cysJIH promoter by using two methods, protection against DNase I digestion and hydroxyl radical cleavage. The positive influence of IHF on in vitro run-off transcription from the cysJIH promoter is shown. The in vivo observations suggest that IHF is necessary for full expression of cysJIH in stationary phase but not during exponential growth.

Effect of DNA gyrase inhibitors on gene expression of the cysteine regulon

SummaryNalidixic acid inhibits the expression of those cysteine genes which are regulated by the cysB product, it has no affect, however, on the constitutively expressed cysE gene. The expression of cysteine genes in a strain carrying a mutation in the nalA locus is resistant to this drug. Novobiocin affects the expression of cysteine genes similarly to nalidixic acid. The effect of nalidixic acid on the expression of genes in a cysteine constitutive mutant was studied.

Constitutive mutation of cysJIH operon in a cysB deletion strain of Salmonella typhimurium.

SummaryIn a cysB deletion strain a new mutation, denoted cys-2332 was isolated, which causes the constitutive expression of the cysJIH operon. cys-2332 is closely linked to cysJIH and presumably is located in the initiator region of this operon, rendering its expression independent of the cysB gene product and the internal inducer O-acetyl-L-serine. The presence of salfite reductase (encoded by cysI and cysJ) activity in a cysB-cys-2332 double mutant indicates that cysG, which is not linked to cysJIH but is required for the synthesis of the sulfite reductase co-factor siroheme, is not controlled by cysB.