Germinating seeds of Arabidopsis thaliana were cocultivated with an Agrobacterium tumefaciens strain (C58Clrif) carrying the pGV3850:pAK1003 Ti plasmid. This Ti plasmid contains the neomycin phosphotransferase II gene (NPT II) which confers resistance to kanamycin and G418. Seeds (T1 generation) imbibed for 12 h before a 24 h exposure to Agrobacterium gave rise to the highest number of transformed progeny (T2 generation). Over 200 kanamycin-resistant T2 seedlings were isolated. Some of the T2 seedlings and T3 families were characterized for genetic segregation of functional NPT II gene(s), NPT II activity, and the presence of T-DNA inserts (Southern analysis). Ninety percent of the T2 individuals transmitted the resistance factor to the T3 families in a Mendelian fashion. Of the T3 families segregating in a Mendelian fashion (n=111), 62% segregated for one functional insert, 29% for two unlinked or linked functional inserts, 5% for three unlinked inserts, 1% for four unlinked inserts, whereas 3% appeared to be homozygous for the insert(s). The 13 families that did not exhibit Mendelian segregation ratios fell into 2 classes, both of which had a deficiency of kanamycin-resistant seedlings. In the Group I T3 families (n=6) only 0%–2% of the seedlings were resistant to kanamycin (100 mg/l), whereas in the Group II families (n=7) 8%–63% of the seedlings were resistant. All of the kanamycin-resistant plants that were tested were found to possess NPT II activity. Southern analysis revealed that all of the resistant plants contained at least one copy of the T-DNA and that the majority of the plants had multiple inserts. Explants from kanamycin-resistant plants survived and formed callus when cultured on callus-inducing medium containg G418.

Agrobacterium -mediated transformation of germinating seeds of Arabidopsis thaliana : A non-tissue culture approach

Publisher Summary This chapter discusses the process of isolation, chromatography, structure, and molecular and catalytic properties of bovine pancreatic ribonuclease. At present there are three simple and widely used chromatographic procedures: (1) Hirs base their method on the carboxyl ion exchange resin IRC-50 with 0.2 M phosphate buffer pH 6.45 as the eluting medium. The principal active component of the enzyme preparation is well retarded and is universally referred to as ribonuclease-A. Several poorly resolved faster running peaks are usually seen, the area having the highest activity and running closest to A normally being called ribonuclease B. The ratio of A to B varies with the preparation but may be as high as 10 to 1. (2) Taborsky has described a system based on carboxymethyl cellulose as the exchanger operated in Tris buffer at pH 8 with a sodium chloride gradient. The excellent and adjustable resolution of this system is frequently useful. The principal peak, labeled D by Taborsky, is indistinguishable from ribonuclease-A in the IRC-50 system. (3) Crestfield found chromatography on sulfoethyl Sephadex valuable. Ribonuclease-A may develop heterogeneity during lyophilization and storage. Aggregation appears to occur. A careful study of the preparation problem has been made by Crestfield by using chromatography on Sephadex G-75, and sulfoethyl Sephadex C-25 as well as IRC-50. These authors recommended that RNase-A be stored as a solution in phosphate buffer at –20°C, that salts be exchanged by dialysis or pre-equilibrated Sephadex columns, and that concentration, if necessary, be effected by ultrafiltration. If lyophilization is necessary, it should be carried out from dilute salt-free solution to minimize aggregate formation. The aggregates can be converted to monomers by heating to 60° for a few minutes at neutral pH. The properties of the ribonuclease dimer are also discussed in the chapter.

24 Bovine Pancreatic Ribonuclease

Ribonuclease inhibitor from human placenta. Purification and properties.

The development of purification processes for protein biopharmaceuticals is challenging due to compressed development timelines, long experimental times, and the need to survey a large parameter space. Typical methods for development of a chromatography step evaluate several dozen chromatographic column runs to optimize the conditions. An efficient batch-binding method of screening chromatographic purification conditions in a 96-well format with a robotic liquid-handling system is described and evaluated. The system dispenses slurries of chromatographic resins into filter plates, which are then equilibrated, loaded with protein, washed and eluted. This paper evaluates factors influencing the performance of this high-throughput screening technique, including the reproducibility of the aliquotted resin volume, the contact time of the solution and resin during mixing, and the volume of liquid carried over in the resin bed after centrifugal evacuation. These factors led to the optimization of a batch-binding technique utilizing either 50 or 100 microL of resin in each well, the selection of an industrially relevant incubation time of 20 min, and the quantitation of the hold-up volume, which was as much as one quarter of the total volume added to each well. The results from the batch-binding method compared favorably to chromatographic column separation steps for a cGMP protein purification process utilizing both hydrophobic interaction and anion-exchange steps. These high-throughput screening tools can be combined with additional studies on the kinetics and thermodynamics of protein-resin interactions to provide fundamental information which is useful for defining and optimizing chromatographic separations steps.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/bit.21904

High-throughput screening of chromatographic separations: I. Method development and column modeling.

