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Biomedical subjects

P Prentki

Publications and source records attributed to P Prentki.

At least 19 recordsLinked to original sources
PubMed1995

In vivo interaction of the Escherichia coli integration host factor with its specific binding sites.

The histone-like protein integration host factor (IHF) of Escherichia coli binds to specific binding sites on the chromosome or on mobile genetic elements, and is involved in many cellular processes. We have analyzed the interaction of IHF with five different binding sites in vitro and in vivo using UV laser footprinting, a technique that probes the immediate environment and conformation of a segment of DNA. Using this generally applicable technique we can directly compare the binding modes and interaction strengths of a DNA binding protein in its physiological environment within the cell to measurements performed in vitro. We conclude that the interactions between IHF and its specific binding sites are identical in vitro and in vivo. The footprinting signal is consistent with the model of IHF-binding to DNA proposed by Yang and Nash (1989). The occupancy of binding sites varies with the concentration of IHF in the cell and allows to estimate the concentration of free IHF protein in the cell.

Amino Acid Sequence↗
PubMed1995

In vivo interaction of the Escherichia coli integration host factor with its specific binding sites.

The histone-like protein integration host factor (IHF) of Escherichia coli binds to specific binding sites on the chromosome or on mobile genetic elements, and is involved in many cellular processes. We have analyzed the interaction of IHF with five different binding sites in vitro and in vivo using UV laser footprinting, a technique that probes the immediate environment and conformation of a segment of DNA. Using this generally applicable technique we can directly compare the binding modes and interaction strengths of a DNA binding protein in its physiological environment within the cell to measurements performed in vitro. We conclude that the interactions between IHF and its specific binding sites are identical in vitro and in vivo. The footprinting signal is consistent with the model of IHF-binding to DNA proposed by Yang and Nash (1989). The occupancy of binding sites varies with the concentration of IHF in the cell and allows to estimate the concentration of free IHF protein in the cell.

Adenosine Triphosphatases↗
PubMed1994

Sequence analysis and transcription of the apxI operon (hemolysin I) from Actinobacillus pleuropneumoniae.

The DNA sequence of the entire apxI operon from Actinobacillus pleuropneumoniae serotype 1 reference strain 4074 has been determined. This 8292-bp fragment of the chromosomal DNA contains four open reading frames (ORFs) of the strongly hemolytic ApxI toxin. These ORFs correspond to the genes apxIC, apxIA, apxIB and apxID, encoding the activator, the structural toxin protein and the two secretion proteins, respectively. Each of the four ORFs is preceded by a consensus sequence for a putative ribosome-binding site (RBS). The region upstream from apxIC contains several sites that could act as promoters. The transcription start point (tsp) of the apxI operon in A. pleuropneumoniae has been determined by primer extension analysis and was found to be located 133-bp upstream from the translation start codon. The tsp is preceded by sequences matching the -10 and -35 consensus sequence of promoters from Escherichia coli. This is the first promoter identified in A. pleuropneumoniae. The same tsp was used when the expression of apxI was induced by a high concentration of free Ca2+ in the growth medium, as well as when the expression of apxI was not induced by growing the cells in medium depleted of free Ca2+ ions. However, the signal strength of the primer extension was approximately tenfold stronger in Ca(2+)-grown cells. The leader sequence of the transcript is unusually long and very A+U rich (75% A+U).

Actinobacillus pleuropneumoniae↗
PubMed1993

Specific interaction of IHF with RIBs, a class of bacterial repetitive DNA elements located at the 3' end of transcription units.

