[Expression of secreted guanyl-specific ribonuclease genes from Bacillus intermedius and Bacillus pumilus in Bacillus subtilis cells]. / Ekspressiia genov sekretiruemykh guanilspetsifichnykh ribonukleaz iz Bacillus intermedius i Bacillus pumilus v kletkakh Bacillus subtilis.

Plasmids with whole genes for ribonucleases from B. intermedius (binase) and B. pumilis (RNase Bp) assembled with the whole gene of barstar, a specific intracellular inhibitor, are constructed. The resultant plasmids pMZ55 and pMZ56 effectively express binase and RNase Bp genes in B. subtilis cells. A medium for maximum expression of RNase genes by recombinant strains is developed. The expression of binase and RNase Bp genes in B. subtilis cells is negatively regulated by exogenic inorganic phosphate.

Expression of the genes for guanyl-specific ribonucleases from Bacillus intermedius and Bacillus pumilus is regulated by the two component signal transduction system PhoP-PhoR in B. subtilis.

Promoters of the genes for guanyl-specific ribonucleases, secreted by B. intermedius (binase) and B. pumilus (Rnase Bp) in phosphate deficient conditions, contain regions similar to appropriate consensus sequences in promoters of the PHO regulated genes of B. subtilis. A number of genes expressed in response to phosphate starvation in B. subtilis are regulated by the two component signal transduction system PhoP-PhoR. Expression of recombinant genes for binase and RNase Bp in B. subtilis strains with mutations in the regulatory protein genes of the PHO regulon was studied. Their expression is strongly regulated by the regulatory proteins of the B. subtilis PHO regulon.

Key role of barstar Cys-40 residue in the mechanism of heat denaturation of bacterial ribonuclease complexes with barstar.

The mechanism by which barnase and binase are stabilized in their complexes with barstar and the role of the Cys-40 residue of barstar in that stabilization have been investigated by scanning microcalorimetry. Melting of ribonuclease complexes with barstar and its Cys-82-Ala mutant is described by two 2-state transitions. The lower-temperature one corresponds to barstar denaturation and the higher-temperature transition to ribonuclease melting. The barstar mutation Cys-40-Ala, which is within the principal barnase-binding region of barstar, simplifies the melting to a single 2-state transition. The presence of residue Cys-40 in barstar results in additional stabilization of ribonuclease in the complex.

Ribonuclease-charged vector for facile direct cloning with positive selection.

Plasmid vectors for positive selection of cloned inserts in Escherichia coli were devised, based on an expression plasmid (pMT416) for the bacterial ribonuclease barnase. In addition to the barnase gene under control of a synthetic tac promoter, these plasmids carry the gene for the barnase inhibitor, barstar, the constitutive expression of which protects the bacterium from the detrimental effects of moderate barnase production. Full expression of the barnase gene overcomes protection by barstar and becomes lethal. Having a unique SmaI/XmaI site in the barnase structural gene, pMT416 itself can be used as a selective vector: uncut or religated pMT416 will preclude growth while plasmids with inserts in the barnase gene will allow the cells to survive. The entire pUC polylinker was inserted into the barnase gene in place of the Val-36 codon. This insert of nineteen largely hydrophilic amino acids does not prevent the lethal effect of full expression of the gene. The resulting plasmid, pMT440, is a generally useful selective cloning vector representing the "kill-the-rest" approach.

Contribution of arginine-82 and arginine-86 to catalysis of RNases from Bacillus intermedius (binase).

To elucidate the functional role of Arg82 and Arg86 in the enzyme activity of binase, the extracellular ribonuclease of Bacillus intermedius, we used site-directed mutagenesis. On cleavage of various substrates the catalytic activity of binase mutant Arg86 Ala is 2.7 x 10(3) - 7.7 x 10(3) times less than that of the native enzyme. The decrease in activity is determined preferentially by the decrease in the molecular rate constant kcat with a relatively small change of enzyme-substrate affinity, characterized by Km. This is the expected result if Arg86 acts to lower the energy of a transition state of the reaction. The replacement of Arg82 by Ala causes a 5-19-fold activity decrease, depending on the substrate. We propose that this residue does not have a direct catalytic function in the molecular mechanism of the binase action and that the activity decrease of binase on the replacement of Arg82 by alanine is mediated by the effect of Arg82 on the pK of catalytic residues.

Conformational stability and thermodynamics of folding of ribonucleases Sa, Sa2 and Sa3.

