An Escherichia coli insertion element (IS2) provides a functional promoter in Bordetella pertussis.

The adenylate cyclase (cyaA) gene of Bordetella pertussis is not expressed in Escherichia coli. Using cya-lac fusions, high-expression spontaneous mutants were isolated and shown to have the insertion element IS2 in orientation II integrated into the reading frame of cyaA. Upon transfer of the IS2-activated cya-lac fusion into B.

Analysis of Bordetella pertussis cya operon regulation by use of cya-lac fusions.

In Bordetella pertussis virulence-associated genes, including adenylate cyclase toxin (Cya), are coordinately regulated in response to environmental signals by proteins coded by the bvg-locus. We have constructed cya-lac fusions in Escherichia coli and have shown that the cya operon is not expressed in E. coli, neither is it activated by bvg, when introduced in trans.

Deletions affecting hemolytic and toxin activities of Bordetella pertussis adenylate cyclase.

The Bordetella pertussis cyaA gene encodes a virulence factor which is a bifunctional protein exhibiting calmodulin-sensitive adenylate cyclase and hemolytic activities (P. Glaser, H. Sakamoto, J.

Virulence dependent and independent regulation of the Bordetella pertussis cya operon.

The Bordetella pertussis adenylate cyclase (cya) operon is composed of four open reading frames, cyaA, B, D and E (Glaser et al., 1988, EMBO J., 7, 3997-4004).

Two different mechanisms for urea action at the LAC and TNA operons in Escherichia coli.

Urea, at concentrations which do not interfere with bacterial growth, specifically inhibits the expression of catabolite sensitive operons. To search for the target and the mechanism of urea action we measured lactose (lac) and tryptophanase (tna) specific mRNA synthesis in vivo and in vitro. We show that urea acts by two different mechanisms at these two catabolite sensitive operons, resembling the manner in which catabolite repression regulates lac and tna.

Identification of a common domain in calmodulin-activated eukaryotic and bacterial adenylate cyclases.

Bordetella pertussis and Bacillus anthracis, two taxonomically distinct bacteria, secrete adenylate cyclase toxins that are activated by the eukaryotic protein calmodulin. The two enzymes contain a well-conserved stretch of 24 amino acid residues [Escuyer et al. (1988) Gene 71, 293-298].

Secretion of cyclolysin, the calmodulin-sensitive adenylate cyclase-haemolysin bifunctional protein of Bordetella pertussis.

The calmodulin-sensitive adenylate cyclase of Bordetella pertussis, a 45 kd secreted protein, is synthesized as a 1706 amino acid precursor. We have shown that this precursor is a bifunctional protein, carrying both adenylate cyclase and haemolytic activities. The 1250 carboxy-terminal amino acids of the precursor showed 25% similarity with Escherichia coli alpha-haemolysin (HlyA) and 22% similarity with Pasteurella haemolytica leucotoxin.

Cloning and expression of the calmodulin-sensitive Bacillus anthracis adenylate cyclase in Escherichia coli.

The adenylate cyclase gene of Bacillus anthracis, encoding the edema factor, a component of anthrax toxin, has been cloned and expressed in Escherichia coli. Clones were selected by their capacity to complement the cyclase deficiency (cya-) of an E. coli strain expressing the eukaryotic protein calmodulin, an essential activator of B.

Affinity-based chromatography utilizing genetically engineered proteins. Interaction of Bordetella pertussis adenylate cyclase with calmodulin.

An engineered calmodulin differs from vertebrate calmodulin in its ability to activate Bordetella pertussis adenylate cyclase, and this difference has been utilized as the basis for a new purification protocol for the adenylate cyclase. VU-8 calmodulin, in which 3 glutamic acid residues (residues 82-84) have been substituted with 3 lysine residues, has a 1000-fold lower apparent affinity for the adenylate cyclase, compared to vertebrate calmodulin, and decreased maximal activity. Because of the relatively calcium-independent nature of the interaction between calmodulin and the cyclase, the use of calmodulin-Sepharose conjugates in the purification of the cyclase requires the use of chaotropic agents for elution.

Immunological relatedness between Bordetella pertussis and rat brain adenylyl cyclases.

A prokaryotic adenylyl cyclase, secreted by Bordetella pertussis, shares a common functional property with eukaryotic adenylyl cyclases, i.e., regulation by the eukaryotic protein calmodulin.

The calmodulin-sensitive adenylate cyclase of Bordetella pertussis: cloning and expression in Escherichia col.

The adenylate cyclase toxin of the prokaryote Bordetella pertussis is stimulated by the eukaryotic regulatory protein, calmodulin. A general strategy, using the adenylate-cyclase-calmodulin interaction as a tool, has permitted cloning and expression of the toxin in Escherichia coli in the absence of any B. pertussis trans-activating factor.

