Analysis of Bordetella pertussis populations in European countries with different vaccination policies.

Despite the widespread use of pertussis vaccines during the last decades, pertussis has remained an endemic disease with frequent epidemic outbreaks. Currently two types of vaccines are used: whole-cell vaccines (WCVs) and recently developed acellular vaccines (ACVs). The long-term aim of our studies is to assess the effect of different vaccination policies on the population structure of Bordetella pertussis and ultimately on the disease burden in Europe. In the present study, a total of 102 B. pertussis isolates from the period 1998 to 2001 from five European countries (Finland, Sweden, Germany, The Netherlands, and France) were characterized. The isolates were analyzed by typing based on variable number of tandem repeats (VNTR); by sequencing of polymorphic genes encoding the surface proteins pertussis toxin S1 and S3 subunits (ptxA and ptxC), pertactin (prn), and tracheal colonization factor (tcfA); and by fimbrial serotyping. The results reveal a relationship between geographic location and VNTR types, the frequency of the ptxC alleles, and serotypes. We have not observed a relationship between the strain characteristics we studied and vaccination programs. Our results provide a baseline which can be used to reveal changes in the B. pertussis population in Europe in the coming years.

DNA binding of polyomavirus large T-antigen: kinetics of interactions with different types of binding sites.

Polyomavirus large T-antigen binds to GRGGC sites in double-stranded viral DNA, regulating transcription and replication. Using surface plasmon resonance to record interactions of large T-antigen with different types of binding sites, we found that the configuration of recognition motifs influenced both the association and dissociation rates. Particularly, the complex formed at the origin of DNA replication was labile. A comparison of the interactions between large T-antigen and binding sites with one, two and four GRGGC motifs in tandem showed a strong preference for dimer binding, without detectable co-operativity between dimers. Sodium chloride stabilised the complexes, whereas the dissociation increased rapidly by increasing pH above 7.0.

Preferred DNA-binding-sites of polyomavirus large T-antigen.

Polyomavirus large T-antigen is a multifunctional protein. Its essential function in virus infection is to control the synthesis of viral RNA and DNA. For this activity specific DNA binding is necessary. Large T-antigen can bind to several sites in the regulatory region of viral DNA, consisting of clustered GRGGC nucleotide motifs. Since large T-antigen also regulates the activity of cellular genes and cellular DNA synthesis, it seemed possible that there are alternative, as yet unrecognized, binding sites. To identify sites preferred by large T-antigen, double-stranded polynucleotides with random sequence were used. These polymers had a 31-bp central segment synthesized from a mixture of all four nucleotides and flanking segments of defined sequence. They were subjected to several cycles of binding to large T-antigen with intervening PCR amplification. Individual polynucleotides with affinity for large T-antigen were then isolated by cloning and their nucleotide sequences were determined. The majority of the polynucleotides contained two or three GRGGC motifs separated by between five and eight variable nucleotides. This result suggests that there are not any alternative high-affinity binding sites of large T-antigen. By comparing all the individual binding motifs an extended consensus sequence was observed. The dinucleotide TG was predominant immediately 5' to the binding pentanucleotide. On the 3'-side, at position +2, C residues were very rare. Although the pentanucleotide motif is the same as in polyomavirus DNA, the extended consensus sequence is not observed in viral DNA. In semi-quantitative experiments, binding of purified large T-antigen to a few of the selected DNA molecules was tested. Stable complexes were formed with DNA substrates containing two or three binding motifs in tandem. Binding to DNA with only one complete motif was weaker, but significantly stronger than non-specific association. This result has implications for the number of large T-antigen binding sites in cellular DNA. When mutant bc1081 large T-antigen, that is defective in specific DNA binding, was used in selection of polynucleotides, a different result was obtained. Neither bc1081 nor wild-type large T-antigen bound strongly to these polynucleotides. However, the presence of the motif TTCGGCTT, or part of it, in five of the six isolated polynucleotides suggested that the T-antigen selection was specific.

Lactose repressor-operator DNA interactions: kinetic analysis by a surface plasmon resonance biosensor.

Lactose repressor binding to operator DNA and subsequent dissociation of the complex was monitored continuously by a biosensor, measuring surface plasmon resonance. In this analysis a synthetic, double-stranded oligonucleotide containing the operator site was immobilized on the sensor surface and repressor protein was passed over the surface. The formation of the repressor-operator complex was specific and could be inhibited by isopropyl-beta-D-thiogalactopyranoside inducer. From the association curve, the apparent kass was determined to be 1.8 x 10(6) M-1 s-1. Dissociation of the complex was, for the first time for the lac repressor, determined as an uncatalyzed reaction and the kdiss was determined to be 3.4 x 10(-4) s-1. As a reference, the repressor-operator interaction was analyzed by electrophoretic mobility shift assay under similar reaction conditions. With this method the equilibrium binding constant was calculated to be 2.4 (+/- 0.2) x 10(8) M-1. The corresponding value calculated from biosensor data was 5.1 x 10(9) M-1.

Apparent heterogeneity between leukemic lymphocyte cell lines.

31P-NMR and polarographic techniques were used to investigate glycolytic versus aerobic oxidative activity in normal and leukemic lymphocytes, and to investigate possible heterogeneity in these parameters between two leukemic cell lines. Molt 3 cells showed a 10-fold higher rate of glutamine-dependent respiration than Molt 4 cells, and an increased level of glutamine-uptake. Molt 3 demonstrated a high intracellular buffering capacity, manifested by constant pHi after addition of glucose, while the same treatment applied to Molt 4 cells induced a change in internal pH of up to 1.23 pH units. This data raises the possibility of heterogeneity of leukemic lymphocytes within the patient from whom the isolation was conducted, or of gross metabolic adaptation by the cell lines in culture.

Loss of DNA-binding and new transcriptional trans-activation function in polyomavirus large T-antigen with mutation of zinc finger motif.

A putative zinc finger in polyomavirus large T-antigen was investigated. We were unable to demonstrate unequivocally a requirement for zinc in specific DNA-binding using the chelating agent 1, 10-phenanthroline. An involvement of the putative zinc finger in specific DNA-binding was nevertheless suggested by the properties of a mutant protein with a cys----ser replacement in the finger motif. Probably as a result of the defective DNA-binding, the mutant protein had lost its activity in initiation of viral DNA-replication and in negative regulation of viral early transcription. However, the trans-activation of the viral late promoter was normal. The analysis also revealed a previously unrecognized activity of large T-antigen. The mutant protein trans-activated the viral early promoter. In the wild-type protein this activity is probably concealed by the separate, negative regulatory function.