Cells of the original line of lymphoma 6C3HED, which regularly prove susceptible to the effects of guinea pig serum in vivo, were cultured in Eagle's medium devoid of L-asparagine; after a latent period of 2 or more weeks, during which time the cell population declined markedly, some of the cells began to proliferate, and thereafter continued vigorous growth. On implantation into mice the proliferating cells were found, however, to have completely and permanently lost their susceptibility to the effects of guinea pig serum. By contrast, when cultures of the original line of 6C3HED cells were prepared in Eagle's medium to which L-asparagine was added in a concentration of 20.0 mg/liter or more, they proliferated vigorously from the beginning; after long periods of growth in the enriched medium in vitro they remained susceptible to the effects of guinea pig serum upon test in vivo. Other amino acids, purines, and pyrimidines were unable to substitute for L-asparagine in this relation. Furthermore, a variant subline of 6C3HED cells which had become insensitive to guinea pig serum under in vivo conditions did not require L-asparagine for growth in tissue culture. It seems plain from the findings as a whole, that in 6C3HED cells, L-asparagine dependence in vitro is associated with the in vivo character of guinea pig serum sensitivity, and conversely L-asparagine independent variants are insusceptible to the effects of guinea pig serum. The implications of the findings complement those of a companion paper in which direct evidence is provided that the L-asparaginase of guinea pig serum is responsible for its antilymphoma effects.

https://rupress.org/jem/article-pdf/118/1/121/1081124/121.pdf

Evidence that the L-asparaginase of guinea pig serum is responsible for its antilymphoma effects. II. Lymphoma 6C3HED cells cultured in a medium devoid of L-asparagine lose their susceptibility to the effects of guinea pig serum in vivo.

THE effects of ribonuclease on ascites tumours have been described recently1. The present communication gives preliminary results obtained on two solid tumours, namely, the spontaneous mammary tumour in C3H or C
+ mice and the Crocker 180 sarcoma in C
+ mice.

Action of ribonuclease on two solid tumours in vivo

It has been shown that when Arabidopsis seeds are incubated with different heteropycnic labelled DNA during the first 4 days of germinating, large fragments of exogenous DNA are taken up by the seedling plant tissues (cotyledons and rootlet) and remain polymerized and double-stranded during the entire growth period. Only a small percentage of the population of exogenous DNA molecules appears to be destroyed and reutilized for DNA synthesis de novo. Most of the exogenous DNA remains free or becomes integrated with the Arabidopsis DNA as double-stranded molecules, not separable by alkali denaturation, but easily separable by ultrasonication.



Analysis of different tissues and organs of growing plants reveals that a large amount of foreign DNA remains in the cotyledons during the vegetative growth phase, but is redistributed at the flowering stage (when cotyledons etiolate). At this time, the foreign DNA leaves the cotyledons and migrates towards the flowering buds where it accumulates as a highly molecular and double-stranded material. In the F1 progeny, radioactive molecules are found which do not correspond to the endogenous material and which have a density depending on that of the foreign DNA used.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1971.tb01596.x

Fate of Exogenous DNA in Arabidopsis thaliana

PREVIOUS papers1 have described the action of ribonuclease on the cells of the Ehrlich. carcinoma in vitro, and the object of the present communication is briefly to record results obtained in vivo, following the administration of ribonuclease to animals bearing various ascites tumours. These experiments involved three hundred animals carrying the Ehrlich or Krebs mouse carcinomata, or the Walker rat carcinoma 256.

Action of ribonuclease on certain ascites tumours.

The reduced form of ribonuclease acts on the cells ofEhrlich's carcinoma by producing important changes in the RNA content of cells:

/pdf/action-de-la-ribonuclease-sur-les-cellules-du-carcinome-d-45m91n08iv.pdf

Lucien Ledoux

Papers

Action of ribonuclease on two solid tumours in vivo

Fate of Exogenous DNA in Arabidopsis thaliana

Action of ribonuclease on certain ascites tumours.

Action de la ribonucléase sur les cellules du carcinome d'Ehrlich

Les groupes actifs de la ribonucléase II. Oxydo-réduction