Biomedical subjects

S Semsey

Publications and source records attributed to S Semsey.

4 recordsLinked to original sources

PubMed2004

Integrative plasmid vector for constructing single-copy reporter systems to study gene regulation in Rhizobium meliloti and related species.

The integrative system of phage 16-3 of Rhizobium meliloti 41 was shown to function in several bacterial species belonging to the Rhizobium, Bradyrhizobium, Azorhizobium, and Agrobacterium genera. It might also function in many other bacterial species provided that both the target site (attB) and the required host factor(s) are present. Here we report on the construction of a new integrative vector that can be utilized in gene regulation studies. It provides an opportunity to create a single-copy set-up for characterizing DNA-protein interactions in vivo, in a wide range of bacteria. To demonstrate the usefulness of the vector, transcription repression by binding of the C repressor protein of phage 16-3 to wild type operators was studied. The assay system provided highly reproducible quantitative data on repression.

Agrobacterium tumefaciens↗

PubMed2000

Footprinting studies of specific complexes formed by RepA, a replication initiator of plasmid pCU1, and its binding site.

The basic replicon of plasmid pCU1 contains three different replication origins. Replication initiated from the oriB origin requires pCU1-encoded protein RepA. Previously, information analysis of 19 natural RepA binding sequences predicted a 20-bp sequence as a RepA binding site. Guanines contacting RepA in the major groove of DNA have also been determined. In this study, we used the missing-nucleoside method to determine all of the bases relevant to RepA binding. The importance of some thymine bases was also confirmed by a missing-thymine site interference assay. Participation of the 5-methyl groups of two thymines (at positions -6 and 7) in RepA binding was pointed out by a missing-thymine methyl site interference assay. Phosphate groups of the DNA backbone which strongly interfered with RepA binding upon ethylation were also identified. The pattern of contacting positions mapped by hydroxyl radical protection footprinting indicates that RepA binds to one face of B-form DNA. The length of the binding site was found to be 20 bp by dissociation rate measurement of complexes formed between RepA and a variety of binding sequences. The symmetry of the binding site and that of the contacting bases, particularly the reacting 5-methyl groups of two thymines, suggest that pCU1-encoded RepA may contact its site as a homodimer.

Bacterial Proteins↗

PubMed1999

Identification of site-specific recombination genes int and xis of the Rhizobium temperate phage 16-3.

Phage 16-3 is a temperate phage of Rhizobium meliloti 41 which integrates its genome with high efficiency into the host chromosome by site-specific recombination through DNA sequences of attB and attP. Here we report the identification of two phage-encoded genes required for recombinations at these sites: int (phage integration) and xis (prophage excision). We concluded that Int protein of phage 16-3 belongs to the integrase family of tyrosine recombinases. Despite similarities to the cognate systems of the lambdoid phages, the 16-3 int xis att system is not active in Escherichia coli, probably due to requirements for host factors that differ in Rhizobium meliloti and E. coli. The application of the 16-3 site-specific recombination system in biotechnology is discussed.

Amino Acid Sequence↗

PubMed1998

Integrative promoter cloning plasmid vectors for Rhizobium meliloti.

A set of integrative 'promoter probe' plasmids were constructed for both translational and transcriptional fusions. The vectors are based on the broad host range, low copy number plasmid pRK290 (IncPl) in which the attachment site of Rhizobium phage 16-3 and the lacZ gene of Escherichia coli were combined. The vectors integrate into the chromosome of Rhizobium meliloti, providing also the advantages of the single copy promoter probe cassettes. Thus they fulfil the prerequisite of the systems used for investigating gene regulation. The plasmids were applied for the study of the transcription regulation of the 16-3 phage. Their versatile use is also demonstrated.

Amino Acid Sequence↗