Ribonucleases Sa, Sa2, and Sa3 are three small, extracellular enzymes produced by different strains of Streptomyces aureofaciens with amino acid sequences that are 50% identical. We have studied the unfolding of these enzymes by heat and urea to determine the conformational stability and its dependence on temperature, pH, NaCl, and the disulfide bond. All three of the Sa ribonucleases unfold reversibly by a two-state mechanism with melting temperatures, Tm, at pH 7 of 48.4 degrees C (Sa), 41.1 degrees C (Sa2), and 47.2 degrees C (Sa3). The Tm values are increased in the presence of 0.5 M NaCl by 4.0 deg. C (Sa), 0.1 deg. C (Sa2), and 7.2 deg. C (Sa3). The Tm values are decreased by 20.0 deg. C (Sa), 31.5 deg. C (Sa2), and 27.0 deg. C (Sa3) when the single disulfide bond in the molecules is reduced. We compare these results with similar studies on two other members of the microbial ribonuclease family, RNase T1 and RNase Ba (barnase), and with a member of the mammalian ribonuclease family, RNase A. At pH 7 and 25 degrees C, the conformational stabilities of the ribonucleases are (kcal/mol): 2.9 (Sa2), 5.6 (Sa3), 6.1 (Sa), 6.6 (T1), 8.7 (Ba), and 9.2 (A). Our analysis of the stabilizing forces suggests that the hydrophobic effect contributes from 90 to 110 kcal/mol and that hydrogen bonding contributes from 70 to 105 kcal/mol to the stability of these ribonucleases. Thus, we think that the hydrophobic effect and hydrogen bonding make large but comparable contributions to the conformational stability of these proteins.

Isolation and purification of two novel streptomycete RNase inhibitors, SaI14 and SaI20, and cloning, sequencing, and expression in Escherichia coli of the gene coding for SaI14.

Two new RNase inhibitors, SaI14 (Mr, approximately 14,000) and SaI20 (Mr, approximately 20,000), were isolated and purified from a Streptomyces aureofaciens strain. The gene sai14, coding for SaI14 protein, was cloned and expressed in Escherichia coli. The alignment of the deduced amino acid sequence of SaI14 with that of barstar, the RNase inhibitor from Bacillus amyloliquefaciens, showed significant similarity between them, especially in the region which contains most of the residues involved in barnase-barstar complex formation.

MR angiography and MR imaging of symptomatic vascular malformations.

PURPOSE: To define the appearance of peripheral vascular malformations at magnetic resonance angiography (MRA) and assess the role of magnetic resonance imaging (MRI) and MRA in the investigation of these lesions. PATIENTS AND METHODS: Fourteen patients (aged 8-51 years) with clinical evidence of a vascular malformation were referred for MRI and MRA, performed on a 0.5T GE Vectra superconducting system (International General Electric, Slough, UK). Multisection T1-weighted spin-echo and T2-weighted fast spin-echo pulse sequences were performed, with an inversion recovery fast spin-echo sequence in two cases. Two-dimensional time of flight (2-D TOF) and/or 2-D phase contrast (PC) MRA was performed in 13 cases. Eleven patients had digital subtraction angiography (DSA) using a Phillips Integris V3000 digital angiographic unit. The findings at MRA and MRI were compared with the catheter angiograms, and the effective diagnostic input of MRA and MRI was determined. RESULTS: MRA demonstrated major feeding vessels and multiple intra-lesional vessels in relation to the high flow lesions, features absent in the low flow lesions. However, small feeding vessels to the AVMs were not clearly identified. MRI gave a clear demonstration of the anatomical extent of all lesions. AVMs (n = 6) and venous malformations (n = 6) were reliably distinguished, the former containing multiple serpentine signal voids on T1- and T2-weighted imaging, the latter being hyperintense to fat on T2-weighted images. Two other high-flow lesions diagnosed clinically as vascular malformations appeared solid on MRI, and were diagnosed histologically as a carotid body tumour and an angiomyolipoma. CONCLUSION: Although 2-D TOF MRA can distinguish AVMs from venous malformations, the technique adds little extra practical information to the diagnostic process, and cannot compete with catheter angiography for the detailed demonstration of AVM feeding vessels. These lesions can also be characterized using spin-echo sequences, though the primary role of MRI is to demonstrate their anatomical extent.

Purification of ribonucleases Sa, Sa2, and Sa3 after expression in Escherichia coli.

The genes for three small ribonucleases from different strains of Streptomyces aureofaciens have been cloned and expressed in Escherichia coli. The purification of these ribonucleases from the periplasmic space is described. The yields range from 10 to 50 mg of protein per liter of culture medium. The molar absorption coefficients, isoelectric pH values, and pH of optimum activity are reported.

Protein-protein interaction: a genetic selection for compensating mutations at the barnase-barstar interface.

