Spiroplasma virus 4: nucleotide sequence of the viral DNA, regulatory signals, and proposed genome organization.

The replicative form (RF) of spiroplasma virus 4 (SpV4) has been cloned in Escherichia coli, and the cloned RF has been shown to be infectious by transfection (M. C. Pascarel-Devilder, J. Renaudin, and J.-M. Bové, Virology 151:390-393, 1986). The cloned SpV4 RF was randomly subcloned and was fully sequenced by the dideoxy chain termination technique, using the M13 cloning and sequencing system. The nucleotide sequence of the SpV4 genome contains 4,421 nucleotides with a G+C content of 32 mol%. The triplet TGA is not a termination codon but, as in Mycoplasma capricolum (F. Yamao, A. Muto, Y. Kawauchi, M. Iwami, S. Iwagani, Y. Azumi, and S. Osawa, Proc. Natl. Acad. Sci. USA 82:2306-2309, 1985), probably codes for tryptophan. With these assumptions, nine open reading frames (ORFs) were identified. All nine are characterized by an ATG or GTG initiation codon, one or several termination codons, and a Shine-Dalgarno sequence upstream of the initiation codon. The nine ORFs are distributed in all three reading frames. One of the ORFs (ORF1) corresponds to the 60,000-dalton capsid protein gene. Analysis of codon usage showed that T- and A-terminated codons are preferably used, reflecting the low G+C content (32 mol%) of the SpV4 genome. The viral DNA contains two G+C-rich inverted repeat sequences. One could be involved in transcription termination and the other in initiation of cDNA strand synthesis. The SpV4 genome was found to contain at least three promoterlike sequences quasi-identical to those of eubacteria. These results fully support the bacterial origin of spiroplasmas

Chez les spiroplasmes le codon UGA n'est pas non sens et semble coder pour le tryptophane

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Gadolinium inhibits Na(+)-Ca(2+) exchanger current in guinea-pig isolated ventricular myocytes

The trivalent cation, gadolinium (Gd(3+)) is commonly used to inhibit stretch-activated channels. In this report, we show that Gd(3+) also inhibits ionic current (I(NaCa)), carried by the Na(+)-Ca(2+) exchanger protein. Under selective recording conditions, Gd(3+) inhibited both outward and inward I(NaCa) from guinea-pig isolated ventricular myocytes in a dose-dependent manner, with half-maximal inhibition concentrations (IC(50)) of 30.0±4.0 μM at +60 mV (Hill-coefficient, h=1.04±0.13) and 20.0±2.7 μM at −100 mV (h=1.13±0.16), respectively (P>0.05, n=5–9). Thus, inhibition was not voltage-dependent. The time from Gd(3+) application to steady-state effect was slow compared to the divalent blocker Ni(2+). The slow time course appeared to reflect gradual Gd(3+) accumulation at its binding site on the exchanger, rather than a use-dependent blocking mechanism. This study indicates that for experiments in which Gd(3+) is used, its inhibitory effect on I(NaCa) should be taken into account

Streptomycin and intracellular calcium modulate the response of single guinea-pig ventricular myocytes to axial stretch

We tested the hypothesis that both stretch-activated channels (SACs) and intracellular calcium ([Ca2+]i) are important in the electrical response of single guinea-pig ventricular myocytes to axial stretch. Myocytes were attached to carbon fibre transducers and stretched, sarcomere length increased by approximately 9 %, and there was a prolongation of the action potential duration. Streptomycin, a blocker of SACs, had no effect upon the shortening, [Ca2+]i transients or action potentials of electrically stimulated, unstretched myocytes, at a concentration of 50 μm, but at 40 μm, prevented any stretch-induced increase in action potential duration. Under action potential clamp, stretch elicited a current with a linear current-voltage relationship that was inward at membrane potentials negative to its reversal potential of −30 mV, in 10 of 24 cells tested, and was consistent with the activation of non-specific, cationic SACs. This current was not seen in any stretched cells that were exposed to 40 μm streptomycin. However, exposure of cells to 5 μm BAPTA-AM, in order to reduce [Ca2+]i transients, also abolished stretch-induced prolongation of the action potential. We conclude that both SACs and [Ca2+]i are important in the electrical response of cardiac myocytes to stretch, and propose that stretch-induced changes in electrical activity and [Ca2+]i may be linked by inter-dependent mechanisms