Identification of invasin: A protein that allows enteric bacteria to penetrate cultured mammalian cells

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Abstract

Bacterial strains harboring the Yersinia pseudotuberculosis inv locus were analyzed in order to investigate the mechanism of host cell penetration by an invasive pathogen. The inv locus was found to be necessary for Y. pseudotuberculosis to enter HEp-2 cells and sufficient to convert E. coli into a microorganism able to penetrate cultured cells. Both E. coli and Y. pseudotuberculosis strains harboring inv mutations were defective for entry into HEp-2 cells. Furthermore, molecular clones containing inv, and little additional DNA, converted E. coli into a microorganism that was indistinguishable from the parental Yersinia strain with regard to the entry of cultured cells. Data from in vitro protein synthesis indicated that a 103 kd protein was synthesized from inv, saturating the coding capacity of the locus. The nucleotide sequence shows an open reading frame corresponding to a protein of similar size. This protein, called invasin, is necessary for the microorganisms to penetrate HEp-2 cells, and is compartmentalized on the outer surface of the bacterium.

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The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers.

J Messing, J. Vieira (1982)

A series of plasmid vectors containing the multiple cloning site (MCS7) of M13mp7 has been constructed. In one of these vectors a kanamycin-resistance marker has been inserted into the center of the symmetrical MCS7 to yield a restriction-site-mobilizing element (RSM). The drug-resistance marker can be cleaved out of this vector with any of the restriction enzymes that recognize a site of the flanking sequences of the RSM to generate an RSM with either various sticky ends or blunt ends. These fragments can be used for insertion mutagenesis of any target molecule with compatible restriction sites. Insertion mutants are selected by their resistance to kanamycin. When the drug-resistance marker is removed with PstI, a small in-frame insertion can be generated. In addition, two new MCSs having single restriction sites have been formed by altering the symmetrical structure of MCS7. The resulting plasmids pUC8 and pUC9 allow one to clone doubly digested restriction fragments separately with both orientations in respect to the lac promoter. The terminal sequences of any DNA cloned in these plasmids can be characterized using the universal M13 primers.

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Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing.

S Henikoff (1984)

A method is described for the rapid generation and cloning of deletion derivatives well-suited for the sequencing of long stretches of DNA. This method is based on two useful features of exonuclease III: (1) processive digestion at a very uniform rate and (2) failure to initiate digestion at DNA ends with four-base 3'-protrusions. The method was applied to a 4570-bp Drosophila genomic DNA fragment cloned in the single-stranded phage vector M 13mp18. An ordered set of deletion clones was made by first cutting replicative form(RF) DNA with two restriction enzymes in the polylinker region of the vector between the Drosophila DNA and the sequencing primer binding site. One enzyme left a four-base 3'-protrusion that protected the remainder of the vector from exonuclease III attack, allowing unidirectional digestion of the insert sequence from the 5'-protruding end left by the other enzyme. Aliquots were removed at uniform intervals, treated with S1 nuclease, Klenow DNA polymerase, T4 DNA ligase, and then used to transfect competent cells. Most of the resulting clones derived from each aliquot were deleted to a predicted extent with only slight scatter, even for deletions of more than 4 kb. The method permits efficient isolation of clusters of deletion breakpoints within small preselected regions of large DNA segments, allowing nonrandom sequence analysis.