Bacillus subtilis SalA is a phosphorylation‐dependent transcription regulator that represses scoC and activates the production of the exoprotease AprE

Bacillus subtilis Mrp family protein SalA has been shown to indirectly promote the production of the exoprotease AprE by inhibiting the expression of scoC, which codes for a repressor of aprE. The exact mechanism by which SalA influences scoC expression has not been clarified previously. We demonstrate that SalA possesses a DNA‐binding domain (residues 1–60), which binds to the promoter region of scoC. The binding of SalA to its target DNA depends on the presence of ATP and is stimulated by phosphorylation of SalA at tyrosine 327. The B. subtilis protein‐tyrosine kinase PtkA interacts specifically with the C‐terminal domain of SalA in vivo and in vitro and is responsible for activating its DNA binding via phosphorylation of tyrosine 327. In vivo, a mutant mimicking phosphorylation of SalA (SalA Y327E) exhibited a strong repression of scoC and consequently overproduction of AprE. By contrast, the non‐phosphorylatable SalA Y327F and the ΔptkA exhibited the opposite effect, stronger expression of scoC and lower production of the exoprotease. Interestingly, both SalA and PtkA contain the same ATP‐binding Walker domain and have thus presumably arisen from the common ancestral protein. Their regulatory interplay seems to be conserved in other bacteria.     

Continent-wide distribution in mycorrhizal fungi: implications for the biogeography of specialized orchids

Background and Aims Although mycorrhizal associations are predominantly generalist, specialized mycorrhizal interactions have repeatedly evolved in Orchidaceae, suggesting a potential role in limiting the geographical range of orchid species. In particular, the Australian orchid flora is characterized by high mycorrhizal specialization and short-range endemism. This study investigates the mycorrhizae used by Pheladenia deformis, one of the few orchid species to occur across the Australian continent. Specifically, it examines whether P. deformis is widely distributed through using multiple fungi or a single widespread fungus, and if the fungi used by Australian orchids are widespread at the continental scale.Methods Mycorrhizal fungi were isolated from P. deformis populations in eastern and western Australia. Germination trials using seed from western Australian populations were conducted to test if these fungi supported germination, regardless of the region in which they occurred. A phylogenetic analysis was undertaken using isolates from P. deformis and other Australian orchids that use the genus Sebacina to test for the occurrence of operational taxonomic units (OTUs) in eastern and western Australia.Key Results With the exception of one isolate, all fungi used by P. deformis belonged to a single fungal OTU of Sebacina. Fungal isolates from eastern and western Australia supported germination of P. deformis. A phylogenetic analysis of Australian Sebacina revealed that all of the OTUs that had been well sampled occurred on both sides of the continent.Conclusions The use of a widespread fungal OTU in P. deformis enables a broad distribution despite high mycorrhizal specificity. The Sebacina OTUs that are used by a range of Australian orchids occur on both sides of the continent, demonstrating that the short-range endemism prevalent in the orchids is not driven by fungal species with narrow distributions. Alternatively, a combination of specific edaphic requirements and a high incidence of pollination by sexual deception may explain biogeographic patterns in southern Australian orchids.     

Modulable Southern and Northern blot hybridization apparatus for routine screening of gene amplification and expression

An apparatus for Northern and Southern blot hybridization is described. It allows from one to twenty-four blots to be processed at the same time, with different probes. All the pre-, post- and hybridization steps are performed without handling the filters and the experimenter is totally protected from beta radiations.

The development of such modulable materials has become necessary since Southern and Northern techniques are becoming routine assays in hospitals, particularly in the field of oncology, in prognosis and for hereditary diseases, as an antenatal diagnostis procedure.     

Estrogen stimulates PTHrP but not PTH/PTHrP receptor gene expression in the kidney of ovariectomized rat

The aim of the present study was to test the hypothesis that the decreased renal tubular reabsorption of calcium observed in estrogen deficiency is associated with a local regulation of either PTHrP or PTH/PTHrP receptor genes in the kidney. Rats were randomly sham-operated (S) or ovariectomized receiving either vehicule (OVX) or 4 μg E2/kg/day (OVX+E4) or 40 μg E2/kg/d (OVX+E40) during 14 days using alzet minipumps. Plasma PTH and calcium levels were lower in untreated OVX animals than in all other groups (P < 0.01). Plasma PTH was higher in OVX+E40 than in OVX+E4 (P < 0.05). PTHrP mRNA expression in the kidney was unaffected by ovariectomy but was increased in OVX+E40 (0.984 ± 0.452 for PTHrP/GAPDH mRNAs expression vs. 0.213 ± 0.078 in sham, P < 0.01). PTH/PTHrP receptor mRNA expression and the cAMP response of renal membranes to PTH were unaffected by ovariectomy and estrogen substitution. In conclusion, renal PTHrP and PTH/PTHrP receptor mRNAs are not modified by ovariectomy. However, 17β-estradiol increases renal expression of PTHrP mRNA without evident changes in its receptor expression and function. This may help to explain the pharmacological action of estrogen in the kidney, especially how it prevents the renal leak of calcium in postmenopausal women. J. Cell. Biochem. 70:84–93, 1998. © 1998 Wiley-Liss, Inc.     

