Characterization of the phospholipase C gene of Pseudomonas aeruginosa cloned in Escherichia coli.

We have cloned a 4.9-kb fragment of Pseudomonas aeruginosa DNA containing the structural gene of phospholipase C (PLC), by inserting it into the BamHI site of plasmid pBR322. Strains of Escherichia coli carrying this recombinant plasmid produce PLC, but expression of the gene differs from that in P.

The cardiotoxicity of eosinophils.

Although an association between high blood eosinophil counts and endomyocardial disease has been known for nearly a hundred years, the reasons for this were not understood. Brockington, Luzzatto and Osunkoya (1970) suggested that eosinophil leucocytes, in susceptible persons, by some unknown mechanisms, cause endomyocardial damage. Evidence to support this possibility has come from three sources: (1) Clinical studies have shown that very high blood eosinophil counts, from any cause, can be associated with endomyocardial disease, and in some patients it has been possible to show that eosinophilia preceded the onset of heart disease.

Clinical features of fifteen patients with the hypereosinophilic syndrome.

Fifteen patients with the hypereosinophilic syndrome were studied during a period of 6.5 years. The mean age at onset was 36 years.

Toxic effects of human eosinophil products on isolated rat heart cells in vitro.

Rat heart cells and mitochondria were incubated with supernatants from eosinophils or neutrophils that had been stimulated with zymosan-C3b. Supernatants from eosinophils, but not neutrophils, were toxic to rat heart cells in a dose-dependent manner. This was associated with an increased O2 uptake, which was blocked by either 1 mM-cyanide or 100 microM-ouabain.

Bacillus licheniformis penicillinase: cleavages and attachment of lipid during cotranslational secretion.

The penicillinase of Bacillus licheniformis is shown to be secreted cotranslationally. In extracts it was formed by membrane-associated but not by free polysomes; and after extracellular labeling of cells, followed by completion of the growing chains on polysomes in vitro, labeled penicillinase could be immunoprecipitated. This product contained electrophoretic peaks of Mr 36,000, 33,000, and 29,000, which correspond to previously reported forms of the enzyme.

Cholera toxin is synthesized in precursor form on free polysomes in Vibrio cholerae 569B.

Membrane-bound and free polysomes have been isolated from Vibrio cholerae 569B. Nacent polypeptide chains were completed in a cell-free translation mixture containing Escherichia coli S-300 extracts and [3H]leucine or [35S]methionine. Cholera toxin-related polypeptides synthesized in vitro were immunologically detected after treatment with either anti-subunit A or anti-subunit B serum.

Enzymes altering the binding capacity of human blood eosinophils for IgG antibody-coated erythrocytes (EA).

Blood eosinophils from some patients with an eosinophilia have a higher capacity to bind to IgC antibody-coated red cells (EA) than blood eosinophils from normal people. Twenty per cent of eosinophils from normal blood bound EA, whereas eight of nine patients with hypereosinophilic syndromes and all nine patients with filariasis who were studied had blood eosinophils with EA rosette-forming capacities of between 42 and 89%. High EA binding capacity was reduced in culture, and prednisolone and cytochalasins A and B inhibited normal blood eosinophil EA binding.

Hypereosinophilia in rats with Trichinella spiralis infections.

Six LOU strain rats developed blood eosinophil counts of over 10 × 10/1. Six months previously they had been infected with 2 larvae/g of , 2 days after being exposed to high ambient temperatures. The morphology of dividing and peripheral eosinophils was normal, but eosinophils in the blood and peritoneum had increased binding capacity for complexed IgG.

The mechanism of protein secretion across membranes.

Many secreted proteins are synthesised as a large precursor with an additional hydrophobic N-terminal signal sequence that is cleaved by a membrane-bound enzyme. The proteins are secreted as nascent chains. The work leading to the current models of protein secretion is reviewed and the value of bacterial systems in the study of protein transfer across membranes is stressed.

Precursor in cotranslational secretion of diphtheria toxin.

By extracellular labeling of peptides of intact Corynebacterium diphtheriae, followed by fractionation of the cells and chain completion by isolated polysomes, it is shown that diphtheria toxin is formed and secreted cotranslationally by membrane-bound polysomes; free polysomes from none. Moreover, when the chains on these polysomes were completed in vitro, in the absence of membrane they were found to include not only diphtheria toxin of a molecular weight of 62,000, but also a larger precursor of a molecular weight of 68,000. The precursor was identified by several properties: immune precipitation; conversion into toxin fragments A and B; adenosine diphosphate ribosyl-transferase activity after activation with trypsin; and cleavage to 62,000 daltons by membrane enzymes.

Triphasic concentration effects of gentamicin on activity and misreading in protein synthesis.

