Przemyslaw Szafranski

In situ detection of tandem DNA repeat length.

A simple method for scoring short tandem DNA repeats is presented. An oligonucleotide target, containing tandem repeats embedded in a unique sequence, was hybridized to a set of complementary probes, containing tandem repeats known lengths. Single-stranded loops structures formed on duplexes containing a mismatched (different) number of tandem repeats. No loop structure formed on duplexes containing a matched (identical) number of tandem repeats. The matched and mismatched loop structures were enzymatically distinguished and differentially labeled by treatment with S1 nuclease and the Klenow fragment of DNA polymerase.

Cloning and analysis of the dnaG gene encoding Pseudomonas putida DNA primase.

The dnaG gene coding for primase, a key enzyme in DNA replication, has been isolated from chromosomal DNA of the soil bacterium Pseudomonas putida. It maps within the putative MMS operon, between the rpsU and rpoD genes. Comparison of the deduced amino acid sequence of P. putida DnaG with sequences of other known bacterial primases reveals the presence of a possible regulatory region which would be unique to pseudomonads. The analysis of nucleotide sequence suggests that stable folding of the dnaG mRNA may significantly contribute to the low level of its expression within a cell.

Streptavidin-based conditional lethal system for biological containment of Pseudomonas putida

The soil bacterium Pseudomonas putida can potentially be applied in bioremediation of areas polluted with aromatic hydrocarbon-based organic solvents and petroleum. However, its use in the open environment has been hindered so far by the lack of reliable survival-control functions. To increase the predictability of P. putida, a novel cell suicide system was constructed, based on triple control of the expression of the lethal Streptomyces avidinii streptavidin gene (stv) coupled with P. putida TOL plasmid-derived Pm/xylS regulatory circuit. In the absence of hydrocarbon pollutants (3-methylbenzoate in described in vitro studies), all but one in 107 to 108 of genetically modified bacteria commits suicide. The stv-based conditional lethal design can thus effectively limit the spread of released microorganisms strictly to polluted localities and keep them alive only as long as the amount of contaminants keeps above a level determined by the sensitivity of their interaction with the XylS protein.