Sporeless mutants of Bacillus thuringiensis. II. mutants derived from var. thuringiensis and var. sotto.

Three sporeless mutants of Bacillus thuringiensis, 2 derived from var. thuringiensis and 1 from var. sotto were selected after mutagenic treatment. They were completely lacking in ability to form spores, yet maintained intact insecticidal activity.     

Superactive mutants of thromboxane prostanoid receptor: functional and computational analysis of an active form alternative to constitutively active mutants

In class A GPCRs the E/DRY motif is critical for receptor activation and function. According to experimental and computational data, R3.50 forms a double salt bridge with the adjacent E/D3.49 and E/D6.30 in helix 6, constraining the receptor in an inactive state. The disruption of this network of interactions facilitates conformational transitions that generate a signal or constitutive activity. Here we demonstrate that non-conservative substitution of either E129^(3.49) or E240^(6.30) of thromboxane prostanoid receptor (TP) resulted in mutants characterized by agonist-induced more efficient signaling properties, regardless of the G protein coupling. Results of computational modeling suggested a more effective interaction between G_q and the agonist-bound forms of the TP mutants, compared to the wild type. Yet, none of the mutants examined revealed any increase in basal activity, precluding their classification as constitutively active mutants. Here, we propose that these alternative active conformations might be identified as superactive mutants or SAM.     

Active-site mutants of class B beta-lactamases: substrate binding and mechanistic study

Increased resistance to β-lactam antibiotics is mainly due to β-lactamases. X-ray structures of zinc β-lactamases unraveled the coordination of the metal ions, but their mode of action remains unclear. Recently, enzymes in which one of the zinc ligands was mutated have been characterized and their catalytic activity against several β-lactam antibiotics measured. A molecular modeling study of these enzymes was performed here to explain the catalytic activity of the mutants. Coordination around the zinc ions influences the way the tetrahedral intermediate is bound; any modification influences the first recognition of the substrate by the enzyme. For all the studied mutants, at least one of the interactions fails, inducing a loss of catalytic efficiency compared to the wild type. The present studies show that the enzyme cavity is a structure of high plasticity both structurally and mechanistically and that local modifications may propagate its effects far from the mutated amino acid. Received 28 August 2002; received after revision 22 October 2002; accepted 24 October 2002 RID="*" ID="*"Corresponding author.     

Sexual behavior mutants revisited: molecular and cellular basis of Drosophila mating

The study of Drosophila melanogaster by a combination of forward genetics with specific mutants, and reverse genetics, in which a given gene is expressed in an appropriate brain area to test its effect on behavior, provides a unique opportunity to explore the causal relationship between a particular gene, its function in the cell and the behavioral outcome at the organismic level. Enhanced male-to-male courtship has been shown to occur as a result of mutations in several different genes. For example, the Voila mutant exhibits intense GAL4 reporter expression in the tarsal gustatory sensilla, suggesting the importance of tapping by a male on the female abdomen with his forelegs. Feminization of parts of the antennal lobe and mushroom body by targeted expression of a female-determining gene transformer ⁺ (tra ⁺) drives the male to court other males. Mutations in the tra target gene fruitless (fru), which is expressed in the antennal lobe as well as the suboesophageal ganglion (the gustatory inputs are processed here), also induce homosexual courtship in males. These results suggest that sensory inputs mediated and/or processed by the tarsal receptors, suboesophageal ganglion, antennal lobe and mushroom body contribute to the regulation of male–female courtship. Mosaic analysis localized the neural center for male courtship behavior to the posterior dorsal brain, in which the sensory information processed by the aforementioned neural structures may be integrated. Another mosaic study mapped the neural center for female sexual behavior, as measured by her receptiveness to copulation, to the anterior dorsal brain. The issue as to how the mutations that reduce female sexual receptiveness, e.g. dissatisfaction (dsf), spinster (spin) and chaste (cht), affect the structure and/or function of this neural center deserves to be addressed urgently. Received 27 April 1999; received after revision 21 June 1999; accepted 8 July 1999     

HPLC of cephalosporins and their oxa-derivatives

Summary The presence of the oxygen atom in place of the sulfur atom has significant impact on the polarity of the -lactam derivative. This has been illustrated by direct comparison of HPLC data of 4 different cephalosporin derivatives and their oxa analogues.Acknowledgment. We gratefully acknowledge the cooperation of Drs Yuji Sendo, Toshiro Konoike, Masayuki Murakami and Mitsuru Yoshioka of Shionogi Research Laboratories, Shionogi and Co., Ltd, Fukushima-ku, Osaka, 553, Japan, who synthesized the oxa--lactams and made them available for this study.     

