Mesenchymal-epithelial conversions induced by 5-Azacytidine: Appearance of cytokeratin Endo-A messenger RNA

When mouse-teratocarcinoma-derived fibroblasts (1246 cell line) are subjected to treatment with the inhibitor of DNA methylation, 5-Azacytidine (5 AzaC), they transiently express at 55-kilodalton intermediate-filament protein recognized by the epithelial-specific monoclonal antibody, TROMA-1, although they retain a fibroblastic morphology. However, rare clones (e.g., the 1339 cell line) that permanently express the antigen recognized by TROMA-1 can be derived from the 5 AzaC-treated 1246 population, and these clones have an epithelial phenotype. In the present study, we used cloned DNA probes to demonstrate that, in 1246 fibroblasts, 5 AzaC induces the appearance of Endo-A mRNA. High levels of Endo-A mRNA were also detected in the epithelial derivative, cell line 1339. In both cases, the capping site of the Endo-A mRNA was found to be the same as that in epithelial cells which normally express this RNA.     

Location of resistance factors in the leaves of potato and wild tuber-bearing Solanum species to the aphid Myzus persicae

Analysis of electrically recorded feeding behaviour of aphids was combined with colony‐development tests to search for sources of resistance to Myzus persicae (Sulzer) (Homoptera: Aphididae) in tuber‐bearing Solanum species (Solanaceae), aiming at a reduction of potato leaf roll virus (PLRV) transmission. Twenty genotypes, originating from 14 gene bank accessions, representing 13 wild tuber‐bearing Solanum spp., three Solanum tuberosum L. (potato) cultivars, and one S. tuberosum breeding line, were selected. Colony‐development tests were carried out in no‐choice experiments by placing adult aphids on plants of each genotype and counting numbers of nymphs and adults on young plants after 8 and 15 days, and on flowering plants after 14 and 30 days. Large differences were observed among genotypes: some developed small colonies and others developed large ones. Also, in a few genotypes, resistance in mature plants was different for leaves of different ages; young leaves were resistant to aphids whereas old senescent leaves were susceptible. The electrical penetration graph (DC‐EPG system) technique was used to study aphid feeding behaviour on each Solanum genotype for 6 h. Electrical penetration graph (EPG) results also showed large differences among the genotypes, indicating resistance at the leaf surface and at three different levels of plant tissue (epidermis, mesophyll, and phloem). Therefore, it was concluded that different mechanisms of resistance to M. persicae exist among the genotypes analysed. EPGs recorded from aphids on Solanum berthaultii Hawkes and Solanum tarijense Hawkes with and without glandular trichomes showed that strong surface resistance can bias EPG parameters associated with resistance located in deeper tissues. Experimental evidence is presented that the resistance to aphids in the genotypes with glandular trichomes strongly depends on these morphological structures.     

Allelism of Endosperm Balance Number (EBN) in Mexican tuber-bearing Solanum species

Summary Endosperm Balance Number (EBN) is a genetic, dose-dependent crossability system functioning in tuber-bearing Solanum species. Each species has been assigned 1EBN, 2EBN, or 4EBN. Species thus designated cross only within their EBN group. Doubling of chromosome number also doubles the EBN. The ploidy: EBN ratio is not consistent among Solanum species. Some diploids are 2EBN while others are 1EBN. Some tetraploids are 4EBN while others are 2EBN. Species from Mexico typically have EBNs that are one-half of their ploidy [e.g. 2x(1EBN), 4x(2EBN)]. Hybrids of Mexican species and a South American species, 2x(1EBN) S. Commersonii, and its 4x(2EBN) colchicine derivative were made and crossed to 1, 2, and 4EBN standard testers to determine the relationship of the genetic organization of EBN among and within these species. Diploid hybrids crossed only to 1EBN standard testers. Hybrids of 4x(2EBN) S. commersonii and 4x(2EBN) Mexican species crossed almost exclusively to 2EBN standard testers. Complex tetraploid hybrids involving S. commersonii, S. stenophyllidium (a Mexican diploid), and Mexican tetraploids of series Longipedicellata also crossed only to 2EBN testers. The apparent lack of recombination and segregation for EBN in these hybrids indicates that the genomes of the Mexican diploid and tetraploid species carry EBN in a way genetically similar to that of the South American species S. Commersonii.Cooperative investigation of the U.S. Department of Agriculture, Agricultural Research Service and the Wisconsin Experiment Station. Supported in part by the USDA/Cooperative States Research Service Competitive Grant No. 83-CRCR-1-1253     

