Biomedical subjects

L Moitoso de Vargas

Publications and source records attributed to L Moitoso de Vargas.

7 recordsLinked to original sources

PubMed1992

Binding of cultured mammalian cells to immobilized bacteria.

The invasin protein of Yersinia pseudotuberculosis binds to integrin receptors on mammalian cells and promotes cellular penetration. We demonstrate here that the cell attachment activity of invasin can be detected in bacterial colonies that have been immobilized on filter membranes. Invasin expressed in either Escherichia coli K-12 or Y. pseudotuberculosis mediated binding to membranes, and as few as 10(5) Y. pseudotuberculosis resulted in detectable attachment of cultured epithelial cells. A similar binding activity was detected in clinical isolates of the related pathogen Y. enterocolitica but not in environmental isolates. Although there exist multiple mechanisms for the binding of wild-type organisms to host cells, efficient mammalian cell binding to immobilized Y. pseudotuberculosis required expression of a functional invasin protein. Several pathogens that are known to bind or penetrate mammalian cells were also tested, and only one of these bound cultured mammalian cells efficiently.

Adhesins, Bacterial↗

PubMed1991

A switch in the formation of alternative DNA loops modulates lambda site-specific recombination.

The virally encoded Xis protein is one of the components in the site-specific recombination reactions of bacteriophage lambda. It is required for excisive recombination and inhibits integrative recombination. The mechanism of Xis inhibition of the integration reaction was investigated by methylation protection assays (footprinting analyses) in conjunction with recombination assays. Xis is shown to mediate the formation of a specific attP looped structure involving cooperative and competitive long-range interactions among integrase, integration host factor, and Xis proteins. This higher-order structure precludes supercoiled attP from engaging in the productive partner interactions that lead to execution of the first strand exchange in integrative recombination. In addition to its previously characterized role in excision, Xis-induced DNA bending is postulated to act as a regulatory switch (in an alternative loop mechanism) that converts the attP intasome from an integrative-competent complex to a nonreactive one.

Bacterial Proteins↗

PubMed1990

Mapping of a higher order protein-DNA complex: two kinds of long-range interactions in lambda attL.

To map the protein-protein and protein-DNA interactions involved in lambda site-specific recombination, Int cleavage assays with suicide substrates, nuclease protection patterns, gel retardation experiments, and quantitative Western blotting were applied to wild-type attL and attL mutants. The results lead to a model in which one IHF molecule bends the attL DNA and forms a higher order complex with the three bivalent Int molecules required for excisive recombination. It is proposed that each of the Int molecules binds in a unique manner: one bridges two DNA binding sites in cis, one is held via its high affinity amino-terminal DNA binding domain, and the third depends upon protein-protein interactions in addition to its low affinity carboxy-terminal DNA binding domain. This protein-DNA complex contains two unsatisfied DNA binding domains, each with a different sequence specificity, and is well suited to specific interactions with an appropriate recombination partner.

Bacteriophage lambda↗

PubMed1989

DNA looping generated by DNA bending protein IHF and the two domains of lambda integrase.

The multiprotein-DNA complexes that participate in bacteriophage lambda site-specific recombination were used to study the combined effect of protein-induced bending and protein-mediated looping of DNA. The protein integrase (Int) is a monomer with two autonomous DNA binding domains of different sequence specificity. Stimulation of Int binding and cleavage at the low affinity core-type DNA sites required interactions with the high affinity arm-type sites and depended on simultaneous binding of the sequence-specific DNA bending protein IHF (integration host factor). The bivalent DNA binding protein is positioned at high affinity sites and directed, by a DNA bending protein, to interactions with distant lower affinity sites. Assembly of this complex is independent of protein-protein interactions.

Bacterial Proteins↗

PubMed1988

Autonomous DNA binding domains of lambda integrase recognize two different sequence families.

The 40 kd lambda Integrase protein is shown to contain two autonomous DNA binding domains with different sequence specificities. Competition experiments in which the binding activity of Int is assayed through nuclease protection demonstrate the functional independence of the two DNA recognition specificities. Proteolytic cleavage of Int and footprinting analysis of the resulting two major peptides allow the physical separation and identification of two DNA binding domains: an amino-terminal peptide that interacts with "arm-type" sites and a carboxy-terminal peptide that binds to "core-type" sequences. In addition, the data suggest that the two domains can bind DNA simultaneously, consistent with a model in which Integrase would link two disparate DNA sequences.

Amino Acid Sequence↗

PubMed1987

Cellular factors couple recombination with growth phase: characterization of a new component in the lambda site-specific recombination pathway.

Here we characterize FIS (factor for inversion stimulation), a new cellular component of the lambda site-specific recombination pathway. This host protein binds to a specific region in the lambda attP overlapping the Xis binding sites and can bind cooperatively with Xis to these sites. FIS stimulates lambda excision up to 20-fold in vitro in the presence of suboptimal Xis concentrations, but has no effect in the presence of saturating Xis; FIS has no effect on integrative recombination. FIS can replace one Xis molecule in a series of cooperative and competitive interactions but cannot carry out excision in the absence of Xis. FIS's role in the regulation of recombination has been inferred from in vivo modification of DNA. In exponentially growing cells the lambda FIS site is fully occupied, whereas in stationary-phase cells this binding site is vacant.

Bacteriophage lambda↗

PubMed1986

Mutations in an integration host factor-binding site: effect on lambda site-specific recombination and regulatory implications.

The manner in which integration host factor (IHF) regulates lambda site-specific recombination has been analyzed by examining the behavior of both wild-type and mutant DNAs in integrative and excisive recombination as well as in protein binding. While integrative recombination of an attP with two base changes in the H1 site required 8-fold more IHF than did wild type, binding to this site was lowered at least 500-fold, suggestive of cooperative interactions. A mutant attP with nine base changes did not integrate at all in vitro, with the defect being less severe in vivo. IHF inhibition of excisive recombination was relieved by both mutations in vitro and in vivo. These results imply that occupancy of the H1 site is critical for determining the direction of recombination. It is proposed that IHF inhibition of excision provides a monitor of the strength of the induction stimulus and the nutritional state of the cell; this would allow the prophage to excise selectively in conditions which favor successful completion of the lytic cycle.

Bacterial Proteins↗