Growth ofNostoc muscorum mutants in the presence of diuron (DCMU) and L-methionine-DL-sulfoximine

Summary Diuron (DCMU) is inhibitory to the photoautotrophic and photoheterotrophic growth of the N₂-fixing blue-green algaNostoc muscorum at concentrations of 1.0×10^–5 M and 2.0×10^–5 M, respectively. A mutant of this organism resistant to 5.0×10^–5 M DCMU under its photoheterotrophic growth conditions, with the ability to utilize DCMU as a carbon and nitrogen source for growth, and complete inability to grow photoautotrophically has been isolated. With the apparent defect in its photosynthetic ability, it is suggested that theDCMU ^r mutant lacks the step inhibited by 1.0×10^–5 M DCMU, and metabolizes DCMU by an existing enzyme system in the absence of such inhibition. That this enzyme may be glutamine synthetase (GS) is explained with the help of a L-methionine-DL-sulfoximine (MSO)-resistant mutant ofN. muscorum which is able to grow faster with 2.0×10^–5 DCMU and is known to contain an altered GS.Thanks are due to the Council of Scientific and Industrial Research, CSIR Complex, Govt. of India, New Delhi-110012, for appointing the author to the Scientists' Pool for undertaking researches on the physiological and genetic controls of nitrogen metabolism in blue-green algae, a part of which is presented in this literature.     

Incorporation of3H-UTP into mitochondria isolated from cytoplasmic and nuclear respiratory-deficient yeast mutants

Zusammenfassung Aus verschiedenen cytoplasmatischen ^–) und nuklearen (p) atmungsdefekten Hefemutanten isolierte Mitochondrien weisen eine Aktino-mycin-empfindliche Inkorporation von³H-UTP in die TCS-unlösliche Fraktion auf. Die Absenz der mitochondrialen Proteosynthese dieser Mutanten könnte daher auf einen Hemmer im Translationsmechanismus zurückgeführt werden.     

Circadian rhythms and their mechanisms

Recent work concerning the number, site(s) and means of adjustment to the 24-h day of internal clocks is reviewed. Work on humans is considered wherever possible though much of the work involving ablation and in vitro techniques necessarily involves other species, particularly rodents. It is concluded that, though recent advances have been impressive and present techniques appear likely to continue to produce results and stimulate discussion, more attention should be directed to considering the circadian system as a whole rather than as an assemblage of individual components.     

Circadian rhythms and their mechanisms

Summary Recent work concerning the number, site(s) and means of adjustment to the 24-h day of internal clocks is reviewed. Work on humans is considered wherever possible though much of the work involving ablation and in vitro techniques necessarily involves other species, particularly rodents. It is concluded that, though recent advances have been impressive and present techniques appear likely to continue to produce results and stimulate discussion, more attention should be directed to considering the circadian system as a whole rather than as an assemblage of individual components.     

Quantitative analysis of absorption spectra and application to the characterization of ligand binding curves

Summary The spectrum of a chromophore may change as a result of perturbations in its environment. The spectral changes resulting from the perturbation are often followed by measurements at just one or two wavelengths but it is usually no more difficult to collect entire spectra. The problem comes in analysing the data from such a series of spectra. In this paper we will suggest a simple procedure in which the spectrum observed under any particular set of conditions may be considered to consist of the sum of two distinct spectral forms. The method, which is free of any assumptions regarding the quantitative relationship between the perturbation and the extent of spectral change, defines any given spectrum in terms of an apparent molar fraction of the contributing spectral forms. The variation of this apparent molar fraction provides information from which a quantitative relationship can be developed to describe the dependence of the spectral change on the perturbant. The method is illustrated using the model system of phenol red protonation and is applied to the characterization of the binding of azide ions to cobalt-substituted carbonic anhydrase.     

Quantitative analysis of absorption spectra and application to the characterization of ligand binding curves

The spectrum of a chromophore may change as a result of perturbations in its environment. The spectral changes resulting from the perturbation are often followed by measurements at just one or two wavelengths but it is usually no more difficult to collect entire spectra. The problem comes in analysing the data from such a series of spectra. In this paper we will suggest a simple procedure in which the spectrum observed under any particular set of conditions may be considered to consist of the sum of two distinct spectral forms. The method, which is free of any assumptions regarding the quantitative relationship between the perturbation and the extent of spectral change, defines any given spectrum in terms of an apparent molar fraction of the contributing spectral forms. The variation of this apparent molar fraction provides information from which a quantitative relationship can be developed to describe the dependence of the spectral change on the perturbant. The method is illustrated using the model system of phenol red protonation and is applied to the characterization of the binding of azide ions to cobalt-substituted carbonic anhydrase.     

Membrane constants and sodium conductance of a single muscle fibre

Riassunto Nella singola fibra muscolare di cavia a 38°C sono state determinate, applicando la «square pulse analysis», la resistenza, la costante di tempo, la costante di spazio e la capacità della membrana a riposo. È stato inoltre possibile calcolare la massima conduttanza per il sodio,gn a, che è risultata di 35,3 mmho/cm².     

The effect of vinblastin and vincristin on single nerve fibres

Summary The perfusion of the node of Ranvier with vinblastin or vincristin reduces the amplitude of the action potential within a few seconds. Vincristins is 10fold more active than vinblastin. Upon withdrawal, the effect is promptly reversible and it is antagonized by acetylcholine.