Southern, Northern and Western blot analysis

Memelink, Johan; Swords, K. M. M.; Staehelin, L. Andrew; Hoge, J. H. C.

doi:10.1007/978-94-011-0511-8_19

Cited by 40 publications

(37 citation statements)

References 19 publications

(9 reference statements)

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“…A total of 9 μg leaf genomic DNA or 10 μg genomic callus DNA was subjected to an overnight digestion using selected restriction enzymes. DNA was then separated by gel electrophoresis and transferred to a nylon membrane as described by Memelink et al (37). The hybridization probes were generated using a PCR-based incorporation of a digoxigenin (DIG)-labeled nucleotide, (DIG-11)-dUTP, to DNA fragments generated by primers specific to the gene elements and other regions from target plasmid.…”

Section: Methodsmentioning

confidence: 99%

Robust crop resistance to broadleaf and grass herbicides provided by aryloxyalkanoate dioxygenase transgenes

Wright¹,

Shan²,

Walsh³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

223

263

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Engineered glyphosate resistance is the most widely adopted genetically modified trait in agriculture, gaining widespread acceptance by providing a simple robust weed control system. However, extensive and sustained use of glyphosate as a sole weed control mechanism has led to field selection for glyphosate-resistant weeds and has induced significant population shifts to weeds with inherent tolerance to glyphosate. Additional weed control mechanisms that can complement glyphosate-resistant crops are, therefore, urgently needed. 2,4-dichlorophenoxyacetic acid (2,4-D) is an effective lowcost, broad-spectrum herbicide that controls many of the weeds developing resistance to glyphosate. We investigated the substrate preferences of bacterial aryloxyalkanoate dioxygenase enzymes (AADs) that can effectively degrade 2,4-D and have found that some members of this class can act on other widely used herbicides in addition to their activity on 2,4-D. AAD-1 cleaves the aryloxyphenoxypropionate family of grass-active herbicides, and AAD-12 acts on pyridyloxyacetate auxin herbicides such as triclopyr and fluroxypyr. Maize plants transformed with an AAD-1 gene showed robust crop resistance to aryloxyphenoxypropionate herbicides over four generations and were also not injured by 2,4-D applications at any growth stage. Arabidopsis plants expressing AAD-12 were resistant to 2,4-D as well as triclopyr and fluroxypyr, and transgenic soybean plants expressing AAD-12 maintained field resistance to 2,4-D over five generations. These results show that single AAD transgenes can provide simultaneous resistance to a broad repertoire of agronomically important classes of herbicides, including 2,4-D, with utility in both monocot and dicot crops. These transgenes can help preserve the productivity and environmental benefits of herbicide-resistant crops.herbicide resistance | weed management | genetically modified crops |

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Section: Methodsmentioning

confidence: 99%

Robust crop resistance to broadleaf and grass herbicides provided by aryloxyalkanoate dioxygenase transgenes

Wright¹,

Shan²,

Walsh³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

223

263

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“…Plaques were lifted on Hybond N ϩ filters (Amersham Pharmacia Biotech, Uppsala), DNA was denatured in 0.5 m NaOH and 1.5 m NaCl, and the filters were neutralized in 0.5 m Tris/HCl (pH 7.5), 1.5 m NaCl, and washed in 2ϫ SSPE (20ϫ SSPE: 3.6 m NaCl; 0.2 m NaH 2 PO 4 , pH 6.5; and 20 mm EDTA). Subsequently, the filters were exposed to UV light for 2 min, prehybridized, hybridized, and washed as described previously (Memelink et al, 1994). The resulting positive plaques were purified by a second and third screening.…”

Section: Cdna Cloningmentioning

confidence: 99%

“…For genomic Southern-blot analysis, 10 g of DNA was digested, electrophoresed on a 0.8% (w/v) agarose gel, blotted, and hybridized as described (Memelink et al, 1994). As probes, randomly labeled cDNA inserts were used.…”

Section: Blotting and Hybridizationmentioning

confidence: 99%

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Glucosylation Activity and Complex Formation of Two Classes of Reversibly Glycosylated Polypeptides

