The minimal sequence needed to define a functional DNA terminator in Bacillus subtilis.

The 47 bp DNA replication terminator (IRI) of Bacillus subtilis, contains two binding sites, A and B, for the replication terminator protein (RTP). Each site binds a dimer of RTP. Removal of the first two base-pairs (bp 1-2) from IRI completely destroyed in vivo terminator (fork arrest) function and was accompanied by loss of RTP binding to the A site, which is distal to the approaching fork that is arrested. Removal of base-pairs 34 to 47 from the other end, proximal to the approaching fork, lowered in vivo function to approximately 50% of the complete IRI. RTP binding appeared to be largely unaffected. Terminator function remained at the approximately 50% level with further deletions that proceeded as far as to include base-pair 28; and RTP binding remained largely unaffected. Removal of more of the sequence beyond base-pair 27 and into the region that makes extensive contact with RTP resulted in a further impairment to in vivo function, and caused altered RTP binding. The base-pairs 1 to 24 segment retained only 16% fork arrest activity and the effect on RTP binding was largely evidenced by an elimination of the ability of this extensively truncated sequence to fill the B site alone. The behaviour of the various terminator deletions emphasize the importance of the previously defined RTP-DNA contacts which allow the binding of RTP to the two overlapping sites, A and B, of IRI for terminator function. A comparison of the affinities of selected truncated terminators for RTP raises the possibility that the overall affinity of RTP for its DNA terminator is not the sole determinant of terminator function.

The Bacillus subtilis DNA replication terminator.

The recent discovery of the Bacillus subtilis plasmid terminator TerLS20 with bidirectional fork arrest activity has provided the opportunity to probe further the structural and functional features of B. subtilis replication terminators in general. The minimal TerI and TerLS20 terminators each comprise two 13 nt segments flanking a central trinucleotide, which is almost completely conserved in all terminators. It corresponds to the region of overlap of the two RTP binding sites (A and B) on the DNA. It has been shown that, despite this conservation, considerable variation in this trinucleotide region still allows fork arrest activity. Thus, the productive interaction of the RTP dimers, which presumably occurs in the vicinity of this trinucleotide region, is not dependent upon stringently defined contacts with the bases in this region. A completely synthetic and highly symmetrical terminator was constructed by replacing the 13 nt segment of the A site of TerI with an opposed segment identical to that in the B site. The efficient bidirectional activity of this new terminator, TerSymB, established more firmly the need for two opposed RTP binding sites in a functional terminator. TerSymB was used to investigate the effect of sequence deviation in one of the 13 nt segments, from that in the B site, on bidirectionality of the terminator. It was found that the deviations introduced converted the terminator significantly towards polarity of action. The partial symmetry within each of the 13 nt segments of TerSymB, and the presumed recognition of this symmetry in the binding of a symmetrical dimer of RTP to each overlapping site, suggest that the bound dimers are centred over positions in the DNA sequence separated by 15 nt. This separation distance has been used in conjunction with the mode of binding of RTP to DNA proposed by Bussiere et al., based on their crystal structure for RTP, to model the interaction of the two dimers of RTP with unbent B-form DNA. Increased separation of the two binding sites of TerSymB was performed by inserting an extra three, seven or ten nucleotides centrally within the TerSymB sequence. The effects of these insertions on RTP binding and fork arrest activity were consistent with the proposed positioning of the RTP dimers within the terminator sequence, and interaction between the dimers bound to TerSymB. A model to account for the generation of RTP-terminator complexes with bidirectional or polar fork arrest activity utilising TerSymB or TerI-VI is presented.

Feasibility of an HA2 domain-based periodontitis vaccine.

In a rat periodontitis model, preinoculation with the Porphyromonas gingivalis HA2 binding domain for hemoglobin provided protection from disease. Protection was associated with induced anti-HA2 immunoglobulin G (IgG) humoral antibodies. The IgG subclass ratios suggested that relatively lower Th2/Th1-driven responses were directly associated with protection when rHA2 was administered in saline.

Protein-nucleoside contacts in the interaction between the replication terminator protein of Bacillus subtilis and the DNA terminator.