Importance of disulfide bonds in receptors for vasoactive intestinal peptide and secretin in rat pancreatic plasma membranes

(1) Vasoactive intestinal peptide (VIP), secretin, and C-terminal octapeptide of cholecystokinin (CCK-8) receptors were identified in rat pancreatic plasma membranes by the ability of these peptides to stimulate adenylate cyclase activity. The membrane preparation procedure was conducted through a series of steps including discontinuous sucrose density gradient fractionation. 5 mM β-mercaptoethanol was added stepwise. Membrane preparations obtained stepwise were preincubated for 10 min at 25°C in the presence of various concentrations of β-mercaptoethanol or dithiothreitol before assaying adenylate cyclase. The use of the reducing agents exerted no effect on p[NH]ppG-, NaF-, and CCK-8- stimulated activities. By contrast, stimulation of adenylate cyclase by low VIP concentrations was specifically altered when β-mercaptoethanol was used during tissue homogeneization at 5°C. (2) In addition, both VIP and secretin responses were highly sensitive towards a preincubation of 10 min at 25°C in the presence of dithiothreitol. (3) These results were likely to reflect alterations at the receptor level. ¹²⁵I-VIP binding was, indeed, reduced after dithiothreitol preincubation, low concentrations of the thiol reagent decreasing the apparent number of high-affinity VIP receptors and higher dithiothreitol concentrations reducing the affinity of VIP receptors.     

Allosteric transition of aspartokinase I-homoserine dehydrogenase I studied by time-resolved fluorescence

The allosteric transition of threonine-sensitive aspartokinase I-homoserine dehydrogenase I from Escherichia coli has been studied by time-resolved fluorescence spectroscopy. Fluorescence decay can be resolved into 2 distinct classes of tryptophan emitters: a fast component, with a lifetime of about 1.5 ns; and a slow component, with a lifetime of about 4.5 ns. The fluorescence properties of the slow component are modified by the allosteric transition. In the T-form of the enzyme stabilized by threonine, the lifetime of the slow component is longer, with a red-shifted spectrum; its accessibility to quenching by acrylamide becomes slightly higher without any decrease of fluorescence anisotropy. These results indicate a change in polarity of the slow component environment. The quaternary structure change associated with the allosteric transition probably involves global movements of structural domains without leading to any local mobility on the nanosecond time-scale. We suggest that the slow component corresponds to the unique tryptophan of the buried kinase domain.     

Clonal analysis of B- and T-cell responses to Ia antigens

Thirty-five Ia^k-specific monoclonal alloantibodies, derived from hybridomas constructed by fusion between mouse myeloma and spleen cells from A.TH alloimmune mice (I ^S anti-I ^k ), have been used to estimate the allotypic polyporphism of the I^k-gene products. Cross-blocking studies using 17 mAb specific for the I-A molecule indicated that six determinants, which were associated with the conventional specificities Ia.2 and Ia.19, were organized in at least three distinct polymorphic areas of the I-A^k molecules. Similarly, another group of six determinants, which did not correspond to previously described conventional Ia specificities, were found to be topologically heterogeneous. By contrast, the five epitopes associated with the Ia. 1 specificity were clustered into a single region of this molecule. In addition the potentiation of binding observed between mAb specific for topologically distinct epitope regions of the I-A^k molecule, suggested that the latter may undergo conformational changes after binding of a given mAb. A similar analysis of 17 mAb specific for the I-E^k molecule indicated that specificity Ia. 7 of the E chain (as defined in this series by eight mAb) was composed of three topologically distinct polymorphic areas, one of which is also spatially related to a complex cluster of eight new determinants of the I-E^k molecule. Finally, one mAb identified a so far undescribed shared determinant of the I-A^k and I-E^k molecules. The present results, which provide a new estimate of the allotypic polymorphism of the Ia^k antigens, are discussed with regard to their functional, biochemical, and evolutionary implications.Abbreviations used in this paper mAb monoclonal antibodies - FCS Fetal calf serum - Con A concanavalin A - H-2 mouse major histocompatibility complex - NMS normal mouse serum - SaCI Staphylococcus aureus Cowan I strain - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Crystallization and Preliminary X-Ray Analysis of Rotavirus Protein VP6

As a first step to gain insight into the structure of the rotavirus virion at atomic resolution, we report here the expression, purification, and crystallization of recombinant rotavirus protein VP6. This protein has the property of polymerizing in the form of tubular structures in solution which have hindered crystallization thus far. Using a combination of electron microscopy and small-angle X-ray scattering, we found that addition of Ca²⁺ at concentrations higher than 100 mM results in depolymerization of the tubes, leading to an essentially monodisperse solution of trimeric VP6 even at high protein concentrations (higher than 10 mg/ml), thereby enabling us to search for crystallization conditions. We have thus obtained crystals of VP6 which diffract to better than 2.4 Å resolution and belong to the cubic space group P4₁32 with a cell dimension a of 160 Å. The crystals contain a trimer of VP6 lying along the diagonal of the cubic unit cell, resulting in one VP6 monomer per asymmetric unit and a solvent content of roughly 70%.