A proline-rich chitinase from Beta vulgaris

A gene (Chl) encoding a novel type of chitinase was isolated from Beta vulgaris. The Ch1 protein consists of an N-terminal hydrophobic prepeptide of 25 amino acids followed by a hevein-like domain of 22 amino acid residues, an unusually long proline-rich domain of 131 amino acid residues with 90 prolines, and finally a catalytic domain of 261 amino acid residues. Proteins with similar proline-rich domains are present in some other plants. The Chl gene shows a transient expression in response to fungal infection.     

Direct effect of glucose on the preproinsulin mRNA level in isolated pancreatic islets

The effects of glucose on the preproinsulin mRNA level and the rate of (pro)insulin biosynthesis were examined in isolated mouse pancreatic islets. Relative concentrations of preproinsulin mRNA were quantitated by a RNA-dot hybridization procedure. The level of preproinsulin mRNA in islets incubated for up to 7 days at 20 mM glucose remained constant. In islets incubated at 3.3 mM glucose the preproinsulin mRNA level decreased and was after 24 h reduced to one tenth of the level at 20 mM glucose. Subsequent incubation at 20 mM glucose completely restored the preproinsulin mRNA level but only after 3 days of culture, while the insulin release was restored within 24 h. The insulin-biosynthetic activity of the islets was correlated to the variation in the level of the preproinsulin mRNA. These results suggest that glucose does have a direct influence on the level of preproinsulin mRNA and that the rate of (pro)insulin biosynthesis is limited by the level of the preproinsulin mRNA.     

Sonication: A new method for gene transfer to plants

Sonication is a novel method for gene transfer into plant protoplasts and intact plant cells. The mode of action of ultrasound and its chemical, biochemical and physiological effects are reviewed. The state of the art of acoustic transformation is presented and possible mechanisms are discussed.     

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.     

Processing, Disulfide Pattern, and Biological Activity of a Sugar Beet Defensin, AX2, Expressed in Pichia pastoris

AX2 is a 46-amino-acid cysteine-rich peptide isolated from sugar beet leaves infected with the fungus Cercospora beticola (Sacc.). AX2 strongly inhibits the growth of C. beticola and other filamentous fungi, but has little or no effect against bacteria. AX2 is produced in very low amounts in sugar beet leaves, and to study the protein in greater detail with respect to biological function and protein structural analysis, the methylotrophic yeast Pichia pastoris was used for large-scale production. The amino acid sequence, processing of the signal peptide, disulfide bridges, and biological activity of the recombinant protein were determined and compared with that of the authentic AX2. In P. pastoris, the protein was expressed with an additional N-terminal arginine. The disulfide bonding was found to be identical to that of the authentic AX2. However, when tested in in vitro bioassay, the biological activity of the recombinant protein was slightly lower than that measured for the authentic protein. Furthermore, the recombinant protein was significantly more sensitive to Ca²⁺ than the authentic protein. This is most probably due to the extra arginine, since no other differences between the two proteins have been found.     

Processing, disulfide pattern, and biological activity of a sugar beet defensin, AX2, expressed in Pichia pastoris.

AX2 is a 46-amino-acid cysteine-rich peptide isolated from sugar beet leaves infected with the fungus Cercospora beticola (Sacc.). AX2 strongly inhibits the growth of C. beticola and other filamentous fungi, but has little or no effect against bacteria. AX2 is produced in very low amounts in sugar beet leaves, and to study the protein in greater detail with respect to biological function and protein structural analysis, the methylotrophic yeast Pichia pastoris was used for large-scale production. The amino acid sequence, processing of the signal peptide, disulfide bridges, and biological activity of the recombinant protein were determined and compared with that of the authentic AX2. In P. pastoris, the protein was expressed with an additional N-terminal arginine. The disulfide bonding was found to be identical to that of the authentic AX2. However, when tested in in vitro bioassay, the biological activity of the recombinant protein was slightly lower than that measured for the authentic protein. Furthermore, the recombinant protein was significantly more sensitive to Ca(2+) than the authentic protein. This is most probably due to the extra arginine, since no other differences between the two proteins have been found.     