The interaction between the DNA replication terminator, IRI, of Bacillus subtilis and its cognate replication terminator protein (RTP) has been examined by the technique of missing nucleoside interference (MNI). IRI contains two adjacent binding sites (A and B) for RTP dimers. The B site is proximal to the replication fork arrest site. The present results have shown that nucleoside contacts with RTP in the two sites are very different. There are more extensive contacts of nucleosides in both strands of the B site with RTP compared with the A site. The data also strongly suggest that filling by RTP of the B site occurs first and is needed for subsequent co-operative filling of an overlapping A site. The A site alone binds RTP poorly. The findings are consistent with interaction occurring between RTP dimers bound to adjacent sites of IRI, which would explain why RTP bound to the B site alone cannot cause replication fork arrest.

Characterization of ryanodine receptors in oligodendrocytes, type 2 astrocytes, and O-2A progenitors.

In this study we have investigated the expression of ryanodine receptors (RyRs), and the ability of caffeine to evoke RyR-mediated elevation of intracellular Ca2+ levels ([Ca2+]i) in glial cells of the oligodendrocyte/type 2 astrocyte lineage. Immunocytochemistry with specific antibodies identified ryanodine receptors in cultured oligodendrocytes, type 2 astrocytes, and O-2A progenitor cells, at high levels in the perinuclear region and in a variegated pattern along processes. Glia acutely isolated from rat brain and in aldehydefixed sections of cortex were similarly found to express RyRs. Caffeine (5-50 mM) caused an increase in [Ca2+]i in most cultured type 2 astrocytes and in 50% of oligodendrocytes. Responses elicited by caffeine were inhibited by pretreatment with ryanodine (10 microM) or thapsigargin (1 microM), and the peak response was unaffected by removal of [Ca2+]o. O-2A progenitor cells, in contrast, were largely unresponsive to caffeine treatment. Pretreatment with kainate (200 microM) to activate Ca2+ entry increased the magnitude of caffeine-evoked [Ca2+]i elevations in type 2 astrocytes and oligodendrocytes, and caused caffeine to activate responses in a significant proportion of previously non-responding O-2A progenitors. In both type 2 astrocytes and oligodendrocytes, caffeine evoked Ca2+ changes which propagated as wavefronts from several initiation sites. These wave amplification sites were characterized by significantly higher local Ca2+ release kinetics. Our results indicate that several glial cell types express RyRs, and that their functionality differs within different cell types of the oligodendrocyte lineage. In addition, ionotropic glutamate receptor activation fills the caffeine-sensitive Ca2+ stores in these cells.

Mechanism of inactivation of ornithine transcarbamoylase by Ndelta -(N'-Sulfodiaminophosphinyl)-L-ornithine, a true transition state analogue? Crystal structure and implications for catalytic mechanism.

The crystal structure is reported at 1.8 A resolution of Escherichia coli ornithine transcarbamoylase in complex with the active derivative of phaseolotoxin from Pseudomonas syringae pv. phaseolicola, N(delta)-(N'-sulfodiaminophosphinyl)-l-ornithine. Electron density reveals that the complex is not a covalent adduct as previously thought. Kinetic data confirm that N(delta)-(N'-sulfodiaminophosphinyl)-l-ornithine exhibits reversible inhibition with a half-life in the order of approximately 22 h and a dissociation constant of K(D) = 1.6 x 10(-12) m at 37 degrees C and pH 8.0. Observed hydrogen bonding about the chiral tetrahedral phosphorus of the inhibitor is consistent only with the presence of the R enantiomer. A strong interaction is also observed between Arg(57) Nepsilon and the P-N-S bridging nitrogen indicating that imino tautomers of N(delta)-(N'-sulfodiaminophosphinyl)-l-ornithine are present in the bound state. An imino tautomer of N(delta)-(N'-sulfodiaminophosphinyl)-l-ornithine is structurally analogous to the proposed reaction transition state. Hence, we propose that N(delta)-(N'-sulfodiaminophosphinyl)-l-ornithine, with its three unique N-P bonds, represents a true transition state analogue for ornithine transcarbamoylases, consistent with the tight binding kinetics observed.