A rapid immunoprecipitation assay for neomycin phosphotransferase II expression in transformed bacteria and plant tissues

1990 ◽  
Vol 68 (6) ◽  
pp. 983-987
Author(s):  
Chris L. Baszczynski
Keyword(s):  
Brassica Napus ◽  
Bovine Serum Albumin ◽  
Kanamycin Resistance ◽  
Plant Tissues ◽  
Nptii Gene ◽  
Neomycin Phosphotransferase ◽  
Kanamycin Resistance Gene ◽  
Dot Blot ◽  
Immunoprecipitation Assay ◽  
Neomycin Phosphotransferase Ii

Anti-kanamycin antibodies produced in rabbits, following coupling of the antibiotic to bovine serum albumin, were used to immunoprecipitate radioactively labelled phosphorylated kanamycin from transformed bacterial or plant extracts in a novel assay system, for the detection of neomycin phosphotransferase II (NPTII) activity. Radioactive counts in the immunoprecipitated pellet give a semiquantitative measure of the kanamycin phosphorylation and hence the amount of NPTII activity. This assay is sensitive, uses very small amounts of radioactivity, and is very rapid, allowing many samples to be processed within a few hours. Immunoprecipitated counts from reactions with bacteria carrying a kanamycin resistance gene or from tobacco and Brassica napus plants transformed with NPTII gene-containing vectors were consistently higher than counts from nontransformed controls. Results obtained with this assay correlate well with those from the previously described gel overlay and dot-blot assays, but can be obtained in an appreciably shorter time frame.Key words: anti-kanamycin antibodies, immunoprecipitation, neomycin phosphotransferase II assay, transformation.

Evaluation of transgenic canola plants under field conditions

Genome ◽  
1992 ◽  
Vol 35 (1) ◽  
pp. 58-63 ◽  
Author(s):  
M. Arnoldo ◽  
C. L. Baszczynski ◽  
G. Bellemare ◽  
G. Brown ◽  
J. Carlson ◽  
...  
Keyword(s):  
Kanamycin Resistance ◽  
Genetically Engineered ◽  
Field Conditions ◽  
Enzyme Assays ◽  
Nptii Gene ◽  
Neomycin Phosphotransferase ◽  
Agrobacterium Mediated Transformation ◽  
Transgenic Canola ◽  
Progeny Analysis ◽  
Transformation Vectors

Eleven independent transgenic canola (Brassica napus ssp. oleifera L. cv. Westar and Regent) lines were evaluated in the field. The plants carried a neomycin phosphotransferase (NPTII) gene for kanamycin resistance that was introduced via Agrobacterium-mediated transformation. NPTII enzyme assays, Southern blot hybridizations and progeny analysis, confirmed the stable, heritable integration and expression of the introduced NPTII gene. A number of agronomic characteristics evaluated under field conditions, including maturity, yield, and oil and protein content, were all statistically comparable between the transformed and nontransformed plants. These results indicate that canola can be genetically engineered successfully, and that the Agrobacterium-based transformation system employed does not induce any adverse effects on the intrinsic agronomic and qualitative traits critical to the agricultural industry.Key words: transgenic field trial, canola, Agrobacterium-mediated transformation, vectors.

A simplified method for the detection of neomycin phosphotransferase II activity in transformed plant tissues

1987 ◽  
Vol 5 (4) ◽  
pp. 380-386 ◽  
Author(s):  
Raymond E. McDonnell ◽  
Robert D. Clark ◽  
Wendy A. Smith ◽  
Maud A. Hinchee
Keyword(s):  
Plant Tissues ◽  
Neomycin Phosphotransferase ◽  
Neomycin Phosphotransferase Ii ◽  
Simplified Method

Asymmetric Somatic Hybridization: Developing a Gene Transfer System for Solanaceous Vegetable Crops

1996 ◽  
Author(s):  
Ken Sink ◽  
Shamay Izhar ◽  
Abraham Nachmias
Keyword(s):  
Lycopersicon Esculentum ◽  
Somatic Hybrid ◽  
Blot Hybridization ◽  
Rflp Analysis ◽  
Kanamycin Resistance ◽  
Nptii Gene ◽  
Vegetable Crops ◽  
Dot Blot ◽  
Hybrid Plants ◽  
Gamma Irradiated

Highly asymmetric somatic hybrid plants were obtained by PEG/DMSO fusion of gamma irradiated (100, 250, 7500 and 1000 Gy) protoplasts of a (KmR-) interspecific hybrid Lycopersicon esculentum x L. pennellii (EP) with protoplasts of eggplant (E). Somatic hybrid calli were selected based on kanamycin resistance and verified by PCR of the NptII gene, RAPD's and Southern's using potato rDNA pTHG2 probes. Flow cytometry indicated all hybrid calli that did not regenerate shoots were 5-9n. Three asymmetric plants regenerated only from callus close to 4n and such calli oly occurred when EP received 100 Gy. The asymmetric plants had eggplant morphology and regenerated from one hybrid callus with 6.29 average size tomato chromosomes. Limited amounts of EP DNA were found in the three somatic hybrid plants H18-1 to -3 by dot-blot hybridization with probe pTHG2, to be equivalent to 6.23, 5.41, and 5.95 % EP, respectively. RFLP analysis of Lycopersicon esculentum and L. pennellii specific chromosomes revealed that only fragments of 8 to 10 out of the 24 EP chromosomes are present in the asymmetric plants. Transgenic plants 2-3, 2-4 and 10-3 were found resistant to verticillium; suggesting successful transfer of the Ve complex from S. torvum to eggplant.

