Replication of the cauliflower mosaic virus: role and stability of the cloned delta 3 discontinuity sequence

A fragment of cauliflower mosaic virus (CaMV) DNA, containing delta 3, one of the three discontinuity sequences, was cloned in various ways into CaMV DNA deleted for the delta 3 sequence. The series of constructions was monitored for the appearance of the typical single-strand (ss) discontinuity after hybrid CaMV replication in plants. The delta 3 discontinuity was observed only if the orientation of inserted DNA sequence was the same as in the wild-type virus. Long polylinker sequences used for insertion of the fragment into cloned viral DNA, affected the stability of the insert in progeny viral DNA in plants by acting as recombination targets.     

Assembly of an antibody and its derived antibody fragment inNicotiana andArabidopsis

The yield and assembly of an IgG1 antibody and its derived F_ab fragment were compared inNicotiana andArabidopsis. The results obtained showed a lot of interclonal variability. For 45% of the primary transgenic calluses, antigen-binding entities represented less than 0.1% of the total soluble protein (TSP). Only two of the 103 analysed transformants contained more than 1% of antigen-binding protein, with 1.26% being the highest yield. Analogous amounts of complete antibody and F_ab accumulated in primary callus tissue. Moreover, yields were in the same range for both species as far as primary callus tissue is concerned. However, the accumulation of the F_ab fragment in leaf tissue of regenerated plants differed significantly betweenNicotiana andArabidopsis. The F_ab fragment accumulated to only 0.044% of TSP inNicotiana leaves but up to 1.3% inArabidopsis leaves. Furthermore, both species showed differences in the assembly pattern of the complete antibody. WhereasArabidopsis contained primarily fully assembled antibodies of 150 kDa,Nicotiana showed an abundance of fragments in the 50 kDa range.     

Strategies to mitigate transgene–promoter interactions

The expression pattern of tissue-specific promoters in transgenes can be influenced by promoter/enhancer elements employed for the expression of selectable marker genes or elements found in DNA flanking the insertion site. We have developed an analytical system in Arabidopsis thaliana to investigate strategies useful in blocking or reducing nonspecific interactions. These experiments confirm that the DNA configuration and the insertion of spacer DNA aid in the appropriate expression of tissue-specific promoters. It is also demonstrated that the novel tobacco cryptic promoter (tCUP), when used to replace the cauliflower mosaic virus (CaMV) 35S promoter/enhancer, does not show nonspecific interactions. Furthermore, it is shown that insulators isolated from yeast and animals may have potential application in plants. Our results may allow the design of strategies that, individually or in combination, can be used to minimize nonspecific interactions and to design vectors for individual tissue-specific promoters.     

Antisense inhibition of a spermidine synthase gene highlights the role of polyamines for stress alleviation in pear shoots subjected to salinity and cadmium

Three transgenic European pear (Pyrus communis L.) lines with reduced spermidine synthase (SPDS) expression and spermidine (Spd) titers were developed using a construct containing an apple SPDS gene (MdSPDS1) in antisense orientation. After exposure to either salt or cadmium stress, growth inhibition was more severe in the antisense lines than in the wild-type (WT). The antioxidant system, as shown by glutathione (GSH) content, activity of glutathione reductase (GR) and superoxide dismutase (SOD), and proline accumulation, was not effectively induced under stress in the antisense lines as compared with the WT. The reduction in antioxidant system function in the antisense lines was accompanied by a greater accumulation of malondialdehyde (MDA), an indicator of lipid peroxidation. Growth inhibition, Spd level, and parameters indicative of the antioxidant system were significantly ameliorated by exogenous Spd application. Under either salt or cadmium stress, GSH content, GR and SOD activity, and proline accumulation were positively correlated with Spd, putrescine (Put), and total polyamine titers. Conversely, MDA level showed a significantly negative correlation with these polyamines under both stress conditions. Thus, the responses to stress treatments were first identified in the SPDS antisense European pears, and the results provide further evidence for the important role of polyamines in both salt and cadmium stress tolerance, in which the polyamines act, at least in part, by influencing the antioxidant system.