et al. 2002

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Reversibly glycosylated polypeptides (RGPs) have been implicated in polysaccharide biosynthesis. In plants, these proteins may function, for example, in cell wall synthesis and/or in synthesis of starch. We have isolated wheat (Triticum aestivum) and rice (Oryza sativa) Rgp cDNA clones to study the function of RGPs. Sequence comparisons showed the existence of two classes of RGP proteins, designated RGP1 and RGP2. Glucosylation activity of RGP1 and RGP2 from wheat and rice was studied. After separate expression of Rgp1 and Rgp2 in Escherichia coli or yeast (Saccharomyces cerevisiae), only RGP1 showed self-glucosylation. In Superose 12 fractions from wheat endosperm extract, a polypeptide with a molecular mass of about 40 kD is glucosylated by UDP-glucose. Transgenic tobacco (Nicotiana tabacum) plants, overexpressing either wheat Rgp1 or Rgp2, were generated. Subsequent glucosylation assays revealed that in RGP1-containing tobacco extracts as well as in RGP2-containing tobacco extracts UDP-glucose is incorporated, indicating that an RGP2-containing complex is active. Gel filtration experiments with wheat endosperm extracts and extracts from transgenic tobacco plants, overexpressing either wheat Rgp1 or Rgp2, showed the presence of RGP1 and RGP2 in high-molecular mass complexes. Yeast two-hybrid studies indicated that RGP1 and RGP2 form homo-and heterodimers. Screening of a cDNA library using the yeast two-hybrid system and purification of the complex by an antibody affinity column did not reveal the presence of other proteins in the RGP complexes. Taken together, these results suggest the presence of active RGP1 and RGP2 homo-and heteromultimers in wheat endosperm.Reversibly glycosylated polypeptides (RGPs) are thought to be involved in polysaccharide metabolism. Polysaccharides are the main components of the plant cell wall. In dicots, 20% of the primary cell wall consists of the polysaccharide xyloglucan, whereas in monocots, this hemicellulose makes up 2% of the primary cell wall (Darvill et al., 1980). Xyloglucan consists of a ␤-1,4-glucan backbone with xylosyl and xylosyl-galactosyl-fucosyl side chains (Hayashi, 1989). The transferases responsible for the addition of these side chains are localized to the Golgi apparatus (Brummell et al., 1990;Driouich et al., 1993;Staehelin and Moore, 1995). RGP1 has been implicated in xyloglucan biosynthesis in pea (Pisum sativum; Dhugga et al., 1997). This protein, a 40-kD doublet, which could be glycosylated with radiolabeled UDP-Glc in the presence of Mn 2ϩ or Mg 2ϩ , is associated with Golgi membranes as shown by density gradient centrifugation (Dhugga et al., 1991). Immunolocalization experiments showed RGP1 to be present in transGolgi dictyosomal cisternae (Dhugga et al., 1997). An Arabidopsis homologue was found to be mostly soluble, but also to be associated with membranes (Delgado et al., 1998). Glycosylation of the pea protein with UDP-Glc, UDP-Xyl, and UDP-Gal in a ratio similar to that of the typical sugar composition of xyloglucan (UDP-Glc:UDP-Xyl:UDP-Gal ϭ 10...

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“…Very weak signals, however, were detected with RNAs 1 and 2 in some autoradiograms exposed for 72 h or more (data not shown). The lack or weakness of cross-hybridization may be explained on the basis that the full-length RNAs share only 45 % sequence similarity (C.-C. Hu and others, unpublished) and that efficient detection of hybridization, under the high stringency conditions we used, requires that sequences have at least 90 % identity to the probe (Memelink et al, 1994).…”

Section: Differentiation Of Psv Strains Into Two Subgroups Based On Wmentioning

confidence: 99%

Evidence for the occurrence of two distinct subgroups of peanut stunt cucumovirus strains: molecular characterization of RNA3.

Aboul-Ata

Naidu

et al. 1997

Journal of General Virology

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Strains of peanut stunt cucumovirus (PSV) were classified into two distinct subgroups, I and II, based on Western and Northern blot analyses using antisera and cloned cDNA probes to strains PSV-ER and PSV-W. These results were corroborated by nucleotide sequence analyses of full-length cDNA clones of RNA3 from representative strains of the two subgroups. Whereas the percentage nucleotide sequence identity between PSV-ER (or PSV-J) and PSV-W RNA3s was determined to be 80 %, the corresponding value between strains ER and J was 91 %, confirming that strains ER and J belong to the same subgroup (subgroup I) whereas strain W belongs to a separate subgroup (subgroup II). PSV-W and PSV-ER RNA3s are 2173 and 2188 nucleotides long, respectively. Each is dicistronic,

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Southern, Northern and Western blot analysis

Cited by 40 publications

References 19 publications

Robust crop resistance to broadleaf and grass herbicides provided by aryloxyalkanoate dioxygenase transgenes

Robust crop resistance to broadleaf and grass herbicides provided by aryloxyalkanoate dioxygenase transgenes

Glucosylation Activity and Complex Formation of Two Classes of Reversibly Glycosylated Polypeptides

Evidence for the occurrence of two distinct subgroups of peanut stunt cucumovirus strains: molecular characterization of RNA3.

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