5-Azacytidine induced myogenesis in a differentiation defective cell line

Abstract. A differentiation defective cell line variant, the T984-15, has lost the capacity to differentiate myogenically. Following treatment with the hypomethylating agent 5-azacytidine, T984-15 cells were induced to differentiate into myogenic colonies containing fused myotubes. Myogenic colonies when cloned, maintained their ability to differentiate after prolonged culture in the absence of further 5-azacytidine treatment. These results indicate that 5-azacytidine treatment resulted in a stable alteration in the capacity of T984-15 cells to differentiate and suggests that the loss of myogenic potential may have occurred as a result of an epigenetic phenomenon rather than a somatic mutational event.     

5-Azacytidine induced myogenesis in a differentiation defective cell line

A differentiation defective cell line variant, the T984-15, has lost the capacity to differentiate myogenically. Following treatment with the hypomethylating agent 5-azacytidine, T984-15 cells were induced to differentiate into myogenic colonies containing fused myotubes. Myogenic colonies when cloned, maintained their ability to differentiate after prolonged culture in the absence of further 5-azacytidine treatment. These results indicate that 5-azacytidine treatment resulted in a stable alteration in the capacity of T984-15 cells to differentiate and suggests that the loss of myogenic potential may have occurred as a result of an epigenetic phenomenon rather than a somatic mutational event.     

Analysis of ribosomal RNA genes in somatic hybrids between wild and cultivated Solanum species

Ribosomal RNA genes were exploited as markers to identify somatic hybrids between Solanum tuberosum cv. Brodick and wild diploid Solanum species, S. megistacrolobum, S. sanctae-rosae and S. sparsipilum and DNA methylation as a possible regulatory factor in gene expression was investigated. Specific restriction enzyme/probe combinations revealed useful polymorphisms in the conserved coding and variable intergenic spacer regions of the ribosomal RNA genes. Some intermediate ribosomal RNA gene profiles indicate hybridity whereas others were characteristic of S. tuberosum cv. Brodick. This evidence is suggestive of somatic exchange/re-arrangement between the NOR region of S. sanctae-rosae and S. tuberosum cv. Brodick. Ribosomal RNA gene copy number analysis of the somatic hybrids did not reveal hexaploid values suggesting that these products are not symmetric hybrids derived from the parental diploid and tetraploid plants. The results indicate site-specific methylation of ribosomal RNA gene sequences for the parental plants; while some somatic hybrids display a reduction, others show an increase. The significance of the findings for somatic cell genetics and plant breeding studies is discussed.     

The utility of NBS profiling for plant systematics: a first study in tuber-bearing Solanum species

Systematic relationships are important criteria for researchers and breeders to select materials. We evaluated a novel molecular technique, nucleotide binding site (NBS) profiling, for its potential in phylogeny reconstruction. NBS profiling produces multiple markers in resistance genes and their analogs (RGAs). Potato (Solanum tuberosum L.) is a crop with a large secondary genepool, which contains many important traits that can be exploited in breeding programs. In this study we used a set of over 100 genebank accessions, representing 49 tuber-bearing wild and cultivated Solanum species. NBS profiling was compared to amplified fragment length polymorphism (AFLP). Cladistic and phenetic analyses showed that the two techniques had similar resolving power and delivered trees with a similar topology. However, the different statistical tests used to demonstrate congruency of the trees were inconclusive. Visual inspection of the trees showed that, especially at the lower level, many accessions grouped together in the same way in both trees; at the higher level, when looking at the more basal nodes, only a few groups were well supported. Again this was similar for both techniques. The observation that higher level groups were poorly supported might be due to the nature of the material and the way the species evolved. The similarity of the NBS and AFLP results indicate that the role of disease resistance in speciation is limited.     

