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.

Regulation of the cysB gene expression in Escherichia coli.

It was found by Northern-type hybridization that the amount of RNA, the transcription of which begins at the cysB regulatory gene promoter, is significantly reduced after cysB gene introduction into Escherichia coli on multicopy plasmid. This result indicates that cysB protein inhibits the transcription of its own gene. O-acetyl-L-serine, an internal inducer of E. coli and Salmonella typhimurin cysteine regulons, has no effect on cysB gene expression.

Properties of cysK mutants of Escherichia coli K12.

Triazole and azaserine resistant mutants of E. coli K12 affecting cysK gene coding for O-acetylserine sulphydrylase were isolated. The cysK gene in E. coli is located in the same region of chromosome as the cycK gene in Salmonella typhimurium. All azaserine and some triazole resistant mutants require cysteine for growth at a normal rate. The cysK mutants have reduced sulphate uptake. Stability and transfer by conjugation of triazole resistant phenotype were checked. Differences in sulphate metabolism between closely related organisms E. coli and S. typhimurium are discussed.

The regulatory cysK mutant of S. typhimurium.

A triazole-resistant mutant, cysK1358, showing novel properties was isolated. Biochemical analysis of this strain suggests a regulatory character of the cysK1358 mutation. The cysK1358 mutation has a pleiotropic effect: the expression of the other gene, cysA, is also altered.

Sulphate permease of Escherichia coli K12.

Some properties of the sulphate transport system and the isolation of sulphate permease mutants in E. coli K12 are described. The gene coding for sulphate permease is located in the same region as the cysA gene in Salmonella typhimurium.

Cloning of cysB mutant alleles of S. typhimurium.

Two cysB mutant alleles of S. typhimurium have been cloned onto pBR vectors. The product of the constitutive cysBc 1352 allele present on the plasmid was found to fulfill regulatory functions: as an activator of the cysteine regulon and as an autorepressor. CysB70 auxotrophic mutation impairs both regulatory functions cysB protein. Transfer of the clones cysBc 1352 allele from E. coli to S. typhimurium and from S. typhimurium to E. coli and biochemical analysis of transformants suggest involvement of a restriction-modification system in the constitutive expression of the cysteine regulon.

Cloning of cysE gene of Escherichia coli with a mini-Mu-lac containing a plasmid replicon.

The restriction map of cysE gene of Escherichia coli was established after cloning a mini-Mu-lac bacteriophage containing a plasmid replicon and recloning on pBR322 vector plasmid. Enzyme assays of transformants indicated the lack of autoregulation of cysE gene.

Identification and mapping of the sulphate permease promoter region in Escherichia coli.

The cys329::lac fusion linking the lacZ gene to the regulatory region of cys locus involved in sulphate transport in E. coli, was cloned and localized within the restriction map of cysA region. The region containing the cysB-dependent promoter governing the expression of sulphate transport system was identified and the direction of transcription from this promoter has been established. Two complementation groups specified by different mutations in cysA region of E. coli were found.

The effect of urea on the expression of the cysteine regulon.

Urea inhibits the expression of those cysteine genes which are regulated by the cysB gene products. It has no effect, however, on the constitutively expressed cysE gene.

Infectious transcripts from cloned cDNA of potato leafroll luteovirus.

Infectious transcripts play a key role in the research on plant viruses at the molecular level. A number of cDNA clones covering the whole genome of the Polish isolate of potato leafroll virus were constructed. Four overlapping clones were selected and assembled using restriction sites. The full copy was positioned between T7 RNA polymerase promoter and unique ScaI site. The full-length capped transcripts of the sequence of the viral genome synthesised in vitro were able to replicate in protoplasts and to produce the viral coat protein.

Studies on the translation mechanism of subgenomic RNA of potato leafroll virus.

The expression of open reading frames located on the subgenomic RNA (sgRNA) has been studied in an in vitro transcription and translation system. The obtained results indicate: a) translation of sgRNA occurs according to the scanning model since the insertion of palindrome (delta G0 = -61 kcal/mol) prevents the initiation of translation; b) ORF6 is translated by suppression of the stop codon separating ORF4 from ORF6 and the presence of suppressor tRNA is necessary for the readthrough; c) the presence of leader sequence of sgRNA (212 nucleotides) decreases the translation efficiency of ORFs located downstream and it affects the ratio of products of ORF4 and ORF5; d) 3'UTR does not influence on an expression of genes located on the sgRNA.

Characterization of the Escherichia coli gene encoding a new member of the short-chain dehydrogenase/reductase (SDR) family.

The nucleotide sequence of a chromosomal DNA fragment located upstream from the cysPTWAM operon of Escherichia coli was established. Sequence analysis indicates the presence of an open reading frame which has been designated ucpA (upstream cys P). The potential protein products exhibits strong sequence homology to the members of a large protein family, short-chain dehydrogenases/reductases. Involvement of Crp, FruR and IHF in the regulation of ucpA transcription in vivo was demonstrated.

Nucleotide sequence of RNA of a Polish isolate of potato leafroll luteovirus.

The nucleotide sequence of the genomic RNA of a Polish isolate of a potato leafroll virus was determined. Some variations between the determined sequences were observed. A comparison of the frequency of sequence variants in particular regions of the genome is presented.