Developmental and pathogen-induced activation of an msr gene,str246C,from tobacco involves multiple regulatory elements

A family of genes, the so-called msr genes (multiple stimulus response), has recently been identified on the basis of sequence homology in various plant species. Members of this gene family are thought to be regulated by a number of environmental or developmental stimuli, although it is not known whether any one member responds more specifically to one stimulus, or whether each gene member responds to various environmental stimuli. In this report, we address this question by studying the tobacco msr gene str246C. Using transgenic tobacco plants containing 2.1 kb of 5′ flanking DNA sequence from the str246C gene fused to the β-glucuronidase (GUS) coding region, the complex expression pattern of the str246C promoter has been characterized. Expression of the str246C promoter is strongly and rapidly induced by bacterial, fungal and viral infection and this induction is systemic. Elicitor preparations from phytopathogenic bacteria and fungi activate the str246C promoter to high levels, as do wounding, the application of auxin, auxin and cytokinin, salicylic acid or copper sulfate, indicating the absence of gene specialization within the msr gene family, at least for str246C. In addition, GUS activity was visualized. histochemically in root meristematic tissues of tobacco seedlings and is restricted to roots and sepals of mature plants. Finally, analysis of a series of 5′ deletions of the str246C promoter-GUS gene fusion in transgenic tobacco plants confirms the involvement of multiple regulatory elements. A region of 83 by was found to be necessary for induction of promoter activity in response to Pseudomonas solanacearum, while auxin inducibility and root expression are apparently not controlled by this element, since its removal does not abolish either response. An element of the promoter with a negative effect on promoter activation by P. solanacearum was also identified.     

Surface distribution and partition during freeze-fracture of CD8 antigens on human lymphocytes and on epithelial transfected cells

Freeze-fracture immunocytochemistry was used to analyse the surface distribution, redistribution induced by antibodies, and partition during freeze-fracture, of CD8 molecules on human T lymphocytes and rat epithelial transfected (FRT-U10) cells. Immunogold labelling of CD8 antigens was uniform over the unfractured cell surfaces of both lymphocytes and epithelial transfected cells. After freeze-fracture, the gold particles were associated with the exoplasmic outer leaflets of the plasma membranes in both cell types. In lymphocytes, incubation with antibodies at 37° C up to 20 min induced patching and capping of the antigens on the unfractured cell surface. After fracture, the patched molecules appeared associated with the protoplasmic inner leaflet of the plasma membranes. Parallel antibody-treatment at 37° C of FRT-U10 cells induced clustering of CD8 molecules but failed to cause further aggregation in larger patches or in caps. After freeze-fracture, the immunola-belling was clustered, but associated with the exoplasmic outer leaflet of the plasma membranes as in untreated cells. The different redistribution induced by antibodies and the different behaviour on fracture of the redistributed molecules in the two cell types may be regulated by CD8 interaction with the cytoskeleton.     

Effect of taurine,l-glutamine and l-histidine addition in an amino acid glucose solution on the cellular bioavailability of zinc

Radioactive zinc was used to study the effect of a binary parenteral nutrient solution, composed of amino acids and glucose, on zinc uptake by fibroblasts. The influence of addition of taurine, l-glutamine and of the increase in l-histidine content of the admixture was assessed. The pure mixture was highly toxic for cells and so it was diluted 1/5 in tyrode buffer with 2% albumin. As compared with cells incubated in the buffer containing albumin, zinc absorption was significantly higher (P < 0.05) in the presence of the amino acids of the mixture. Amino acids thus increased bioavailability by displacing zinc bound to albumin. When the histidine concentration in the nutrient medium (4.2 mm) was doubled, inhibition was noted after 30 min of incubation and zinc uptake thereafter remained comparable to that in histidine-free medium. The addition of glutamine (4.2 mm), usually not present in binary mixtures, resulted in significant differences as compared with glutamine-free control medium. Taurine (0.8 mm), led to a constant increase in zinc uptake by fibroblasts as compared with that obtained with taurine-free mixture. However, ultrafiltration showed that taurine was not able to displace zinc from albumin.     