The prokaryotic REP (repetitive extragenic palindromes) or PU (palindromic units) sequences are often associated with other repetitive elements, forming arrangements which have been called 'BIMEs' (bacterial interspersed mosaic elements). It is estimated that the Escherichia coli chromosome carries approximately 300-500 BIMEs, whose biological role is at present unknown. We have identified a family of BIMEs consisting of two converging REP sequences flanking a 35 bp conserved segment which carries a static DNA bend and a binding site for IHF, the integration host factor of E.coli. We estimate that the E.coli genome harbors approximately 100 copies of this module, which we name 'RIB' (reiterative ihf BIME). We have analyzed by gel retardation and by footprinting the in vitro interaction of IHF with individual RIBs, and shown that the protein binds strongly and specifically to their center. We have also demonstrated binding of IHF to the chromosomal population of RIBs, using a new approach which combines two-dimensional bandshift and Southern blotting. RIB elements are at the end of transcription units, and thus define a new class of ihf sites. Possible implications for genome structure and DNA topology are discussed.

Bacterial Proteins↗
PubMed1993

Two-dimensional gel selection of protein binding sites on DNA.

We have developed a gel electrophoretic approach for visualizing and cloning protein binding sites from complete genomes. This system consists of a simple two-dimensional band shift, in which protein-DNA complexes are retarded in the first dimension, performed at low temperature, and disrupted in the second dimension, performed at high temperature. We present here results obtained with the integration host factor (IHF) and cAMP receptor protein (CRP) proteins of Escherichia coli, and discuss some of the important methodological aspects of the technique.

Amino Acid Sequence↗
PubMed1992

Use of gel retardation to analyze protein-nucleic acid interactions.

Protein-nucleic acid interactions are crucial in the regulation of many fundamental cellular processes. The nature of these interactions is susceptible to analysis by a variety of methods, but the combination of high analytical power and technical simplicity offered by the gel retardation (band shift) technique has made this perhaps the most widely used such method over the last decade. This procedure is based on the observation that the formation of protein-nucleic complexes generally reduces the electrophoretic mobility of the nucleic acid component in the gel matrix. This review attempts to give a simplified account of the physical basis of the behavior of protein-nucleic acid complexes in gels and an overview of many of the applications in which the technique has proved especially useful. The factors which contribute most to the resolution of the complex from the naked nucleic acid are the gel pore size, the relative mass of protein compared with nucleic acid, and changes in nucleic acid conformation (bending) induced by binding. The consequences of induced bending on the mobility of double-strand DNA fragments are similar to those arising from sequence-directed bends, and the latter can be used to help characterize the angle and direction of protein-induced bends. Whether a complex formed in solution is actually detected as a retarded band on a gel depends not only on resolution but also on complex stability within the gel. This is strongly influenced by the composition and, particularly, the ionic strength of the gel buffer. We discuss the applications of the technique to analyzing complex formation and stability, including characterizing cooperative binding, defining binding sites on nucleic acids, analyzing DNA conformation in complexes, assessing binding to supercoiled DNA, defining protein complexes by using cell extracts, and analyzing biological processes such as transcription and splicing.

Base Sequence↗
PubMed1991

Plasmid vectors for selecting IS1-promoted deletions in cloned DNA: sequence analysis of the omega interposon.

We have constructed two plasmid vectors which allow selection for in vivo deletions within cloned DNA fragments. The plasmids are derivatives of pBR322 which carry the Escherichia coli rpsL (strA) gene, known to confer a dominant streptomycin (Sm)-sensitivity phenotype to the host cell, and a copy of the IS1 transposable element. Sm-resistant strains that harbor these plasmids display sensitivity to Sm. Spontaneous IS1-promoted deletions across the rpsL gene can be isolated simply by selection for Sm resistance. Hence, nested sets of deletions of a cloned DNA can be obtained and sequenced with an IS1-specific primer. Using this approach, we have determined the complete nucleotide sequence of the omega interposon [Prentki and Krisch, Gene 29 (1984) 303-313].

Amino Acid Sequence↗
PubMed1991

Identification of protein binding sites in genomic DNA by two-dimensional gel electrophoresis.

We describe a simple two-dimensional electrophoresis procedure to identify the recognition sites of DNA-binding proteins within large DNA molecules. Using this approach, we have mapped E. coli IHF (Integration Host Factor) binding sites within phage Lambda (48 kb) and phage Mu (39 kb) DNA. We are also able to visualize IHF binding sites in E. coli chromosomal DNA (4,700 kb). We present an extension of this technique using direct amplification by PCR of the isolated restriction fragments, which should permit the cloning of a collection of recognition sequences for DNA binding proteins in complex genomes.

