Nucleotide sequence of the type A streptococcal exotoxin (erythrogenic toxin) gene from Streptococcus pyogenes bacteriophage T12.

The gene specifying type A streptococcal exotoxin (speA), also known as erythrogenic toxin, was cloned from the Streptococcus pyogenes bacteriophage T12 genome and analyzed by nucleotide sequencing. The speA gene consists of 753 base pairs and codes for a 29,244-molecular-weight protein. The speA gene product contains a putative 30-amino acid signal peptide, resulting in a molecular weight of 25,787 for the secreted protein.

Muscle adenylate kinase catalyzes adenosine 5'-tetraphosphate synthesis from ATP and ADP.

Muscle adenylate kinases from rabbit and porcine sources were found by NMR to catalyze the formation of adenosine tetraphosphate (5'AdoP4) from adenosine triphosphate (ATP) and adenosine diphosphate (ADP). The reaction was completely reversible, with an equilibrium constant of approx. 0.

Active streptokinase from the cloned gene in Streptococcus sanguis is without the carboxyl-terminal 32 residues.

The streptokinase expressed by the cloned gene in Streptococcus sanguis has a molecular weight of about 44 000 [Malke, H., Gerlach, D., Kohler, W.

The streptokinase gene.

The subject of this paper is the molecular cloning, nucleotide sequencing, and expression in heterologous hosts of the streptokinase gene (skc) from the group C streptococcal strain H46A. The skc gene shows no extended regions homologous to the staphylokinase gene.

Long-term effects of high or low Ca intakes and of lack of parathyroid function on rat femur biomechanics.

In order to assess the repercussion of chronically affected parathyroid function on bone biomechanics, 3-point flexion tests were carried out with fresh, whole femurs of young, intact rats fed diets with low, normal, or high Ca contents, and thyroparathyroidectomized (TPTX) rats fed normal Ca diet. Ca-restriction reduced, and TPTX augmented, inertial parameters and load-resistance of the whole femurs, not affecting the bending stress or the modulus of elasticity of the bone material, suggesting that parathyroid status affected bone mass and architecture without biomechanical alteration of bone tissue. High-Ca feeding enhanced tissue strength and stiffness as a direct effect, not altering bone geometry.

Two-dimensional J-resolved nuclear magnetic resonance spectral study of two bromobenzene glutathione conjugates.

The application of two-dimensional J-resolved nuclear magnetic resonance spectroscopy to determine the structure of two bile metabolites isolated from rats injected interperitoneally with bromobenzene is described. The structures of the two molecules are obtained unambiguously from the proton-proton spin coupling constants. This paper discusses the fundamentals of the technique and demonstrates the resolution of small long-range coupling constants.

Expression in Escherichia coli of streptococcal plasmid-determined erythromycin resistance directed by the cat gene promoter of pACYC 184.

The streptococcal erythromycin resistance (Emr) plasmid pSM7 (6.4 kb) and the E. coli vector pACYC184 (4.

Streptococcus-Escherichia coli shuttle vector pSA3 and its use in the cloning of streptococcal genes.

A shuttle vector that can replicate in both Streptococcus spp. and Escherichia coli has been constructed by joining the E. coli plasmid pACYC184 (chloramphenicol and tetracycline resistance) to the streptococcal plasmid pGB305 (erythromycin resistance).

Nucleotide sequence of the streptokinase gene from Streptococcus equisimilis H46A.

The entire nucleotide sequence of a cloned 2568-bp PstI fragment from the genome of Streptococcus equisimilis H46A encoding the streptokinase gene (skc) has been determined. The longest open reading frame comprises 1320 bp which code for streptokinase. The protein is synthesized with a 26-amino acid residue N-terminal extension having properties characteristic of a signal peptide.

The gene for type A streptococcal exotoxin (erythrogenic toxin) is located in bacteriophage T12.

The infection of Streptococcus pyogenes T25(3) with the temperate bacteriophage T12 results in the conversion of the nontoxigenic strain to type A streptococcal exotoxin (erythrogenic toxin) production. Although previous research has established that integration of the bacteriophage genome into the host chromosome is not essential for exotoxin production, the location of the gene on the bacteriophage or bacterial chromosome had not been determined. In the present investigation, recombinant DNA techniques were used to determine whether the gene specifying type A streptococcal exotoxin (speA) production is located on the bacteriophage chromosome.

Kinetics of creatine phosphokinase and adenylate kinase. A two-dimensional NMR analysis.

The kinetics of creatine phosphokinase and adenylate kinase catalyzed reactions were studied at equilibrium by two-dimensional Fourier transform phosphorus-31 nuclear magnetic resonance. For the creatine phosphokinase reaction, a pseudo-first-order rate constant of 0.29 s-1 was determined for the transfer of a phosphate group from adenosine triphosphate to creatine phosphate.

Streptokinase: cloning, expression, and excretion by Escherichia coli.

Genomic DNA from Streptococcus equisimilis strain H46A was cloned in Escherichia coli by using the bacteriophage lambda replacement vector L47 and an in vitro packaging system. A casein/plasminogen overlay technique was used to screen the phage bank for recombinants carrying the streptokinase gene ( skc ). The gene was present with a frequency of 1 in 836 recombinants, and 10 independent clones containing skc were isolated and physically characterized.

Expression of a streptokinase gene from Streptococcus equisimilis in Streptococcus sanguis.

