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  • 1
    Electronic Resource
    Electronic Resource
    Direct screening for high-level expression of an introduced α-amylase gene in plants (1992)
    Springer
    Plant molecular biology 18 (1992), S. 1133-1139 
    Details
    ISSN: 1573-5028
    Keywords: α-amylase ; microcalli ; expression level ; screening ; Nicotiana tabacum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A method is described for obtaining transgenic plants with a high level of expression of the introduced gene. Tobacco protoplasts were transformed with an expression construct containing a translational fusion between mature α-amylase from Bacillus licheniformis and the signal peptide of the tobacco PR-S protein. A total number of 5200 transformed protoplasts was cultured to microcalli and screened for α-amylase expression by incubation on media containing starch followed by staining with iodine. The calli were divided into four classes, based on the resulting halo sizes on the plates. The halo sizes were found to correlated with the expression levels in transgenic plants regenerated from the calli. The expression levels varied between 0 and 0.5% of soluble leaf protein in the regenerated transgenic plants. Wider implications of this method are discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00047716
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  • 2
    Electronic Resource
    Electronic Resource
    Non-recombinant background in gene targeting: illegitimate recombination between a hpt gene and a defective 5′ deleted nptII gene can restore a Kmr phenotype in tobacco (1994)
    Springer
    Plant molecular biology 25 (1994), S. 721-733 
    Details
    ISSN: 1573-5028
    Keywords: direct gene transfer ; gene targeting ; homologous recombination ; illegitimate recombination ; Nicotiana tabacum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Previously we have demonstrated gene targeting in plants after Agrobacterium-mediated transformation. In these initial experiments a transgenic tobacco line 104 containing a T-DNA insertion with a defective neomycin phosphotransferase (nptII) gene was transformed with a repair construct containing an otherwise defective nptII gene. Homologous recombination between the chromosomally located target and the incoming complementary defective nptII construct generated an intact nptII gene and led to a kanamycin-resistant (Kmr) phenotype. The gene targeting frequency was 1×10−5. In order to compare direct gene transfer and Agrobacterium-mediated transformation with respect to gene targeting we transformed the same transgenic tobacco line 104 via electroporation. A total of 1.35×108 protoplasts were transformed with the repair construct. Out of nearly 221 000 transformed cells 477 Kmr calli were selected. Screening the Kmr calli via PCR for recombination events revealed that in none of these calli gene targeting had occurred. To establish the origin of the high number of Kmr calli in which gene targeting had not occurred we analysed plants regenerated from 24 Kmr calli via PCR and sequence analysis. This revealed that in 21 out of 24 plants analysed the 5′-deleted nptII gene was fused to the hygromycin phosphotransferase (hpt) gene that was also present on the repair construct. Sequence analysis of 7 hpt/nptII gene fusions showed that they all contained a continuous open reading frame. The absence of significant homology at the fusion site indicated that fusion occurred via a process of illegitimate recombination. Therefore, illegitimate recombination between an introduced defective gene and another gene present on the repair construct or the chromosome has to be taken into account as a standard byproduct in gene targeting experiments.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00029609
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  • 3
    Electronic Resource
    Electronic Resource
    Extracellular targeting of the vacuolar tobacco proteins AP24, chitinase and β-1,3-glucanase in transgenic plants (1993)
    Springer
    Plant molecular biology 21 (1993), S. 583-593 
    Details
    ISSN: 1573-5028
    Keywords: post-translational processing ; targeting ; AP24 ; chitinase ; glucanase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The Nicotiana tabacum ap24 gene encoding a protein with antifungal activity toward Phytophthora infestans has been characterized. Analysis of cDNA clones revealed that at least three ap24-like genes are induced in tobacco upon infection with tobacco mosaic virus. Amino acid sequencing of the purified protein showed that AP24 is synthesized as a preproprotein from which an amino-terminal signal peptide and a carboxyl-terminal propeptide (CTPP) are cleaved off during post-translational processing. The functional role of the CTPP was investigated by expressing chimeric genes encoding either wild-type AP24 or a mutant protein lacking the CTPP. Plants expressing the wild-type construct resulted in proteins properly sorted to the vacuole. In contrast, the proteins produced in plants expressing the mutant construct were secreted extracellularly, indicating that the CTPP is necessary for targeting of AP24 to the vacuoles. Similar results were obtained for vacuolar chitinases and β-1,3-glucanases of tobacco. The extracellularly targeted mutant proteins were shown to have retained their biological activity. Together, these results suggest that within all vacuolar pathogenesis-related proteins the targeting information resides in a short carboxyl-terminal propeptide which is removed during or after transport to the plant vacuole.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00014542
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