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  • 1
    Electronic Resource
    Electronic Resource
    Transformation of the oomycete pathogen Phytophthora megasperma f. sp. glycinea occurs by DNA integration into single or multiple chromosomes (1993)
    Springer
    Current genetics 23 (1993), S. 211-218 
    Details
    ISSN: 1432-0983
    Keywords: Transformation ; Oomycete ; Phytophthora megasperma ; Chromosome separationex
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A procedure for stable transformation was developed for Phytophthora megasperma f. sp. glycinea, an oomycete pathogen of soybean. Transformants were obtained using a bacterial hygromycin resistance gene fused to a promoter and terminator from the ham34 gene of another oomycete, Bremia lactucae. Vector DNA, alone or complexed to cationic liposomes, was introduced into protoplasts using polyethylene glycol and CaCl2. DNA and RNA hybridization, and phosphotransferase assays, confirmed the presence and expression of vector DNA in the transformants. Hybridization to electrophoretically separated chromosomes of P. m. glycinea showed that vector DNA had integrated into only one chromosome in four transformants, and into multiple chromosomes in one transformant.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00351498
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  • 2
    Electronic Resource
    Electronic Resource
    Inactivation of transgenes in Phytophthora infestans is not associated with their deletion, methylation, or mutation (1995)
    Springer
    Current genetics 28 (1995), S. 571-579 
    Details
    ISSN: 1432-0983
    Keywords: Transgene inactivation ; Silencing ; Oomycete ; Phythophthora infestans
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The mitotic and meiotic stabilities of transgenes were evaluated in the oomycete, Phytophthora infestans. Genes encoding β-glucuronidase (GUS), neomycin phosphotransferase (NPT) and hygromycin phosphotransferase (HPT), fused to one of six promoters from P. infestans or other oomycetes, were usually stably expressed during continued asexual culture and transmitted to progeny. However, the activity of these genes became undetectable in many strains during asexual or sexual propagation. Over 33 months of growth, transgene expression stopped each month in 1–3% of the transformants. Silencing of the genes was not associated with their deletion, mutation, or hypermethylation. The conformation of the integrated sequences was similar in strains destined to continue or terminate expression of the transgenes. Expression of the genes was not associated with a loss of fitness during growth in vitro and in planta, which might otherwise have selected for silencing events.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00518171
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  • 3
    Electronic Resource
    Electronic Resource
    Intermolecular ligation mediates efficient cotransformation in Phytophthora infestans (1993)
    Springer
    Molecular genetics and genomics 239 (1993), S. 241-250 
    Details
    ISSN: 1617-4623
    Keywords: β-Glucuronidase ; Oomycete ; Recombination ; Transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The processing of DNA molecules during transformation was characterized in the oomycete Phytophthora infestans. Linear and circular forms of nonreplicating transformation vectors supported similar rates of stable transformation. Remarkably, digestion of plasmids within the selectable marker genes neomycin phosphotransferase (npt) or hygromycin phosphotransferase (hpt) had little effect on the recovery of drug-resistant transformants, and the cleaved sites were shown to be reconstituted in the transformants. An assay for the transient expression of β-glucuronidase (GUS) in protoplasts treated with partial or disrupted GUS genes demonstrated that active genes could be reconstituted through intramolecular and/or intermolecular ligation between compatible ends, while incompatible ends were inefficiently joined. Stable transformation studies also demonstrated that complementing portions of incomplete npt or hpt genes joined through homologous recombination. Based on the indication of efficient ligation between DNA molecules during transformation, an efficient procedure for cotransformation was developed. The frequency of cotransformation between vectors expressing selected genes (npt or hpt) and nonselected sequences (GUS, β-galactosidase, or streptomycin phosphotransferase) approached unity when the plasmids were linearized with the same restriction enzyme before transformation. In contrast, cotransformation between circular plasmids or those cut with different enzymes occurred infrequently (10%). Hybridization analysis of DNA from cotransformants demonstrated that linearized plasmids became colocalized within genomic DNA, while circular plasmids typically inserted at unliked sites.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00281624
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