Microprojectile-mediated DNA delivery to the Salicaceae family

1993 ◽  
Vol 71 (11) ◽  
pp. 1458-1466 ◽  
Author(s):  
Yvonne A. Devantier ◽  
Barbara Moffatt ◽  
Catherine Jones ◽  
Pierre J. Charest
Keyword(s):  
Gene Expression ◽  
Populus Deltoides ◽  
Expression System ◽  
Transient Gene Expression ◽  
Dna Delivery ◽  
Kanamycin Resistance ◽  
Cell Suspensions ◽  
Neomycin Phosphotransferase ◽  
Salix Alba ◽  
Neomycin Phosphotransferase Ii

A transient gene expression system was developed for the Salicaceae family using microprojectile-mediated DNA delivery (Biolistic™) to cell suspensions. Using Populus nigra × Populus maximowiczii line NM1, 10 variables were optimized. Optimum transient gene expression under the 10 conditions tested was obtained with a 7- to 9-day-old cell suspension. Highest transient levels were observed with samples positioned 13.5 cm from the stopping plate and bombarded with 1.6-μm gold particles coated with 1 μg of DNA precipitated with CaCl2. Assaying for β-glucuronidase gene expression was performed 1 day after bombardment. The fate of the microparticles in the bombarded cells was studied, showing that 58.9% of cells expressing the β-glucuronidase gene had microparticles located in the nucleus or its vicinity and the remaining cells had microparticles in the cytoplasm. Cell suspensions from five different lines (P. nigra × P. maximowiczii lines NM1 and NM6, Populus deltoides × P. nigra line DN106, Populus tremula × Populus alba line 7171-B4, and Salix alba sanguined line SA-2) yielded transient gene expression. The relative strengths of β-glucuronidase expression in the lines tested were NM1 = 7171-B4 > NM6 > DN106 > SA-2. Six plasmid constructions were also tested in line NM1 for transient β-glucuronidase gene expression. The x-glucuronide histochemical assay did not reveal any differences, but the methyl umbelliferone glucuronide fluorescent assay yielded the following relative levels of transient gene expression with the different promoter sequences: 35S-35S-AMV enhancer = 35S-AMV enhancer > 35S-35S = 35S-35S-AMV enhancer with the β-glucuronidase – neomycin phosphotransferase fusion > 35S. Four transgenic cell lines of P. nigra × P. maximowiczii were characterized for kanamycin resistance and neomycin phosphotransferase II gene activity. Polymerase chain reaction and Southern hybridization analyses indicated the presence of the β-glucuronidase and neomycin phosphotransferase II genes in the genome of three of these transgenic lines. Key words: microprojectile, particle bombardment, Salicaceae, poplar, willow.

scholarly journals Direct introduction of neomycin phosphotransferase II protein into apple leaves to confer kanamycin resistance

2016 ◽  
Vol 33 (5) ◽  
pp. 403-407 ◽  
Author(s):  
Keiji Numata ◽  
Yoko Horii ◽  
Yoko Motoda ◽  
Narumi Hirai ◽  
Chikako Nishitani ◽  
...  
Keyword(s):  
Kanamycin Resistance ◽  
Neomycin Phosphotransferase ◽  
Direct Introduction ◽  
Neomycin Phosphotransferase Ii ◽  
Apple Leaves

scholarly journals Reduced Virulence of a Bordetella bronchiseptica Siderophore Mutant in Neonatal Swine

2001 ◽  
Vol 69 (4) ◽  
pp. 2137-2143 ◽  
Author(s):  
Karen B. Register ◽  
Thomas F. Ducey ◽  
Susan L. Brockmeier ◽  
David W. Dyer
Keyword(s):  
Nasal Cavity ◽  
Resistance Gene ◽  
Parent Strain ◽  
Virulent Strain ◽  
Clinical Signs ◽  
Kanamycin Resistance ◽  
Live Vaccine ◽  
Bordetella Bronchiseptica ◽  
Kanamycin Resistance Gene ◽  
Phosphate Buffered Saline

ABSTRACT One means by which Bordetella bronchiseptica scavenges iron is through production of the siderophore alcaligin. A nonrevertible alcaligin mutant derived from the virulent strain 4609, designated DBB25, was constructed by insertion of a kanamycin resistance gene into alcA, one of the genes essential for alcaligin biosynthesis. The virulence of the alcA mutant in colostrum-deprived, caesarean-delivered piglets was compared with that of the parent strain in two experiments. At 1 week of age, piglets were inoculated with phosphate-buffered saline, 4609, or DBB25. Two piglets in each group were euthanatized on day 10 postinfection. The remainder were euthanatized at 21 days postinfection. Clinical signs, including fever, coughing, and sneezing, were present in both groups. Nasal washes performed 7, 14, and 21 days postinoculation demonstrated that strain DBB25 colonized the nasal cavity but did so at levels that were significantly less than those achieved by strain 4609. Analysis of colonization based on the number of CFU per gram of tissue recovered from the turbinate, trachea, and lung also demonstrated significant differences between DBB25 and 4609, at both day 10 and day 21 postinfection. Mild to moderate turbinate atrophy was apparent in pigs inoculated with strain 4609, while turbinates of those infected with strain DBB25 developed no or mild atrophy. We conclude from these results that siderophore production by B. bronchiseptica is not essential for colonization of swine but is required for maximal virulence. B. bronchiseptica mutants with nonrevertible defects in genes required for alcaligin synthesis may be candidates for evaluation as attenuated, live vaccine strains in conventionally reared pigs.

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