Bordetella pertussis adenylate cyclase: the gene and the protein.

Using the adenylate cyclase-calmodulin interaction as a tool, the B. pertussis cya gene was cloned in a cya defective E. coli strain harbouring a plasmid which expressed high levels of calmodulin.

The calmodulin-sensitive adenylate cyclase of Bordetella pertussis: cloning and expression in Escherichia coli.

The adenylate cyclase toxin of the prokaryote Bordetella pertussis is stimulated by the eukaryotic regulatory protein, calmodulin. A general strategy, using the adenylate-cyclase-calmodulin interaction as a tool, has permitted cloning and expression of the toxin in Escherichia coli in the absence of any B. pertussis trans-activating factor.

Properties of cyclic AMP-independent catabolite gene activator proteins of Escherichia coli.

The protein products of two crp alleles encoding mutationally altered catabolite gene activator proteins CAP and CAPc, which are functionally active in vivo in the absence of cAMP, were purified by an immunoaffinity purification procedure. These proteins bind cAMP with the same affinity as does the wild-type catabolite gene activator protein. From their susceptibility to the proteolytic enzyme subtilisin, we conclude that the two mutationally altered proteins adopt structural features adequate for biological activity and similar to the conformation that cAMP elicits or stabilizes in wild-type catabolite gene activator protein.

A gerontology internship program for medical students.

Although the aged are the country's major users of medical care and social services, traditional medical school curricula devote little time to the aged and their social needs. This article reports on a community-based gerontology internship established for pre-clinical medical students to sensitize them to the needs of the elderly in the community. Students were assigned to community agencies servicing the elderly; working with elderly clients, they learned about needs and services.

Catabolite repression 1985.

The present status of catabolite repression is summarized with respect to the involvement of cyclic AMP and other mediators. A model is presented which may account for the relationship between positive control of gene expression exerted by cAMP and its receptor, CAP, and negative control of catabolite repression mediated by specific metabolites.

One-step purification of hybrid proteins which have beta-galactosidase activity.

A one-step purification method of hybrid proteins exhibiting beta-galactosidase activity, based on affinity chromatography in the presence of high salt concentration, is described. Starting from crude bacterial extracts, several milligrams of near-homogeneous proteins can be obtained in a few hours with an overall yield of 85 to 95%. The purified hybrid proteins can be used to obtain antibodies against the foreign portion of the protein fusion.

Identification of the Escherichia coli cya gene product as authentic adenylate cyclase.

The Escherichia coli cya gene has been fused in the same register with the lacZ gene. The corresponding hybrid cya-lacZ gene is expressed as a bifunctional protein that exhibits both adenylate cyclase and beta-galactosidase activities, thus proving that cya is the structural gene for adenylate cyclase. The hybrid protein was purified to homogeneity and has been used to raise antibodies that recognize wild-type adenylate cyclase.

Regulation of cyclic AMP synthesis in Escherichia coli K-12: effects of the rpoD800 sigma mutation, glucose, and chloramphenicol.

An immediate 12-fold inhibition in the rate of beta-galactosidase synthesis occurs in Escherichia coli cells containing the mutant sigma allele rpoD800 after a shift to 42 degrees C. In the present study we characterize the nature of the inhibition. The severe inhibition of beta-galactosidase synthesis was partly relieved by cyclic AMP (cAMP).

A social work perspective on ethical practice in end-stage renal disease.

Social workers increasingly are defining problems that they encounter in practice in health settings as ethical dilemmas. A distinction is made between those practice questions that can best be answered by an appeal to theoretical or empirical knowledge and expertise (clinical questions) and those which invoke values and ethical imperatives (ethical questions). End-stage renal disease poses in high relief the practice dilemmas that are encountered by social workers and offers an opportunity to explore and clarify issues in resolving them.

Transcriptional control of polarity in Escherichia coli by cAMP.

In Escherichia coli, 3'5'-adenosine cyclic monophosphate (cAMP) and its receptor protein (CAP) are known to be involved in the control of transcription initiation of catabolic operons. In previous papers we have shown that the cAMP-CAP complex is also involved as a modulator of polarity in polycistronic transcription units. Furthermore we showed that there exists a functional relationship between this complex and the transcription termination protein, Rho.

Two functional domains in adenylate cyclase of Escherichia coli.

To study the regulation of Escherichia coli adenylate cyclase, plasmids that carry part of the cya gene, as well as plasmids containing hybrid genes having part of cya fused to part of lacZ, were constructed. This allowed us to propose that the enzyme is made of at least two well-defined domains. The NH2 terminus carries the ATP leads to cAMP catalytic activity, whereas the COOH-terminal end corresponds to a regulatory polypeptide domain.