A protein that activates expression of a multidrug efflux transporter upon binding the transporter substrates.

Multidrug transporters are membrane proteins which, by an unknown mechanism, recognize diverse toxic compounds and efflux them from cells. We found that two substrates of the Bacillus subtilis multidrug transporter Bmr, rhodamine 6G and tetraphenylphosphonium (TPP), enhance Bmr expression at the level of transcription. Gene knock-out experiments demonstrated that an open reading frame located immediately downstream of the bmr gene is required for this enhancement. The protein product of this open reading frame, BmrR, shows distinct sequence homology to several known bacterial transcription activator proteins, such as MerR and TipAL. Gel-mobility shift and DNase protection assays indicated that BmrR binds specifically, as a dimer, to the bmr gene promoter. Furthermore, the affinity of this binding was enhanced by rhodamine and TPP, thus suggesting that these structurally dissimilar molecules interact directly with BmrR. Indeed, we found that BmrR bound rhodamine 6G stoichiometrically, one rhodamine molecule/BmrR dimer, and that TPP competed with rhodamine for this binding. Our results indicate that the enhancement of Bmr expression by some of its substrates is due to the ability of the regulatory protein, BmrR, to bind structurally dissimilar compounds resulting in enhanced transcription of the transporter gene.

Mutants of the Bacillus subtilis multidrug transporter Bmr with altered sensitivity to the antihypertensive alkaloid reserpine.

The Bacillus subtilis multidrug transporter Bmr effluxes structurally diverse toxic compounds out of bacterial cells. Antihypertensive alkaloid reserpine reverses Bmr-mediated multidrug resistance by inhibiting drug transport. We have obtained a mutant of the bmr gene that provides a normal level of multidrug resistance, which can, however, only be reversed by very high concentrations of reserpine. Reduction of Bmr sensitivity to reserpine has been caused by the substitution of Leu for Val286 in the Bmr molecule. This mutation also led to a dramatic decrease of [3H]reserpine binding to membrane vesicles prepared from the Bmr-overexpressing bacteria. Leucine is larger than valine by one methylene group. Substitution of Val286 with a smaller residue, glycine, had an opposite effect. It led to increased sensitivity of Bmr to reserpine and increased affinity of reserpine binding to the membranes prepared from Bmr-overexpressing bacteria. Neither of the mutations significantly changed the sensitivity of Bmr to rescinnamine, a structural analog of reserpine. The results suggest that Val286 is involved in the formation of the reserpine-binding site of the Bmr molecule.

Fluoroquinolone resistance protein NorA of Staphylococcus aureus is a multidrug efflux transporter.

The gene of the Staphylococcus aureus fluoroquinolone efflux transporter protein NorA confers resistance to a number of structurally dissimilar drugs, not just to fluoroquinolones, when it is expressed in Bacillus subtilis. NorA provides B. subtilis with resistance to the same drugs and to a similar extent as the B. subtilis multidrug transporter protein Bmr does. NorA and Bmr share 44% sequence similarity. Both the NorA- and Bmr-conferred resistances can be completely reversed by reserpine.

Exon encoding the antigen-binding site of MHC class II beta-chains is divided into two subregions with different evolutionary histories.

The evolution of the Ag-binding site of polymorphic class II molecules was investigated by comparing the pattern of silent and replacement substitutions in the first domain exon of DQB and DRB alleles in two distantly related mammalian species, man and cattle. We show that the first domain can be subdivided into two regions, corresponding to the beta-strand and alpha-helical regions, with distinct evolutionary histories. The data for the alpha-helical region are in conflict with the standard phylogeny of mammalian class II genes. In this region, there is only weak locus divergence at the protein level and the frequency of silent substitutions is extremely low between nonorthologous genes (i.e., DQB-DRB) within species. We propose that the major underlying cause for the observed sequence similarity in the alpha-helical region is due to a selective constraint restricting the sequence divergence at the protein level. This selective constraint may be related to the interaction between different isotypic forms of polymorphic class II beta-chains and a common ligand. The extremely low frequencies of silent substitutions between nonorthologous genes within species are most likely due to the transfer of sequence information between loci by the occurrence of gene conversion-like events in the particular gene segment.

Gene duplications and sequence polymorphism of bovine class II DQB genes.