Bacterial Proteins↗
PubMed1991

Incompatibility between pSC101 and lambda dv replicons.

Escherichia coli cells are unable to co-maintain a lambda dv and a minimal pSC101 replicon. Selection for both plasmids results in a much reduced growth rate of the cells, whereas selection for one plasmid results in a rapid loss of the other. A chromosomal mutant has been isolated, which restores the normal growth rate of cells harbouring both plasmids, but does not alleviate the incompatibility. The nature of this new case of incompatibility is discussed.

Bacteriophage lambda↗
PubMed1990

Functional organization of the ends of IS1: specific binding site for an IS 1-encoded protein.

The IS 1-encoded protein InsA binds specifically to both ends of IS1, and acts as a repressor of IS1 gene expression and may be a direct inhibitor of the transposition process. We show here, using DNasel 'foot-printing' and gel retardation, that the InsA binding sites are located within the 24/25 bp minimal active ends of IS1 and that InsA induces DNA bending upon binding. Conformational modification of the ends of IS1 as a result of binding of the host protein integration host factor (IHF) to its site within the minimal ends has been previously observed. Using a collection of synthetic mutant ends we have mapped some of the nucleotide sequence requirements for InsA binding and for transposition activity. We show that sequences necessary for InsA binding are also essential for transposition activity. We demonstrate that InsA and IHF binding sites overlap since some sequence determinants are shared by both InsA and IHF. The data suggest that these ends contain two functional domains: one for binding of InsA and IHF, and the other for transposition activity. A third region, when present, may enhance transposition activity with an intact right end. This 'architecture' of the ends of IS1 is remarkably similar to that of IS elements IS10, IS50 and IS903.

Bacterial Proteins↗
PubMed1989

Omegon-Km: a transposable element designed for in vivo insertional mutagenesis and cloning of genes in gram-negative bacteria.

To combine the features of the omega interposons with the advantages of in vivo transposition mutagenesis, we have constructed an artificial transposon, called Omegon-Km. The Omegon-Km transposon is carried on the plasmid pJFF350 which can be conjugally mobilized into a broad range of Gram-negative bacteria. Omegon-Km is flanked, in inverted orientation, by synthetic 28-bp repeats derived from the ends of IS1. In addition, each end of Omegon-Km has the very efficient transcription and translation terminators of the omega interposon. Internally, Omegon-Km carries the selectable kanamycin (Km)-neomycin resistance gene (alph A) which is expressed well in many Gram-negative bacteria. The IS1 transposition functions are located on the donor plasmid but external to Omegon-Km. Thus, insertions of Omegon-Km are very stable because they lack the capacity for further transposition. Omegon-Km mutagenesis is performed by conjugal transfer of pJFF350 from Escherichia coli into any Gram-negative recipient strain in which this plasmid is unable to replicate. Those cells which have had a transposition event are selected by their resistance to Km. Very high frequencies of Omegon-Km transposition were observed in Pseudomonas putida. Preliminary experiments with other Gram-negative soil and water bacteria (Rhizobium leguminosarum, Paracoccus denitrificans) yielded mutants at reasonable levels. The presence of an E. coli-specific origin of replication (ori) within Omegon-Km allows the rapid and easy cloning, in E. coli, of the nucleotide sequences flanking the site of the transposition event.

Blotting, Southern↗
PubMed1988

Processing of unstable bacteriophage T4 gene 32 mRNAs into a stable species requires Escherichia coli ribonuclease E.

Gene 32 from bacteriophage T4 is transcribed as precursor transcripts which are processed to a stable product. This processing of the gene 32 mRNA was observed in RNase III or P-deficient strains of Escherichia coli. However, after infection of an RNase E-deficient strain, the amount of processed transcript was significantly reduced while the levels of the precursor transcripts remained high. RNase E therefore appears to have an essential role in the processing of the gene 32 mRNA. We have mapped the exact 5' end of the processed transcript by primer extension. The cleavage occurs near a stem-loop structure at a site which shows some similarity to other known RNase E cleavage sites. The effects of the processing on the differential stability of the upstream and downstream sequences, and on gene expression, are discussed.

