ALBERT

Notes: Abstract. We examined the structure, intranuclear distribution and activity of ribosomal DNA (rDNA) in Nico-tiana sylvestris (2n=2x=24) and N. tomentosiformis (2n=2x=24) and compared these with patterns in N. tabacum (tobacco, 2n=4x=48). We also examined a long-established N. tabacum culture, TBY-2. Nicotiana tabacum is an allotetraploid thought to be derived from ancestors of N. sylvestris (S-genome donor) and N. tomentosiformis (T-genome donor). Nicotiana sylvestris has three rDNA loci, one locus each on chromosomes 10, 11, and 12. In root-tip meristematic interphase cells, the site on chromosome 12 remains condensed and inactive, while the sites on chromosomes 10 and 11 show activity at the proximal end of the locus only. Nicotiana tomentosiformis has one major locus on chromosome 3 showing activity and a minor, inactive locus on chromosome 11. In N. tabacum cv. 095-55, there are four rDNA loci on T3, S10, S11/t and S12 (S11/t carries a small T-genome translocation). The locus on S12 remains condensed and inactive in root-tip meristematic cells while the others show activity, including decondensation at interphase and secondary constrictions at metaphase. Nicotiana tabacum DNA digested with methylcytosine-sensitive enzymes revealed a hybridisation pattern for rDNA that resembled that of N. tomentosiformis and not N. sylvestris. The data indicate that active, undermethylated genes are of the N. tomentosiformis type. Since S-genome chromosomes of N. tabacum show rDNA expression, the result indicates rDNA gene conversion of the active rDNA units on these chromosomes. Gene conversion in N. tabacum is consistent with the results of previous work. However, using primers specific for the S-genome rDNA intergenic sequences (IGS) in the polymerase chain reaction (PCR) show that rDNA gene conversion has not gone to completion in N. tabacum. Furthermore, using methylation-insensitive restriction enzymes we demonstrate that about 8% of the rDNA units remain of the N. sylvestris type (from ca. 75% based on the sum of the rDNA copy numbers in the parents). Since the active genes are likely to be of an N. tomentosiformis type, the N. sylvestris type units are presumably contained within inactive loci (i.e. on chromosome S12). Nicotiana sylvestris has approximately three times as much rDNA as the other two species, resulting in much condensed rDNA at interphase. This species also has three classes of IGS, indicating gene conversion has not homogenised repeat length in this species. The results suggest that methylation and/or DNA condensation has reduced or prevented gene conversion from occurring at inactive genes at rDNA loci. Alternatively, active undermethylated units may be vulnerable to gene conversion, perhaps because they are decondensed and located in close proximity within the nucleolus at interphase. In TBY-2, restriction enzymes showed hybridisation patterns that were similar to, but different from, those of N. tabacum. In addition, TBY-2 has elevated rDNA copy number and variable numbers of rDNA loci, all indicating rDNA evolution in culture.

Notes: Abstract. Phylogenetic schemes based on changing DNA sequence have made a major impact on our understanding of evolutionary relationships and significantly built on knowledge gained by morphological and anatomical studies. Here we present another approach to phylogeny, using fluorescent in situ hybridisation. The phylogenetic scheme presented is likely to be robust since it is derived from the chromosomal distribution of ten repetitive sequences with different functions and evolutionary constraints [GRS, HRS60, NTRS, the Arabidopsis-type telomere repeat (TTTAGGG)n, 18S-5.8S-26S ribosomal DNA (rDNA), 5S rDNA, and four classes of geminiviral-related DNA (GRD)]. The basic karyotypes of all the plant species investigated Nicotiana tomentosiformis, N. kawakamii, N. tomentosa, N. otophora, N. setchellii, N. glutinosa (all section Tomentosae), and N. tabacum (tobacco, section Genuinae) are similar (x=12) but the distribution of genic and non-genic repeats is quite variable, making the karyotypes distinct. We found sequence dispersal, and locus gain, amplification and loss, all within the regular framework of the basic genomic structure. We predict that the GRD classes of sequence integrated into an ancestral genome only once in the evolution of section Tomentosae and thereafter spread by vertical transmission and speciation into four species. Since GRD is similar to a transgenic construct that was inserted into the N. tabacum genome, its fate over evolutionary time is interesting in the context of the debate on genetically modified organisms and the escape of genes into the wild. Nicotiana tabacum is thought to be an allotetraploid between presumed progenitors of N. sylvestris (maternal, S-genome donor) and a member of section Tomentosae (T-genome donor). Of section Tomentosae, N. tomentosiformis has the most similar genome to the T genome of tobacco and is therefore the most likely paternal genome donor. It is known for N. tabacum that gene conversion has converted most 18S-5.8S-26S rDNA units of N. sylvestris origin into units of an N. tomentosiformis type. Clearly if such a phenomenon were widespread across the genome, genomic in situ hybridisation (GISH) to distinguish the S and T genomes would probably not work since conversion would tend to homogenise the genomes. The fact that GISH does work suggests a limited role for gene conversion in the evolution of N. tabacum.