Barnase and barstar are trivial names of the extracellular RNase and its intracellular inhibitor produced by Bacillus amyloliquefaciens. Inhibition involves the formation of a very tight one-to-one complex of the two proteins. With the crystallographic solution of the structure of the barnase-barstar complex and the development of methods for measuring the free energy of binding, the pair can be used to study protein-protein recognition in detail. In this report, we describe the isolation of suppressor mutations in barstar that compensate for the loss in interaction energy caused by a mutation in barnase. Our suppressor search is based on in vivo selection for barstar variants that are able to protect host cells against the RNAse activity of those barnase mutants not properly inhibited by wild-type barstar. This approach utilizes a plasmid system in which barnase expression is tightly controlled to keep the mutant barnase gene silent. When expression of barnase is turned on, failure to form a complex between the mutant barnase and barstar has a lethal effect on host cells unless overcome by substitution of the wild-type barstar by a functional suppressor derivative. A set of barstar suppressors has been identified for barnase mutants with substitutions in two amino acid positions (residues 102 and 59), which are critically involved in both RNase activity and barstar binding. The mutations selected as suppressors could not have been predicted on the basis of the known protein structures. The single barstar mutation with the highest information content for inhibition of barnase (H102K) has the substitution Y30W. The reduction in binding caused by the R59E mutation in barnase can be partly reversed by changing Glu-76 of barstar, which forms a salt bridge with the Arg-59 in the wild-type complex, to arginine, thus completing an interchange of the two charges.

A plasmid vector with positive selection and directional cloning based on a conditionally lethal gene.

A plasmid vector with a multiple cloning site (MCS) for positive selection of cloned inserts in Escherichia coli (Ec) has been devised, based on the expression plasmid (pMT416) for the bacterial ribonuclease barnase (Barn). The host is protected from the lethal effect of moderate expression of barn by expression of the gene bars, encoding its inhibitor, barstar (Bars), placed on the same plasmid. Full expression, however, is lethal. Induction is also lethal with the derived plasmid, pMT440, which has the pUC19 MCS inserted into barn. Under inducing conditions, transformation by the vector is lethal unless the product of the modified barn is inactivated by insertion of cloned DNA fragments into the MCS. Plasmid pMT440 is, therefore, a generally useful selective cloning vector not requiring any special strain of Ec.

Dissociation constants and thermal stability of complexes of Bacillus intermedius RNase and the protein inhibitor of Bacillus amyloliquefaciens RNase.

Binase, the extracellular ribonuclease of Bacillus intermedius, is inhibited by barstar, the natural protein inhibitor of the homologous RNase, barnase, of B. intermedius. The dissociation constants of the binase complexes with barstar and its double Cys40,82Ala mutant are about 10(-12) M, only 5 to 43 times higher than those of the barnase-barstar complex. As with barnase, the denaturation temperature of binase is raised dramatically in the complex. Calorimetric studies of the formation and stability of the binase-barstar complex show that the binase reaction with barstar is qualitatively similar to that of barnase but some significant quantitative differences are reported.

In vivo system for the detection of low level activity barnase mutants.

We report the design of a new tightly controlled barnase system which allows the existence of the barnase gene in host cells without a signal sequence. When expression of barnase is turned on by gene inversion in vivo, the lethal effect of barnase (or its mutants) is not compromised either by coexpression of its polypeptide inhibitor (barstar), or by extracellular secretion. This serves as a rapid, sensitive in vivo test for the detection of any very low residual activity of the barnase mutants. Active-site mutants His102Lys, Glu73Asp and Arg87Lys, and a mutant which greatly reduces the stability and yield of protein, Arg83Lys, produce enough activity to be detectable by this test. In contrast, when expressed on a secretion vector, these mutants do not yield detectable activity in a solution assay. Truly inactive mutants, such as those of His102 to Gly, Ala or Leu, were completely harmless when expressed in this system.

Phosphate regulation of biosynthesis of extracellular RNases of endospore-forming bacteria.

The gene for the extracellular ribonuclease of B. pumilus KMM62 (RNase Bp) has been cloned and sequenced. The structural gene for this enzyme is similar to those of the extracellular ribonucleases of B. intermedius 7P (binase) and B. amyloliquefaciens H2 (barnase), as are the regulatory regions of binase and RNase Bp. The regulatory region of the barnase gene, however, is quite different from the other two. In the promoter of the genes for binase and RNase Bp, but not in that for barnase, is a region similar to the Pho box of E. coli. We have established that inorganic phosphate suppresses the synthesis of the binase and RNase Bp, but does not effect the synthesis of barnase.

An improved system for ribonuclease Ba expression.

The extracellular ribonuclease from Bacillus amyloliquefaciens (barnase, RNase Ba) is a well-characterized enzyme extensively used in structure-function studies. A new system for efficient expression and purification of barnase has been developed. The strong regulated expression cassette with the Pr promoter of lambda phage and the cooperative expression of barnase and barstar under its control have been applied to expression of these proteins in Escherichia coli. The expression cassette containing the Pr promoter of E. coli lambda phage under cI repressor regulation and the nucleotide sequence coding for barnase and barstar structural genes were merged into the plasmid pTN441, which was used for large-scale barnase production. The phoA signal peptide was used to express the target protein into cell periplasm. The purification of RNase Ba was carried out in two steps: the initial sample was concentrated followed by RP-HPLC. The system provides a stable yield of homogeneous protein of about 100-150 mg per liter of culture medium.