Autoradiography↗
PubMed1988

Mutational analysis of the open reading frames in the transposable element IS1.

IS1 is one of the smallest transposable elements found in bacteria (768 bp). It contains eight overlapping open-reading-frames (ORFs) greater than 50 codons, designated insA to insG and insB'. To determine which of the ORFs actually code for proteins involved in transposition, we have introduced amber codons into each ORF by site-directed mutagenesis which make neutral changes in the overlapping ORFs. Each mutant IS1 was then tested for its ability to mediate cointegrate formation in Su+ and Su- backgrounds. The mutant elements were also tested for trans-complementation in an IS1-free Salmonella background. Our results show that the products of the insA and insB genes are the only ones essential for cointegrate formation. We suggest that other ORFs may, however, encode accessory proteins.

Amino Acid Sequence↗
PubMed1987

Escherichia coli integration host factor binds specifically to the ends of the insertion sequence IS1 and to its major insertion hot-spot in pBR322.

We report here that the ends of IS1 are bound and protected in vitro by the heterodimeric protein integration host factor (IHF). Under identical conditions, RNA polymerase binds to one of these ends (IRL) and protects a region that includes the sequences protected by IHF. Other potential sites within IS1, identified by their homology to the apparent consensus sequence, are not protected. Footprinting analysis of deletion derivatives of the ends demonstrates a correspondence between the ability of the end sequence to bind IHF and its ability to function as an end in transposition. Nonetheless, some transposition occurs in IHF- cells, indicating that IHF is not an essential component of the transposition apparatus. IHF also binds and protects four closely spaced regions within the major hot-spot for insertion of IS1 in the plasmid pBR322. This striking correlation of hot-spot and IHF-binding sites suggests a possible role for IHF in IS1 insertion specificity.

Bacterial Proteins↗
PubMed1987

Sense and antisense transcription of bacteriophage T4 gene 32. Processing and stability of the mRNAs.

Analysis of bacteriophage T4 gene 32 transcription has revealed a multiplicity of mRNAs. In plasmids, gene 32 is expressed primarily from a strong promoter that is shut off after phage infection. In a wild-type infection, gene 32 is initially transcribed from prereplicative polycistronic and monocistronic promoters; subsequently, a monocistronic late mRNA predominates. This transcript, as well as a post-transcriptionally processed product of the earlier mRNA, can be stable. The eventual degradation of the stable mRNAs is temporally regulated by the phage. Finally, the transcription termination region of gene 32 can function as an antisense promoter both in vitro and in vivo.

Base Sequence↗
PubMed1987

Escherichia coli integration host factor bends the DNA at the ends of IS1 and in an insertion hotspot with multiple IHF binding sites.

The integration host factor of Escherichia coli (IHF) is a small, histone-like protein which participates in the integration of bacteriophage lambda into the E. coli chromosome and in a number of regulatory processes. Our recent footprinting analysis has shown that IHF binds specifically to the ends of the transposable element IS1, as well as to several sites within a short segment of the plasmid pBR322. We have extended our studies of the binding of the IHF molecule to these sites in vitro using a gel retardation assay. We report here that IHF bends the DNA upon binding, as judged from the strong cyclic dependence of the protein-induced mobility shift on the position of the binding site. Using cloned, synthetic ends of IS1 as substrates, we have found that some mutations within the conserved bases of the IHF consensus binding sequence abolish binding, and that alterations of the flanking sequences can greatly reduce IHF binding. The presence of multiple IHF sites on a single DNA fragment increases binding very little, indicating that IHF does not bind cooperatively in this complex. We discuss the possibility that DNA bending is related to the role IHF plays in forming and stabilizing nucleoprotein complexes, and suggest that bending at the IHF sites may be important to its diverse effects in the cell.

Bacterial Proteins↗