Changes in the initial phase of lipid peroxidation induced by elicitor from Phytophthora infestans in Solanum species

The initial phase of the lipid peroxidation process in leaves of Solanum nigrum var. gigantea, Solanum tuberosum cv Bzura and clone H-8105, which represent non-host resistance, field resistance and susceptibility, respectively, against Phytophthora infestans, was investigated. Based on quantitative and qualitative high-performance liquid chromatography (HPLC) analyses of free and esterified fatty acid hydroperoxides (FAHs), we characterized the lipid peroxidation process induced by the pathogen-derived elicitor, culture filtrate (CF), in leaves of the studied genotypes. In all plants, FAHs generated due to 13-lipoxygenase (LOX) action dominated over those from the non-enzymatic pathway. The FAHs derived from 9-LOX activity were found only in CF-treated leaves of the non-host resistant S. nigrum. However, experiments in vitro and in planta with exogenous linoleic acid (LA) as a substrate for LOX revealed high constitutive activity of 9-LOX in all genotypes, which increased in response to CF treatment. The time course changes in polyunsaturated fatty acid (PUFA) pools in the total lipid fractions as well as the degree of their oxidation suggested that CF-induced PUFA peroxidation was enhanced mostly in S. nigrum, less so in Bzura and least in the susceptible clone H-8105. The obtained results are discussed in light of the overall biochemical cell status of plants in the studied interactions.     

AFLP data support the recognition of a new tuber-bearing Solanum species but are uninformative about its taxonomic relationships

Solanum section Petota, containing the cultivated potato and its wild relatives, is a group of around 200 species. Many of these species are morphologically very variable with unclear boundaries, and the group as a whole appears to be somewhat over-classified. Describing a new species in this group should only be undertaken with caution, and molecular data can be used to test the distinctness of any putative new taxon. AFLP markers have shown the ability to reliably distinguish species in several groups within the genus Solanum. We tested the distinctness of a new tuber-bearing Solanum species using morphological and AFLP data, and tried to establish its affiliation to the series within the section. There was clear support for the species status of the material known as Solanum hannemanii in genebank collections, but the AFLP data were inconclusive about its relationships to the other investigated species. Also, the distinction of the series Tuberosa and Megistacroloba, to which these species belong, was not supported.     

5-Azacytidine enhances transient expression of a transfected gene in cultured mammalian cells

The level of transient expression of the Escherichia coli β-galactosidase (β-Gal) gene transfected into three mammalian cell lines was enhanced by culturing the cells with 1–5 μM 5-azacytidine (5-azaC) after the transfection. This enhancement was similarly observed in four different transfection procedures tested. The enhancement of β-Gal expression was primarily due to the increase in the level of its mRNA. Since 5-azaC did not exhibit any obvious effect on the levels of endogenous gene products examined, it is suggested that 5-azaC may preferentially affect the transient expression of an exogenous gene.     

Changes in free polyamine levels induced by salt stress in leaves of cultivated and wild tomato species

The effects of N_aCl on endogenous free levels of the poluamines putrescine, spermi dine and spermine, and the relationships between polyamines, K⁺ levels and Na⁺ accumulation were determined in leaves of the cultivated tomato ( Lycopersicon esculentum Mill.) and its wild, salt-tolerant relative L. pennellii (Correll) D' Arcy at different exposure times during a 32-day period. Both stress treatments (100 and 200 m M NaCl) decreased the levels of putrescine and spermidine, although to a different degree for the cultivated and wild tomato species. The spermine levels did not decrease with salinity in L. pennellii over the salinization period, whereas they decreased in L. esculentum , except at the first application of the 100m M NaCl treatment. In both species, the changes induced by salinity in total polyamines and K⁺ were very similar, with the accumulation of Na⁺ in the leaf being concomitant with a decrease in both total polyamines and K⁺. This suggests that the main role of the polyamines in the leaf tissues. In this sense, a direct relationship between total polyamines and K⁺, and inverse relationship between polyamines and Na⁺ and between K⁺ and Na⁺ were found for both species. In the short term (up to 4 days) a peculiar physiological behavior was found in L. pennellii , as the total polyamine and K⁺ levels decreased at 100 m M but not at 200 m M NaCl, while after this time the latter plants had values lower than those of the 100 m M NaCl-treated plants at day 11.     