Gentamicin is shown to exert a triphasic concentration effect on peptide synthesis in vitro with natural messengers. Low concentrations (up to 2 micron) caused slowing and a decrease in total synthesis, but little misreading (assayed with extracts lacking Glu-tRNA); the inhibition was greater with an initiating system (with phage RNA as messenger) than with pure chain elongation on purified endogenous polysomes of Escherichia coli. Moderate concentrations (up to 100 micron) slowed synthesis less, markedly increased its duration in the noninitiating system, and strongly stimulated misreading; at optimal concentrations total synthesis was even greater than normal.

Extracellular labeling of growing secreted polypeptide chains in Bacillus subtilis with diazoiodosulfanilic acid.

Studies of the mechanism of protein secretion in a Gram-positive bacterium, Bacillus subtilis, yielded results very similar to those previously obtained with a Gram-negative organism: nascent chains protruding from protoplasts could be labeled extracellularly; the labeled chains could be recovered on polysomes isolated from the membrane--polysome fraction; they could be released by puromycin, low Mg2+, or chain completion; the completed chains include a known secreted protein (alpha-amylase); and their ribosomes appear to be attached to membrane solely by their nascent chains. The reagent used for extracellular labeling, [1252]diazoiodosulfanilic acid, yielded severalfold more specific labeling of the nascent chains (7--10% of the total cellular labeling and one-fourth to one-third of that of the membrane--polysome fraction) than was obtained earlier with another nonpenetrating reagent.

Interaction of secreted nascent chains with surrounding membrane in Bacillus subtilis.

To determine the length of secreted nascent polypeptide chain that is surrounded by membrane, we digested labeled nascent chains protruding from protoplasts of Bacillus subtilis with Pronase and isolated the residual ribosome-attached chains from the membrane-polysome fraction. Gel chromatography revealed a sharp major peak that had been protected by membrane plus bound ribosomes. The ribosomes themselves protected half as great a length.

Nascent peptide as sole attachment of polysomes to membranes in bacteria.

When membrane-polysome complexes from Escherichia coli were treated with puromycin, at various Mg2+ and K+ concentrations, the bulk of the ribosomes were released from the membrane. Moreover, many released ribosomes released ribosomes remained attached to mRNA (pseudopolysomes). These results suggest that ribosomes are attached to the membrane in bacteria solely by their nascent chain.

Streptomycin causes misreading of natural messenger by interacting with ribosomes after initiation.

The induction of misreading by streptomycin in vitro, previously observed with synthetic messengers, is now demonstrated with natural (endogenous or viral) messenger by the use of extracts of temperature sensitive mutants lacking Glu--tRNA or Val--tRNA synthetase. With chain-elongating but noninitiating ribosomes (i.e.

DNA-directed synthesis in vitro of beta-galactosidase: requirement for a ribosome release factor.

The DNA-directed synthesis of beta-galactosidase in Escherichia coli extracts has been investigated in a partially fractionated system. A dependency was obtained for 3',5'-cyclic AMP receptor protein and also for a factor, from the salt wash of ribosomes, that has been purified to near homogeneity. This factor has been identified with a ribosome release factor previously purified from the supernatant fraction by A.

Extracellular labeling of nascent polypeptides traversing the membrane of Escherichia coli.

To provide direct evidence for the hypothesis that secreted proteins may traverse membranes as growing chains, we labeled spheroplasts of Escherichia coli with a reagent (acetyl[35S]methionyl methylphosphate sulfone) that reacts with amino groups but does not cross the membrane. After fractionation, about 6% of the label in the membrane-polysome fraction was found to be attached to the polysomes. This attachment was via peptidyl-tRNA, as shown by several tests: release of most of the label from purified polysomes at low Mg2+; subsequent loss of about 25,000 daltons on cleavage by dilute alkali; release by puromycin; and release, accompanied by a marked increase in average molecular weight, on peptide chain completion.

Purification of normal human eosinophils using the different binding capacities of blood leucocytes for complexed rabbit IgG.

A method has been developed for separating eosinophils from other types of leucocyte in normal individuals. Erythrocytes are sedimented with dextran, and mononuclear cells are removed on an isotonic density gradient of ficoll and sodium diatrizoate. The eosinophils and neutrophils are then washed and sedimented onto plastic petri dishes coated with human IgG and rabbit anti-human IgG antibody; As neutrophils and monocytes have Fc-binding sites for complexed rabbit IgG they attach to the dishes, and the unabsorbed normal eosinophils which lack this binding site are eluted in a purified suspension.

Genetic relationships among three chlorophyll-deficient mutants in peanut, Arachis hypogaea L.

The genetic relationships of three chlorophyll-deficient mutant peanuts, lutescens (lu), aureus (au), and virescent (v) were studied under field and greenhouse conditions. The F1 plants from crosses between these mutants produced phenotypically normal green. In F2, aureus X virescent segregated 675 normal green : 225 virescent : 45 aureus : 15 virescent aureus : 64 seedling lethal, and lutescens X virescent segregated 45 normal green : 15 virescent : 3 lutescens : 1 seedling lethal.