In this report the first example of functional expression of a fimbrial gene cluster of a non‐enteric human pathogen in Escherichia coli is described. This is shown for Haemophilus influenzae fimbriae which mediate adherence to oropharyngeal epithelial cells. A genomic library of H.influenzae type b, strain 770235f+bo, was constructed using a cosmid vector and screened with a synthetic oligonucleotide probe derived from the N‐terminal sequence of the fimbrial subunit of H.influenzae. Four cosmid clones were found which hybridized to this oligonucleotide probe. Escherichia coli strains harbouring these clones expressed the H.influenzae fimbriae at their cell surface, as was demonstrated in a whole‐cell ELISA and by immunogold electron microscopy using a monoclonal antibody specific for the H.influenzae fimbriae. Surface expression could be maintained during subcloning until a minimal H.influenzae DNA insert of approximately 8.1 kb was obtained. Escherichia coli strains harbouring the 8.1 kb H. influenzae DNA were able to cause a mannose‐resistant adherence to oropharyngeal epithelial cells and a mannose‐resistant haemagglutination of human AnWj‐positive erythrocytes. The nucleotide sequence of hifA, the gene encoding the major fimbrial subunit, was determined. The predicted amino acid sequence shows a significant homology with a number of E.coli fimbrial subunits.
Short communications235 79"69 _+ 3"60 × IO 6 were calculated. This mol. wt. is lower than the values of 92 × IO 6 reported by Rapp et al. (1977) and I IO × lO 6 reported by lltis et al. (I977). In their studies the mol.wt. was determined by co-sedimentation in a neutral sucrose gradient of tritium-labelled VZV DNA from the Hirt supernatant with T4 DNA. The tool. wt. of the two DNA isolates appeared indentical. However preliminary results from analysis by endonuclease cleavage have indicated differences in base sequences between the DNA of the two virus strains. More isolates should be analysed to permit further conclusions.
Gemeentel(ike Geneeskundige en Gezondheidsdienst,
From an undiluted passaged virus stock, two size classes of defective simian virus 40 (SV40) DNA were isolated from which two evolutionary variants were cloned. By means of restriction enzyme and heteroduplex analysis, physical maps of the mutants have been constructed. Both mutants contained the region of SV40 DNA coding for the early proteins plus some adjacent sequences (the region from 0.120 to 0.685 map unit, clockwise, on the standard SV40 DNA map). Furthermore, each mutant contained, in the form of two inverted repeats, four times the sequences from the region 0.625 to 0.685 map unit, clockwise. Some biological properties of the mutant DNA were examined, and we found that the mutant DNA (i) has, as compared with SV40 DNA, an impaired ability to induce T antigen in permissive and nonpermissive cells; (ii) does not complement a thermosensitive A mutant of SV40; (iii) replicates very inefficiently without a helper; and (iv), as an apparent contradiction, transforms nonpermissive baby rat kidney cells as well as SV40 DNA. A hypothetical mechanism for the expression of the mutant DNA that might explain the observed biological properties is presented.
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