Notes: Application of an external electric field induces birefringence in a solution of polydiacetylene (poly-4BCMU) in toluene due to orientation of the macromolecules. The field induced orientation in the red phase indicates an anisotropic polarizibility tensor characteristic of a rod-like conformation. The birefringence due to the polymer in its coil conformation (yellow phase) is extremely weak. The transient response of the anisotropic light scattering was studied in the red phase after switching the electric field on or off. We find a free rotational relaxation time of ∼0.1 s, consistent with the rotational diffusion constant expected for rod-like polydiacetylene macromolecules. These results rule out the large aggregate interpretation proposed to explain the color change transition. The field induced birefringence data independently demonstrate the existence of the rod-coil conformational transition for polydiacetylene 4BCMU macromolecules in solution.

Notes: A rod-to-coil conformational transition has been demonstrated for polydiacetylene, 4-butoxy-carbonyl-methylurethane (4BCMU) in solution. The transition can be induced by changing either the temperature or the quality of the solvent. The light scattering and spectroscopic data as a function of polymer concentration have shown that the transition is a single chain (intramolecular) phenomenon. However, because of the large end-to-end length (L(approximately-equal-to)1.2 μm) of the fully extended polymer, the dilute limit is not reached until concentrations below 10−5 g/cm3. At higher concentrations evidence of cluster growth and aggregation are observed prior to gelation which occurs above a critical concentration c0(approximately-equal-to)5×10−4 g/cm3. This cluster growth occurs as a result of the rod-like conformation of the individual molecules, but it is not the cause of the transition. The large increase in scattering intensity (at fixed polymer concentration) on going from coil to rod follows directly from the change in dielectric constant due to the spectral shift of the π–π* absorption; no significant increase in molecular weight is implied by the data. A theoretical model of the transition has been developed in which the ordered rod-like conformation is the low temperature phase. Conformational kinks (to a coil phase) cost energy through interruption of the π-electron delocalization and through the breaking of H bonds between R groups. Nevertheless, the increase in entropy associated with the many degrees of freedom of the coil-like conformation is sufficient to lead to the observed transition.

Notes: : The efficacy of 2% molecular weight 240, 2% molecular weight 360 polylactic acid (PLA), and an equal mix of both at reducing numbers of Escherichia coli O157:H7 and Lactobacillus plantarum on raw beef was determined. Fresh beef cubes inoculated with either organism were dipped in PLA solutions or wrapped in PLA-sprayed films. Samples were vacuum packaged and stored at 4°C for 42 d. Treated samples maintained a significantly lower pH than controls. Growth of E. coli O157:H7 was totally inhibited by both PLA treatments by up to 7.29 log10 CFU/cm2 when the spray method was used. However, PLA treatments against L. plantarum were not very effective.

Notes: : Glycoprotein of Solanum nigrum Linne (SNL glycoprotein) was isolated and identified using SDS-PAGE. SNL glycoprotein's antioxidative effects were tested in vitro and its cytotoxicity effects were tested using MCF-7 cells. One glycoprotein was isolated from SNL fruits, the other from stems and leaves. SNL glycoprotein is reactive with oxygen radicals, such as the -OH and O2- in vitro. Moreover, the SNL glycoprotein's radical scavenging activity is sensitive to the superoxide anion radical and the hydroxyl radical. In the MCF-7 cell, SNL glycoprotein I had a cytotoxic effect at 1 μg/mL and SNL glycoprotein II at 100 μg/mL. When MCF-7 cells were treated with SNL glycoproteins, nitrite oxide production was induced.