Electroporation: Mechanism and transient expression, stable transformation and biological effects in plant protoplasts

The present state of knowledge about the mechanistic and theoretical aspects of electroporation is summarized. Parameters affecting the efficiency of transient expression and stable transformation of electroporated plant protoplasts are rewieved. Biological effects of electroporation on plant protoplasts are described.     

Relationship between promoter strength and transformation frequencies using mannose selection for the production of transgenic sugar beet

The relationship between the expression level of the selectable marker gene and transformation frequency was investigated in transgenic sugar beets with five different promoters, a modified cauliflower mosaic virus 35S RNA promoter (E35S), an enhanced nopaline synthase promoter (ENOS), a modified mannopine synthase promoter (SMAS), a heat shock protein promoter (HSP80) and a chlorophyll a/b-binding protein promoter (CAB3), to drive the expression of the selectable marker gene. The selection system employed was based on the Escherichia coli phosphomannose isomerase (PMI) gene as selectable marker gene and mannose as selective agent. The selected transgenic shoots were analysed for PMI activity and the average activity for each promoter was found to be 5.9 (HSP80), 31 (SMAS), 38 (E35S), 49 (ENOS) and 61 (CAB3) mU/mg. The weakest promoter, HSP80, resulted in the lowest transformation frequency (0.30%), suggesting that this promoter was too weak to confer sufficient resistance to mannose. On the other hand, the strongest promoters, ENOS and CAB3, only gave intermediate transformation frequencies, 0.44% and 0.47% respectively, while the somewhat weaker SMAS promoter produced the highest transformation frequency, 0.89%. Thus, these data suggest that the activity of the selectable PMI gene should be above a certain threshold level; however, above this level, no simple correlation between the PMI activities, calculated as averages, and transformation frequencies could be deduced. However, extended data analysis by dividing the transgenic shoots into 4 groups according to their PMI activities (low(<10 mU/mg), medium (10–50 mU/mg), high (50–100 mU/mg) and very high (>100 mU/mg) expressers) revealed a significant positive correlation between the relative number of shoots having medium levels of expression and transformation frequency. This indicated that promoters that predominanthly give rise to intermediate expression levels of the selectable PMI gene result in high transformation frequencies.     

Transformation of the endospermous legume guar (Cyamopsis tetragonoloba L.) and analysis of transgene transmission

A procedure for transformation of the large-seeded endospermous legume guar (Cyamopsis tetragonoloba L.) and a study on transmission of the transgenes to offspring generations are presented. Using Agrobacterium tumefaciens with a T-DNA construct harbouring a -glucuronidase gene (uidA) and a neomycin phosphotransferase gene (nptII), maximum transformation frequencies of cotyledonary explants were obtained using 145 mg/l kanamycin sulfate as selective agent. Carbenicillin and cefotaxime, used for the elimination of Agrobacterium after co-culture, displayed considerable toxicity to guar tissues but replacing most of these -lactams by the non-phytotoxic -lactamase inhibitor sulbactam as well as addition of thidiazuron and silver thiosulfate increased transformation frequencies up to 10-fold in total. The presence of the transgenes in the primary transformants was demonstrated by genomic DNA analysis of GUS-positive shoots. Chimaeric plants (5–10%) were identified by GUS analysis at the flowering stage and were discarded. Analysis of the R₁ offspring from 17 independent transformants showed that in 41% of those, the uidA gene(s) was expressed and stably inherited consistent with Mendelian genetics. This was also found for the R₂ and R₃ generations of single copy transformants. On the other hand, a large proportion (47%) of the primary transformants gave R₁ offspring in which 100% of the plants were GUS-negative. Analysis of these plants by PCR revealed that, at least, most of the transgene sequences were absent, suggesting that they had not been transmitted from the parent transformants. This occurred at similar high frequencies (40–50%) irrespective of the estimated copy number of the transgenes. Thus, major parts of the transgenes, even when present in multiple copies, displayed aberrant transmission, at a high frequency, in the process of going from the primary transformants to the first offspring generation.