The influence of alkyl pyridinium sponge toxins on membrane properties, cytotoxicity, transfection and protein expression in mammalian cells

The ability of two alkyl pyridinium sponge toxin preparations (poly-APS and halitoxin) to form transient pores/lesions in cell membranes and allow transfection of plasmid cDNA have been investigated using HEK 293 cells. Poly-APS and halitoxin preparations caused a collapse in membrane potential, reductions in input resistance and increased Ca²⁺ permeability. At least partial recovery was observed after poly-APS application but recovery was more rarely seen with halitoxin. The transfection with plasmid cDNAs for an enhanced green fluorescent protein (EGFP) and human tumour necrosis factor receptor 2 (TNFR2) was assessed for both toxin preparations and compared with lipofectamine. Stable transfection was achieved with poly-APS although it was less efficient than lipofectamine. These results show that viable cells transfected with alien cDNA can be obtained using novel transient pore-forming alkyl pyridinium sponge toxins and a simple pre-incubation protocol. This provides the first proof of principle that pore-forming alkyl pyridinium compounds can be used to deliver cDNA to the intracellular environment without permanently compromising the plasma membrane.     

Controlled partial restriction digestions of DNA by competition with modification methyltransferases

Competitive reactions, using defined ratios of DNA restriction methyltransferase to endonuclease, are shown to result in reliable partial restriction digests of DNA. This method is suitable over a wide range of DNA concentrations and works on DNA in liquid or embedded in agarose. Simultaneous methylase/endonuclease reactions using endonucleases that cleave human DNA very infrequently, such as ClaI or NotI, should generate very large discrete partial DNA fragments suitable for physical mapping in the million base-pair range. Another possible application of methylase/endonuclease competitive reactions is the production of defined partial digests for making cosmid, lambda, or other genomic libraries.     

Non-invasive image analysis evaluation of growth during plant micropropagation

Microcomputerized video image analysis was adapted for rapid, objective, and non-intrusive quantification of shoot growth and development for plants growing in vitro. Custom-developed staging arrangements were essential to insure accurate viewing and representation of the plants in each of three standard culture vessels. Shoot length measurements from digitized culture images were strongly correlated with length measured manually ex vitro. Image analysis weighted density measurements of proliferating microcultures (even with irregular growth habits) provided a reliable indicator of shoot culture fresh weight. Non-destructive time course evaluations of growth rate and quality were demonstrated.Abbreviations FW fresh weight - WD weighted density     

Analysis of rice (Oryza sativa L.) genome using pulsed-field gel electrophoresis and rare-cutting restriction endonucleases

TheOryza sativa (rice) genome is small (600 to 900 megabase pairs) when compared to that of other monocotyledonous plants. Rice was the first of the major cereals to be successfully transformed and regenerated. An RFLP map with approximately 300 markers is readily available, and the DNA content per map unit is only two to three times that ofArabidopsis thaliana. Rice is also the main staple food for the majority of peoples in the world. We developed techniques for the preparation of intact genomic DNA from Indica and Japonica subspecies of rice, used statistical methods to determine which restriction endonucleases are rare-cutting, and used pulsed-field gel electrophoresis (PFE) to separate large fragments of rice DNA. Southern hybridization to blotted rice PFE gels was used to show that the digests were complete. The long-term goal of our work is to generate an integrated genetic/physical map for the rice genome, as well as helping to establish rice as a model for map-based gene cloning and genome analysis.     

Parentage determination in maize hybrids using the arbitrarily primed polymerase chain reaction (AP-PCR)

Summary Using a novel procedure based on the polymerase chain reaction, we have developed a rapid, efficient, and economical method for identifying plant genotypes. The arbitrarily primed polymerase chain reaction (AP-PCR) generates reproducible fingerprints from any organism, without the need for DNA sequence information. These fingerprints include DNA fragment polymorphisms that can be (1) used for varietal identification and parentage determination, (2) followed in segregating populations produced by crosses, (3) used as markers for the construction of genetic maps, and (4) used to generate dendograms of phylogenetic relationships, especially at the intraspecific level. AP-PCR requires only minute quantities of DNA (10–25 ng per reaction) and therefore can be used in situations in which DNA is limiting. We demonstrate the use of AP-PCR to identify inbred parents of hybrid maize plants in double-blind experiments.     

PCR with 5-methyl-dCTP replacing dCTP.

When dCTP is replaced by methyl5-dCTP in the polymerase chain reaction some templates cannot be efficiently amplified by Taq polymerase or Vent polymerase using standard cycling parameters. However, this phenomenon can be overcome by increasing the temperature of the denaturation steps to 100 degrees C, or by adding dITP to destabilize the m5dC:dG base pairs. Once the block to amplification of m5dC-substituted DNA was overcome, methylated DNA from the 'superpolylinker' of the plasmid pSL 1180 was used as a substrate to check the methyl-sensitivity of a variety of restriction endonucleases. The m5dC-substituted DNAs should also be valuable substrates for defining the specificity of methyl-dependent endonucleases.     

Direct analysis of root zone data in a microculture system

Disks were isolated from young leaves of winter rape plants and grown in vitro at ambient (15°C) or low (2°C) temperatures for two weeks. In the control disks the growth cessation and beginning of chlorophyll degradation were observed after 1 week of culture. In the low-temperature treated disks the expansion of cells was slower than that in the control material but it continued for two weeks and was accompanied by a marked accumulation of dry matter. Practically, no chlorophyll degradation was observed. The low temperature treatment brought about the decrease in the frost killing temperature of the tissue which was associated with its increased capacity to subcool water. A short (18 h) exposure of the cold-grown leaf disks to slight frost (–5°C) increased further their resistance to freezing, despite the fact that the subcooling capacity of disks decreased in result of the treatment. Therefore, the two stages of hardening, observed previously for the whole plants, can also be detected in the isolated material. In the cold-grown disks, a transient accumulation of reducing sugars but a steady decrease in ATP and water-soluble protein contents were observed. These observations indicate that tissue isolation might affect processes involved in the functional adaptation of cells to cold.Abbreviations DTA differential thermal analysis - T_k50 frost killing temperature - T_in ice nucleation temperature