Isolation and characterisation of transposon-induced mutants of Erwinia carotovora subsp. atroseptica exhibiting reduced virulence

Summary The blackleg pathogen Erwinia carotovora subsp. atroseptica (Eca) causes an economically important disease of potatoes. We selected a genetically amenable Eca strain for the genetic analysis of virulence. Tn5 mutagenesis was used to generate nine mutants which exhibited reduced virulence (Rvi^-) of strain SCRI1043. Following physiological characterisation, mutants were divided into three classes: (1) auxotrophs; (2) extracellular enzyme mutants; and (3) a growth rate mutant. The isolation of these Rvi^- mutants has allowed us to consider some factors that affect Eca virulence.     

Water use efficiency and carbon isotope composition of plants in a cold desert environment

Summary The effects of the availabilities of water and nitrogen on water use efficiency (WUE) of plants were investigated in a sagebrush steppe. The four species studied wereArtemisia tridentata (shrub),Ceratoides lanata (suffrutescent shrub),Elymus lanceolatus (rhizomatous grass), andElymus elymoides (tussock grass). Water and nitrogen levels were manipulated in a two-by-two factorial design resulting in four treatments: control (no additions), added water, added nitrogen, and added water and nitrogen. One instantaneous and two long-term indicators of WUE were used to testa priori predictions of the ranking of WUE among treatments. The short-term indicator was the instantaneous ratio of assimilation to transpiration (A/E). The long-term measures were 1) the slope of the relationship between conductance to water vapor and maximum assimilation and 2) the carbon isotope composition (¹³C) of plant material. Additional water decreased WUE, whereas additional nitrogen increased WUE. For both A/E and ¹³C, the mean for added nitrogen alone was significantly greater than the mean for added water alone, and means for the control and added water and nitrogen fell in between. This ranking of WUE supported the hypothesis that both water and nitrogen limit plant gas exchange in this semiarid environment. The short- and long-term indicators were in agreement, providing evidence in support of theoretical models concerning the water cost of carbon assimilation.     

Abscisic Acid ELISA: Organic Acid Interference

Consideration must be exercised in determination of buffers and solutions used when carrying out enzyme-linked immunosorbent assays (ELISAs). A commercial monoclonal antibody kit for abscisic acid (Idetek, Inc.) gives significant false-positives with tricarboxylic acid cycle intermediates. The organic acids or contaminants interfered with ELISA assays for ABA as indicated by deviations in the slopes of standard curves of ABA in the organic acids. The interference, in the case of α-ketoglutarate, was caused by a contaminant. Of the organic buffers tested—Tris, Tricine, and Hepes—only Hepes showed false-positive ABA. In addition, we present data indicating the presence of ABA in commercial mannitol and provide a simple procedure for removal of the ABA.     

Forskolin stimulation of pepsinogen secretion from frog esophageal mucosa is partly mediated by intrinsic cholinergic neurons

Forskolin was found to stimulate pepsinogen secretion from frog esophageal mucosa. The stimulation was dose-dependent and was accompanied with a great increase in tissue cAMP content. The response to forskolin mimicked the action of bethanechol and was not additive with bethanechol. The stimulatory effect of forskolin was inhibited by 50% in the presence of either atropine or tetrodotoxin. On the other hand, incubation in a calcium-free medium not only reduced the response to forskolin by 45% but also eliminated the influence of atropine and tetrodotoxin. These results indicate that forskolin may stimulate pepsinogen secretion from the frog esophageal mucosa via activating adenylate cyclase, and part of its effect may arise from eliciting acetylcholine release from the intrinsic neurons.     

Infectivity-deficient vesicular stomatitis virus produced in the presence of interferon has a functional virion core

Interferon induces two antiviral actions against vesicular stomatitis virus by (i) inhibiting viral protein synthesis which leads to a reduction in virion production, and (ii) producing progeny which are deficient in infectivity (VSVIF). At low or physiological concentrations of interferon, while the virion production was decreased by less than 10-fold, the virion infectivity yield was suppressed more than 1000-fold. The VSVIF was found to be deficient (quantitatively) in envelop glycoprotein G and protein M. Tryptic peptide mapping indicated that there was no detectable structural abnormality in the G, M, and N proteins of VSVIF. The virion cores, lacking only the envelop G protein, isolated from VSVIF and control VSV have essentially identical specific infectivity. This indicated that the virion proteins L, N, NS, and M, as well as viral RNA that make up the virion core, must be functionally normal, and the observed deficiency in G protein was likely to be the cause of the functional deficiency of the virion. Low concentrations of DEAE-dextran, which is known to partially overcome the virion's dependence on the G protein for adsorption to the cell during infection, were found to enhance the infectivity of VSVIF more than the control virion. These results together indicated that the loss of infectivity in the VSVIF was due to the deficiency of the surface glycoprotein G.     

Agarose gel electrophoresis in isozyme separation and visualization

Isozyme analysis of rodent-human somatic cell hybrids has been used frequently to detect specific human chromosomes. The majority of these isozyme systems employs starch gels, the use of which can be laborious when screening large numbers of cell lines. We describe the development of two procedures to detect the long arms of human chromosomes 1 and 2 in Chinese hamster-human cell hybrids by a rapid and reproducible method using 1-mm-thick agarose gels. Detection of human chromosome 1q was accomplished by screening for human fumarate hydratase activity, whose gene has been mapped to 1q42.1. Detection of chromosome 2q was performed by screening for the isozyme isocitrate dehydrogenase 1, which has been localized to 2q32-qter. These systems provide a basis for the further development of procedures for detecting chromosome-specific isozyme markers in agarose gels.     

Scanning motion,ocellar morphology and orientation mechanisms in the phototaxis of the nematode Mermis nigrescens

Summary The putative ocellus of Mermis females consists of a hollow cylinder of dense hemoglobin pigmentation located in the anterior tip. The exact location of the photoreceptive nerve endings, however, is unknown. During phototaxis a continual bending or scanning motion of the head (anterior 2 mm) causes the orientation of the tip to swing about the direction of the source. By turning off (shuttering) the light source whenever the tip orientation was to one side of the source direction, the average orientation of the base of the head, and eventually the body orientation, was caused to be biased about 28° to the opposite side. Because the shuttering was synchronized with the scanning motion, the scanning motion must be involved in the maintenance of orientation to light. The direction of the bias rules out a two-signal comparison mechanism of orientation and demonstrates that a deviation of the tip from the source direction must decrease, rather than increase, the illumination of the photoreceptors. These findings, and the ocellar morphology, require that the photoreceptors be located inside the hollow tube of pigmentation where they can be shadowed by the pigment during deviations of the tip. Focusing by the curved anterior end should cause a similar modulation of the illumination at this location. The occasional episodes of transverse phototaxis can be explained by the leakiness of the pigment walls to transverse illumination. Analysis of the motion of the anterior in the presence and absence of shuttering indicates that the orientation of the base of the head, due to the motion of the neck, is controlled by the signals generated during one or more cycles of the scanning motion of the head. The orientation may be regulated by the phase relationship between the photoreceptor signal and putative proprioceptive signals that indicate the bending in the head.