The genetic diversity of bovine class II DQB genes was investigated by polymerase chain reaction amplification and DNA sequencing. The first domain exon was amplified from genomic DNA samples representing 14 class II haplotypes, defined by restriction fragment length polymorphism (RFLP) analysis. The presence of a polymorphism in the copy number of DQB genes was confirmed since two DQB sequences were isolated from certain haplotypes. Four subtypes of bovine DQB genes were found. DQB1 is the major type and was found in almost all haplotypes. DQB2 is very similar to DQB1 but was found only in the duplicated haplotypes DQ9 to 12. DQB3 and DQB4 are two quite divergent genes only present in certain duplicated haplotypes. The bovine DQB complexity thus resembles that in the human DRB region. Bovine DQB genes were found to be highly polymorphic as ten DQB1 alleles and four DQB2 alleles were identified. The observed sequence polymorphism correlated well with previously defined DQB RFLPs. Bovine and human DQB alleles show striking similarities at the amino acid level. In contrast, the frequency of silent substitutions is much higher in comparisons of DQB alleles between species than within species ruling out the possibility that any of the contemporary DQB alleles have been maintained since the divergence of humans and cattle. The frequency of silent substitutions between DQB alleles was markedly lower in cattle than in humans, in agreement with a previous comparison of human and bovine DRB alleles.

[Microcolpohysteroscopy]. / Die Mikrokolpohysteroskopie (MCH).

Microcolpohysteroscopy (MCH) is a method of contact microscopic examination of the ecto- and endocervical epithelium after in vivo staining. To investigate the capability of this procedure, to visualise exactly and identify the margins of cervical intraepithelial neoplasias (CIN), 85 patients with pathological pap smears and two patients with suspicious colposcopy were examined by MCH, microbiopsy from suspicious areas of epithelium and subsequent histology. The ability of MCH to identify and demarcate CIN lesions was verified. It would appear, that using this method, the therapy of higher-grade CIN can be tailored more adequately to the size of the lesion without loss of reliability in respect of the completeness of excision.

Cloning and sequence analysis of 14 DRB alleles of the bovine major histocompatibility complex by using the polymerase chain reaction.

The genetic diversity in the first domain exon of a bovine class II DRB gene was investigated by PCR amplification and DNA sequencing. Genomic DNA samples representing 14 different class II haplotypes, defined by RFLP analysis, were used. The analysis revealed an extensive polymorphism and 14 alleles at a single locus, designated DRB3, were identified. Multiple amino acid substitutions were found in all pairwise comparisons of alleles; 5 to 21 substitutions in the 83 positions compared. The genetic diversity at the amino acid level found in cattle matches the one previously found in the DRB1 locus in man. The significantly higher frequency of replacement substitutions compared with the frequency of silent substitutions provides strong evidence that there is selection for genetic diversity in the bovine DRB3 first domain exon. A comparison of the DRB polymorphism in man and cattle reveals a striking similarity as regards the location of polymorphic positions in the DRB molecule and the degree of polymorphism at polymorphic positions. The majority of polymorphic positions in both species are found in the proposed antigen recognition site of the class II molecule. In addition, there are eight positions which are polymorphic in both species but have not been assigned to the antigen recognition site. The possible functional significance of the polymorphism of these latter positions is discussed.

Evolution of MHC polymorphism: extensive sharing of polymorphic sequence motifs between human and bovine DRB alleles.

The evolution of MHC polymorphism has been studied by comparing the amino acid and nucleotide sequences of 14 bovine and 32 human DRB alleles. The comparison revealed an extensive sharing of polymorphic sequence motifs in the two species. Almost identical sets of residues were found at several highly polymorphic amino acid positions in the putative antigen recognition site. Consequently, certain bovine alleles were found to be more similar to certain human alleles than to other bovine alleles. In contrast, the frequencies of silent nucleotide substitutions were found to be much higher in comparisons between species than within species implying that none of the human or bovine DRB alleles originated before the divergence of these distantly related species. The results suggest that the observed similarity in DRB polymorphism is due to convergent evolution and possibly the sharing of short ancestral sequence motifs. However, the relative role of the latter mechanism is difficult to assess due to the biased base composition in the first domain exon of polymorphic class II beta genes. The frequency of silent substitutions between DRB alleles was markedly lower in cattle than in man suggesting that the DRB diversity has evolved more rapidly in the former species.