DNA fingerprinting of red clover (Trifolium pratense L.) with Jeffrey's probes: detection of somaclonal variation and other applications

Summary DNA fingerprints generated by the Jeffreys' probes, 33.6 and 33.15, indicated the presence of minisatellite-like sequences in the red clover genome. The fingerprints generated by probe 33.6 gave less background and fewer but better defined bands than those obtained with probe 33.15. Assay of a regenerative somaclonal variant (F49R) by DNA fingerprinting with probe 33.6 detected mutation that was unlinked to the regenerative trait. The fingerprints obtained under the applied conditions also demonstrated genetic stability of consecutive generations of the regenerants in tissue culture. DNA fingerprints of F₁ plants revealed that each polymorphic band was inherited from either one or the other parent. Both probes distinguished individual-specific genotypes in seven cultivars of red clover. Greater variability in DNA fingerprints was detected between (V=0.899) than within (0.417≤V≤0.548) cultivars.     

Transfer of a grapevine stilbene synthase gene to rice (Oryza sativa L.)

A gene derived from grapevine (Vitis vinifera) coding for stilbene synthase has been transferred into protoplasts of the commercially important japonica rice cultivar Nipponbare using PEG-mediated direct gene transfer. Transgenic plants were regenerated from calli selected on kanamycin. Southern blot analysis of genomic DNA isolated from regenerants and progeny plants demonstrated that the stilbene synthase gene is stably integrated in the genome of transgenic rice plants and inherited in the offspring. The transient formation of stilbene-synthase-specific mRNA shortly after inoculation with the fungus of the rice blast Pyricularia oryzae has demonstrated that the grapevine stilbene synthase promoter is also active in monocotyledonous plants. Preliminary results indicate an enhanced resistance of transgenic rice to P. oryzae. Received: 1 July 1996 / Revision received: 5 November 1996 / Accepted: 30 November 1996     

Pores in the outer membrane of Escherichia coli K12: involvement of proteins b and e in the functioning of pores for nucleotides.

Summary Mutants lacking outer membrane proteins were studied in order to investigate the role of these proteins in the functioning of aqueous pores through the outer membrane. Protein b is involved in the functioning of pores through which low concentrations of adenosine-monophosphate (AMP), guanosine-monophosphate (GMP), bis(paranitrophenyl)-phosphate (bis-PNPP) and, although less pronounced, also cytidine-monophosphate (CMP) enter the cell. In the absence of the receptor protein of phage T6, which facilitates the permeation of various nucleosides (Hantke, 1976), protein b plays a major role in the uptake of adenosine. Proteins c and d and the receptor proteins of phages lambda and T6 do not have a pore function for AMP, GMP, CMP and bis-PNPP. However, a newly discovered peptidoglycan-associated protein, protein e, can also mediate the permeation of the latter four components, but not of adenosine, through the outer membrane.Protein b does not play a major role in the uptake of higher concentrations of AMP. Obviously mainly other pores are used under those conditions. The results strongly suggest that at low concentrations of nucleoside monophosphates (and possibly of other nutrients) protein b functions as a specific pore.     

Application of wide-spectrum light-emitting diodes in micropropagation of popular ornamental plant species: a study on plant quality and cost reduction

In the present study, the applicability of four wide-spectrum light-emitting diodes (LEDs) emitting warm light (AP67, AP673L, G2, and NS1) was determined for the micropropagation of five popular ornamental plant species: Chrysanthemum × grandiflorum, Gerbera jamesonii, Heuchera × hybrida, Ficus benjamina, and Lamprocapnos spectabilis. Plantlets were grown in a growth room with a 16-h photoperiod. The photosynthetic photon flux density was set at 62–65 μmol m⁻² s⁻¹. The composition of the media and subculture timing were adjusted to the needs of each species tested. The results were compared to the cool daylight-emitting fluorescent (FL) control (TLD 36W/54). In most of the species studied (except for F. benjamina), the highest propagation ratios, or ratios similar to the FL control, were observed under the red- and far-red-abundant G2 LEDs. NS1 spectrum (with the highest proportion of blue and green light) was also efficient for G. jamesonii and L. spectabilis, and it provided a similar propagation ratio as the FL control. Light quality affected shoot length, number of leaves, callus regeneration, and the biosynthesis of chlorophyll. This influence, however, was species-dependent. Lighting conditions did not affect the dry matter and rooting in most of the species tested, except for G. jamesonii. The substitution of FLs with G2 LEDs can result in a 50% reduction of annual electricity costs, while the application of NS1 lamps can generate savings of up to 75%. In conclusion, the G2 LED lighting system seemed to be the most suitable in terms of propagation efficiency, plantlet quality, and cost reduction.

     

Quantitative analysis of cell surface antigen-antibody interaction using Gaussia princeps luciferase antibody fusion proteins

Cell surface antigen-specific antibodies are of substantial diagnostic and therapeutic importance. The binding properties of such antibodies are usually evaluated by cell-free assays, in particular surface plasmon resonance (SPR) analysis, or flow cytometry. SPR analyses allow the detailed quantitative and dynamic evaluation of the binding properties of antibodies, but need purified, typically recombinantly produced antigens. It can, however, be difficult to produce the required antigen. Furthermore, cellular factors influencing the antigen-antibody interaction are not considered by this method. Flow cytometry-based analyses do not have these limitations, but require elaborated calibration controls for absolute quantification of bound molecules. To overcome the limitations of SRP and flow cytometry in the characterization of cell surface antigen-specific antibodies, we developed Fn14-specific antibody 18D1 as an example of an antibody fusion protein format that includes the luciferase of Gaussia princeps (GpL), which enables very simple and highly sensitive cellular binding studies. We found that GpL-tagging of the C-terminus of the antibody light chain does not affect the interaction of 18D1-IgG1 with its antigen and Fc-gamma receptors (FcγRs). In accordance with this, the GpL_(LC-CT)-18D1-IgG1 antibody fusion protein showed basically the same FcγR-dependent agonistic properties as the parental 18D1 antibody. Similar results were obtained with isotype switch variants of 18D1 and antibodies specific for CD95, LTβR and CD40. In sum, we demonstrate that antibody GpL fusion proteins are easily manageable and versatile tools for the characterization of cell surface antigen-antibody interactions that have the potential to considerably extend the instrumentarium for the evaluation of antibodies.