OFF Phase Research Articles

The pupillary system of noctuid moth eyes: Dynamic changes in relation to control of pupil size

AbstractEye pupil‐size changes, in response to repetitive ON‐OFF light stimulation, of 3 species of noctuid moths have been observed by infrared reflection photometry. For moderate ON stimulus intensities (2.5·109 quanta·cm−2·s−1), eye reflectance changed exponentially with time, in both the ON and OFF phases. For low ON stimuli (2.5·108 quanta·cm−2·s−1), however, the dark adaptation response in the OFF phase was composed of an initial exponential and a subsequent linear term. For repetitive low‐intensity ON‐OFF stimuli, the response amplitude changed in a diurnal way. The shape of the response to each ON‐OFF stimulus was, however, independent of diurnal time. Finally, in eyes that were spontaneously entering a light‐adapted state, pure exponential dark adaptation was elicited by intense light pulses. It is suggested that the substances controlling light and dark adaptation are interrelated. The substances constitute a chemical cycle running at constant speed, in the absence of light, and the substance causing pigment dispersion (light adaptation) is a photoproduct of the other substance. The concentration of the substance causing pigment contraction (dark adaptation) is controlled by the rate of the back reaction, which is composed of an exponential term, related to accumulation of the photoproduct during light stimulation, and a constant term, due to the speed of the cycle. The cycle speed is subject to diurnal changes. © 1992 Wiley‐Liss, Inc.

Molecular basis of Mycoplasma surface antigenic variation: a novel set of divergent genes undergo spontaneous mutation of periodic coding regions and 5′ regulatory sequences.

Antigenic diversity is generated in the wall-less pathogen Mycoplasma hyorhinis by combinatorial expression and phase variation of multiple, size-variant membrane surface lipoproteins (Vlps). The unusual structural basis for Vlp variation was revealed in a cluster of related but divergent vlp genes, vlpA, vlpB and vlpC, which occur as single chromosomal copies. These encode conserved N-terminal domains for membrane insertion and lipoprotein processing, but divergent external domains undergoing size variation by loss or gain of repetitive intragenic coding sequences while retaining a motif with distinctive charge distribution. Genetic analysis of phenotypically switched isogenic lineages representing ON or OFF expression states of Vlp products ruled out chromosomal rearrangement or frameshift mutations as mechanisms for Vlp phase variation. However, highly conserved vlp promoter regions contain a tract of contiguous A residues immediately upstream of the -10 box which is subject to frequent mutations altering its length in exact correspondence with the ON and OFF phase states of specific genes. This suggests a mechanism of transcriptional control regulating high frequency phase variation and random combinatorial expression of Vlps. The multiple levels of diversity embodied in the vlp gene cluster represents a novel adaptive capability particularly suited for this class of wall-less microbe.

Suppression of charge movement by calcium antagonists is not related to calcium channel block

The calcium channel-inhibiting drugs nitrendipine and diltiazem represent two important classes of organic calcium antagonists. In the present study, the effect of these drugs on calcium currents and charge displacement currents in bullfrog semitendinosus muscle fibers was examined using a vaseline gap voltage clamp. Nitrendipine (10 microM) reduced the quantity of charge that moved both during the ON phase (QON) and the OFF phase (QOFF) of charge movement. This action appeared to be most selective for QON. However, at this same concentration, nitrendipine had no blocking action on inward calcium currents. In contrast to these findings, diltiazem blocked calcium currents in a concentration-dependent manner, while slightly increasing the quantity of charge moved during QON and QOFF. The enhancement of charge movement by diltiazem resulted from two actions. First, diltiazem shifted the voltage-dependence of charge movement to more negative potentials. Second, diltiazem increased the maximum amount of charge moved.

Control of cloned gene expression by promoter inversion in vivo: construction of improved vectors with a multiple cloning site and the ptac promoter

We have constructed three gene-expression plasmids which contain (an) invertible promoter(s) and a multiple cloning site. We used either the plac promoter or the P tac- P lac tandem promoters, the latter directing a more than fourfold increase in expression of the gulK reporter gene in Escherikhiu coli host. All these plasmids were derived from the pNH7a expression plasmid of Podhajska et al. [Gene 40 (1985) 163–1681. Like pNH7a, these vectors have three novel properties: (i) in the ‘OFF phase’, the promoter is facing away from the gene to be expressed, (ii) the ‘ON phase’ is attained by the rapid and efficient inversion of the promoter mediated by the phage I Int product and the flanking attP and attB sites, which have a divergent orientation, and (iii) only a short heat pulse is required for the efficient inversion of the promoter and switching from the OFF to the ON phase. As for the pNH7a vector, the present plasmids contain the nut-N transcriptional antitermination system, which permits efficient gene expression even if terminator(s) happen to be present between the promoter(s) and the expressed gene. The promoter inversion is rapid and over 95 % efficient, as assayed by restriction analysis and galactokinase assay. Many genes could be conveniently cloned in the multiple cloning site, and then either kept totally silent or expressed in a rigidly controlled manner. Moreover, the pNH8, pNH16 and pNH18 plasmids, with already inverted promoters, could be used for expression of cloned genes, either in an unregulated manner or regulated by the luc repressor. They would be particularly useful for genes associated with terminators affecting their expression.

Control of cloned gene expression by promoter inversion in vivo: construction of the heat-pulseactivated att-nutL- p- att- N module

We have constructed a prototype of gene-expression plasmids with three novel properties: (i) its “OFF phase” is absolute in all common hosts because the expression promoter is facing away from the studied gene and is blocked by a strong terminator; (ii) the “ON phase” is attained by the rapid and efficient inversion of the promoter; (iii) only a short heat pulse or exposure to other inducing agents is required to initiate this two-stage process. In the first stage, synthesis of the phage λ Int protein is induced by the transient derepression of the properly engineered λ xis −cIts857 prophage. In the immediately following second stage, Int causes inversion of a promoter cloned between the inverted P′OP phage att site and the normally oriented ΔPOΔP′ pseudo-bacterial att site. The inverted promoter can now control the expression of the studied gene and also of the λ N gene cloned in tandem. The N product, in conjunction with the nutL site placed downstream of the promoter, permits efficient antitermination of any terminators present in the att sites, in the plasmid or in the cloned DNA, making this system efficient and of practical value. Employing the promoter-inverting plasmid, it was possible to obtain rapid onset and a high level of galactokinase synthesis from the cloned galK gene. Only a transient, 10-min induction at 42°C was employed, permitting protein synthesis at 30° C, which might be of importance for thermosensitive products. Furthermore, the entire promoter-inversion module can be transferred to any plasmid as a 1.3-kb AvaI-Cla I fragment (see Fig. 1). The promoter-inverting plasmid provides also a simple method for measuring the Int function by assaying the galactokinase activity.