Analysis of mannose selection used for transformation of sugar beet

Various factors affecting mannose selection for the production of transgenic plants were studied using Agrobacterium tumefaciens-mediated transformation of sugar beet (Beta vulgaris L.) cotyledonary explants. The selection system is based on the Escherichia coli phosphomannose isomerase (PMI) gene as selectable gene and mannose as selective agent. Transformation frequencies were about 10-fold higher than for kanamycin selection but were only obtained at low selection pressures (1.0–1.5 g/l mannose) where 20–30% of the explants produced shoots. The non-transgenic shoots were eliminated during the selection procedure by a stepwise increase in the mannose concentration up to 10 g/l. Analysis of the transformed shoots showed that the PMI activity varied from 2.4 mU/mg to 350 mU/mg but the expression level was independent of the selection pressure. Complete resistance to mannose of transformed shoots was observed already at low PMI activities (7.5 mU/mg). Genomic DNA blot analysis confirmed the presence of the PMI gene in all transformants analysed. The possible mode of action of mannose selection compared to other selection methods is discussed.     

The effect of vaccination and sea water entry on immunocompetence and susceptibility to Kudoa thyrsites in Atlantic salmon (Salmo salar L.)

The effect of intraperitoneal (IP) vaccination and sea water entry (SWE) on the immunocompetence of Cascade Atlantic salmon was investigated. Smolts were IP injected with Aqua Health's Forte trade mark vaccine (Listonella (Vibrio) anguillarum, Listonella ordalii, Vibrio salmonicida and Aeromonas salmonicida) at four times (42, 238, 433 and 630 degree days, DD) prior to SWE and were examined for immunocompetence. Immune response measurements included mitogen-driven proliferation of peripheral blood leukocytes (PBL), head kidney leukocyte respiratory burst activity and alternative complement hemolytic titres and were measured 24h prior to SWE, 72 h post-SWE and again 3.5 weeks post-SWE. A 50% reduction in the number of PBL was observed 3 days post-vaccination. At this time LPS-driven proliferation was low (stimulation index, SI, 1.5-2.9) in all groups prior to SWE compared with that of PBL from freshwater-reared Atlantic salmon parr (6.7). By 72 h and 3.5 weeks post-SWE, the LPS-driven SI from unvaccinated salmon and those vaccinated 630 and 433 DD prior to SWE increased 3-fold. In contrast, SI from salmon vaccinated 42 and 238 DD prior to SWE remained low. A similar pattern was observed for cultured PBL stimulated with PHA, although unlike LPS-stimulated PBL, the SI of cells from parr and unvaccinated control smolts remained low following SWE but increased in fish vaccinated 433 and 630 DD prior to SWE. The respiratory burst activity of head kidney leukocytes was not affected by SWE but showed a transient 50% depression 3 days post-vaccination. The alternative complement activity (ACH50) was similar for all treatment groups prior to and at 72h post-SWE. By 3.5 weeks post-SWE, ACH50 values in salmon vaccinated 42 and 238 DD prior to SWE doubled to 874 and 860 U/ml, respectively. The prevalence and severity of Kudoa thyrsites infections, detected in all treatment groups approximately 2400 DD following SWE, were not significantly different among groups. Atlantic salmon parr should be IP vaccinated no earlier than 433 DD before SWE to avoid an enhanced risk of acquiring pathogens because of transient depression in some immune mechanisms.     

Pectin methyl esterase from orange fruit: characterization and localization by in-situ hybridization and immunohistochemistry

Pectin methyl esterase (PME) from orange (Citrus sinensis L.) fruit peels has been purified by ammonium sulphate precipitation, and ion-exchange and gel-filtration chromatography. Characterization of the enzyme revealed a 36-kDa protein with an isoelectric point >9, a pH optimum at 7 and temperature optimum at 50 °C. The substrate specificity and kinetic experiments showed that the affinity of PME for pectin was highly dependent on the degree of esterification (DE) of the pectin, with K _m values of 0.7 mg ml^-1 for pectin with a DE of 70% and 17 mg ml^-1 for pectin with a DE of 25%. The sequences of the NH₂-terminal end of digested peptides from the mature protein were obtained. A DNA fragment of 501 bp was cloned by polymerase chain reaction amplification using degenerate primers and was further used for screening of a cDNA library. Two cDNA clones were isolated encoding PMEs of 584 amino acids and 362 amino acids, respectively, including a putative signal peptide. The deduced amino acid sequence showed full identity to the sequenced peptides. Polyclonal antibodies raised against orange peel PME were used for immunohistochemistry. The main localization of PMEs was in the outer cell layers of the juice vesicles, in the outer cell layers of the lamellae between the segments and in the inner cell layers of the albedo in the peel. In-situ hybridization showed that the mRNA is very abundant in the fruit and was found in the same cell layers as the native enzyme. A very intensive staining for PME mRNA was also seen in the core and in the flavedo close to the oil glands. Received: 15 November 1997 / Accepted: 7 April 1998