Using recombinant DNA techniques, we introduced a previously cloned streptokinase gene from Streptococcus equisimilis into the Challis strain of S. sanguis (group H). The gene was expressed in the new host under the control of its own promoter and the gene product had biological properties identical to authentic streptokinase.

In vivo flux between phosphocreatine and adenosine triphosphate determined by two-dimensional phosphorous NMR.

The in vivo unidirectional flux between creatine phosphate and ATP was determined in the leg and head of the anesthetized rat using a two-dimensional NMR technique. The unidirectional flux of creatine phosphate to ATP was 13 mumol/s/g-weight in the leg and 2 mumol/s/g-weight in the head. The unidirectional flux between ATP and inorganic phosphate was too slow to be detected and was estimated to be less than 0.

Rates of enzyme-catalyzed exchange determined by two-dimensional NMR: a study of glucose 6-phosphate anomerization and isomerization.

The application of two-dimensional (2D) Fourier-transform NMR to the determination of rate constants of complex enzyme-catalyzed reactions in the steady state is described. The yeast phosphoglucose isomerase (EC 5.3.

Stereoselective formation of bromobenzene glutathione conjugates.

Two bromobenzene-glutathione conjugates have been detected as both in vivo and in vitro metabolites of bromobenzene. Separation and purification by high pressure liquid chromatography (HPLC) and analysis by 13C and 1H-NMR spectroscopy indicated that the metabolites are trans-3-bromo-6-(glutathion-S-yl)-cyclohexa-2,4-dien-1-ol and trans-4-bromo-6-(glutathion-S-yl)-cyclohexa-2,4-dien-1-ol. The two conjugates are formed in unequal amounts; over a dose range of 25-500 mg/kg the ratio of the two conjugates excreted into bile in 6 h was 1.

Phage influence on the synthesis of extracellular toxins in group A streptococci.

Phage conversion of group A streptococci to produce streptococcal exotoxins was shown to occur more widely than has been previously reported. Toxigenic conversion was found in 19 newly constructed lysogenic and pseudolysogenic strains resulting in synthesis of exotoxin types A and B. Conversion was accomplished by a positive conversion effector, which was a phage characteristic expressed by the prophage and vegetatively reproducing phage.

Examination of Streptococcus mutans for immunoglobulin G Fc reactivity.

Representative strains of oral streptococci were tested for Fc immunoglobulin G (IgG) reactivity by two different techniques, agglutination of rabbit-IgG sensitized sheep erythrocytes and uptake of human and rabbit 125I-radiolabeled IgG. None of the S. mutans serotypes a through e reacted with the Fc region of either human or rabbit IgG.

Phage-host interactions and the production of type A streptococcal exotoxin in group A streptococci.

The infection of Streptococcus pyogenes nontoxigenic strain T 253 with bacteriophage T12 to form lysogen T 253 (T12) resulted in the production of type A streptococcal exotoxin (erythrogenic toxin or streptococcal pyrogenic exotoxin). Two lines of evidence indicated that lysogeny per se was not sufficient to promote toxigenic conversion of strain T 253. First, a virulent mutant of phage T12, unable to form stable lysogens, was able to affect type A exotoxin production by strain T 253.

Enzyme-linked immunosorbent assay for detection of type A streptococcal exotoxin: kinetics and regulation during growth of Streptococcus pyogenes.

We describe the detection and quantitation of type A streptococcal exotoxin (erythrogenic toxin, streptococcal pyrogenic exotoxin) by an enzyme-linked immunosorbent assay. This sensitive and specific technique detected microgram amounts of type A exotoxin and was useful for studying the kinetics and regulation of type A exotoxin production during the growth of Streptococcus pyogenes NY5. Maximum production of type A exotoxin was observed during the mid-log phase of growth, similar to the production of other streptococcal extracellular products.

Plasmid pGB301, a new multiple resistance streptococcal cloning vehicle and its use in cloning of a gentamicin/kanamycin resistance determinant.

Streptococcal plasmid pGB301 is an in vivo rear ranged plasmid with interesting properties and potential for the molecular cloning of genes in streptococci. Transformation of S. sanguis (Challis) with the group B streptococcal plasmid pIP501 (29.

Molecular cloning of an erythromycin resistance determinant in streptococci.

The erythromycin resistance determinant of plasmid pDB102, a derivative of plasmid pSM19035, was cloned into the single HindIII site of the 3.6-megadalton cryptic Streptococcus mutans plasmid pVA318 and introduced into Streptococcus sanguis strain Challis by transformation. Plasmid pDB201, which was isolated from one of the transformants, consisted of the vector plasmid and the 1.

Cross-reactivity of Streptococcus mutans antigens and human heart tissue.

Rocket and two-dimensional immunoelectrophoreses were used to demonstrate that antisera from rabbits immunized with Streptococcus mutans strain B13 cross-reacted with human heart tissue. Absorption of the anti-S. mutans serum with S.

Physical mapping of plasmid pDB101: a potential vector plasmid for molecular cloning in streptococci.

A physical map of the streptococcal macrolides, lincomycin, and streptogramin B (MLS) resistance plasmid pDB101 was constructed using six different restriction endonucleases. Ten recognition sites were found for HindIII, seven for HindII, eight for HaeII, and one each for EcoRI, HpaII, and KpnI. The localization of the restriction cleavage sites was determined by double and triple digestions of the plasmid DNA or sequential digestions of partial cleavage products and isolated restriction fragments, and all sites were aligned with a single EcoRI reference site.