Citrate metabolism in 16 Leuconostoc mesenteroides subsp. mesenteroides and subsp. dextranicum strains

Citrate metabolism was studied in non-growing cells of Leuconostoc mesenteroides subsp. mesenteroides and subsp. dextranicum with respect to energetics, formation of degradation products and stoichiometry. The use of selective ionophores and uncoupler showed that citrate utilization was coupled to the proton motive force generated by ATP hydrolysis. Differences in citrate metabolism observed in 20 Leuconostoc strains were related to strains but not to the species or subspecies studied. Citrate metabolism was stimulated by glucose up to a concentration of 25 mmol 1^-1 and decreased at higher concentrations. The main degradation products resulting from the co-metabolism of citrate (10 mmol 1^-1) and glucose (2 mmol 1^-1) were acetate, lactate and pyruvate. Only four Leuconostoc strains produced low levels of acetoin and diacetyl. No strains produced ethanol or acetaldehyde. Citrate degradation ability was stable for at least 130 generations in 81% of the Leuconostoc strains.     

The evolution of long interspersed repeated DNA (L1, LINE 1) as revealed by the analysis of an ancient rodent L1 DNA family

Summary All modern mammals contain a distinctive, highly repeated (⩾50,000 members) family of long interspersed repeated DNA called the L1 (LINE 1) family. While the modern L1 families were derived from a common ancestor that predated the mammalian radiation ∼80 million years ago, most of the members of these families were generated within the last 5 million years. However, recently we demonstrated that modern murine (Old World rats and mice) genomes share an older long interspersed repeated DNA family that we called Lx. Here we report our analysis of the DNA sequence of Lx family members and the relationship of this family to the modern L1 families in mouse and rat. The extent of DNA sequence divergence between Lx members indicates that the Lx amplification occurred about 12 million years ago, around the time of the murine radiation. Parsimony analysis revealed that Lx elements were ancestral to both the modern rat and mouse L1 families. However, we found that few if any of the evolutionary intermediates between the Lx and the modern L1 families were extensively amplified. Because the modern L1 families have evolved under selective pressure, the evolutionary intermediates must have been capable of replication. Therefore, replicationcompetent L1 elements can reside in genomes without undergoing extensive amplification. We discuss the bearing of our findings on the evolution of L1 DNA elements and the mammalian genome.     

Combined effects of sulfites, temperature, and agitation time on production of glycerol in grape juice by Saccharomyces cerevisiae.

Analysis of variance was used to evaluate the simultaneous effects of strain, incubation temperature (15 to 25 degrees C), agitation time (0 to 24 h), and initial sulfite concentration (100 to 300 ppm) on glycerol production in grape juice by Saccharomyces cerevisiae. Fourteen strains were studied to determine their growth patterns in the presence of sulfites and ethanol. Baker's yeast strains were more sensitive to sulfite than wine strains, and little growth occurred at initial sulfite levels greater than 150 ppm. Sensitivity to sulfite increased with increasing levels of ethanol. Three strains exhibiting the best growth in the presence of sulfites and ethanol were selected for interaction studies. Fermentations were carried out until the solids content had decreased to less than 6 degrees Brix, which was the point that glycerol content became stable. For the three strains used, the greatest level of glycerol production was observed in the presence of 300 ppm of sulfite for most incubation temperatures and agitation times. There was significant interaction between the strain, incubation temperature, and agitation time parameters for glycerol synthesis, and a response surface method was used to predict the optimal conditions for glycerol production. Under static conditions, the highest level of glycerol production was observed at 20 degrees C, while incubation at 25 degrees C gave the best results when the cultures were agitated for 24 h. Response surface equations were used to predict that the optimum conditions for glycerol production by S. cerevisiae Y11 were a temperature of 22 degrees C, an initial sulfite concentration of 300 ppm, and no agitation, which yielded 0.68 g of glycerol per 100 ml.     

Cucumber mosaic virus genome is encapsidated in alfalfa mosaic virus coat protein expressed in transgenic tobacco plants

The expression of viral coat protein (CP) in transgenic plants has been shown to be very effective in virus plant protection. However, the introduction of CP genes into plants presents the potential risk of the encapsidation of a superinfecting viral genome in the transgenic protein, an event which could change the epidemiology of the disease. To detect the potential heterologous encapsidation of the cucumber mosaic virus (CMV) genome by alfalfa mosaic virus (AIMV) CP expressed in transgenic tobacco plants, a system of immunocapture (IC) and amplification by polymerase chain reaction (PCR) was optimized. This provided high sensitivity and reliable selection of the heterologously encapsidated CMV genome in the presence of natural CMV particles. As little as 2 pg of virus could be detected by immunocapture/polymerase chain reaction (IC/PCR) technique. Evidence for heterologous encapsidation of the CMV genome was found in 11 of the 33 transgenic plants tested two weeks after CMV inoculation. This demonstrates a significant rate of heterologous encapsidation events between two unrelated viruses in transgenic plants. Since CP is involved in the interactions of the virus particle with its vector, the release in the field of such transgenic plants could alter the transmission properties of some important viruses.