Two CAPS markers predict Verticillium wilt resistance in wild Solanum species

Verticillium wilt of potato is a persistent problem in the USA and worldwide. The disease, which is caused primarily by the fungus Verticillium dahliae, is difficult to manage, causes yield losses, and contaminates soil for subsequent plantings. Control strategies based on host resistance are seen as long-term, stable solutions, but difficult to achieve given the genetic nature of the host and the challenges associated with resistance evaluations. To provide breeders with marker-assisted selection opportunities, we generated a pair of cleaved amplified polymorphic sequence molecular markers within the coding region of Ve2, a potato gene with homology to the tomato Ve1 gene that confers resistance to V. dahliae. The position of the marker was determined according to the consensus sequences of Ve2 homologs of wild Solanum species with resistance to V. dahliae. Marker testing indicated their broad applicability, being able to track the resistance to V. dahliae in progeny containing genetic information derived from species S. chacoense, S. brevicaule, S. berthaultii, S. tarijense, and S. tuberosum. Furthermore, the two isolates of V. dahliae used in our inoculation experiments differed in virulence and demonstrated specificity for some wild potato species. Experimentation leading to the development of the markers and tests of their usefulness against a wide range of diploid potato germplasm is presented.     

Agrobacterium-mediated transformation of five wild Solanum species using in vitro microtubers

Summary A genetic transformation method, using in vitro microtubers and Agrobacterium-mediated transformation has been developed for five wild Solanum species: S. verrucosum, S. hjertingii, S. papita, S. stoloniferum, S. demissum, which range in ploidy from diploid to hexaploid. A disarmed A. tumefaciens strain, C58 harbouring the co-integrate vector pGV3580::pKU2 with the genes of neomycin phosphotransferase (NPTII) and hygromycin phosphotransferase (HPTII) as selectable markers, was used. Selection of putative transformants was based on their ability to grow and produce roots on a medium containing 150 mg/l kanamycin. The transgenic nature of the putative transformants was confirmed by Polymerase Chain Reaction analysis and by NPTII dotblot assay to show the expression of the NPTII gene. Additionally, the transmission of transgenes, NPTII and HPTII in selfed-sexual progeny of some transgenic plants was also determined.Abbreviations MS Murashige and Skoog - NPTII neomycin phosphotransferase - HPTII hygromycin phosphotransferase - PCR polymerase chain reaction - GA₃ gibberellic acid - CCC chlorocholine chloride - BAP benzylaminopurine - NAA naphthalene acetic acid - ZR zeatin riboside - IAA indole acetic acid - LB Luria Broth     

Secondary metabolite profile in induced tetraploids of wild Solanum commersonii Dun

The main aim of this work was to study the leaf secondary metabolite profiles of artificially induced tetraploids (2n=4x=48) of Solanum commersonii, a diploid (2n=2x=24) wild potato species. The tetraploid genotypes of S. commersonii were produced by oryzalin treatment. Both HPLC-UV and LC/MS analyses revealed that there were no qualitative differences in the metabolite profiles between the diploid S. commersonii and its tetraploids. By contrast, the results showed that the phenylpropanoid content was generally significantly higher in the tetraploids than in the diploid S. commersonii. Concerning the glycoalkaloids (GAs), the results provided evidence that the content of minor GAs (solanidenediol triose, solanidadienol lycotetraose, and solanidenol lycotetraose) was higher in tetraploids than in the diploid progenitor, while the content of major GAs (dehydrodemissine and dehydrocommersonine) was significantly higher in diploid S. commersonii than in its tetraploid genotypes. The results are discussed from the practical perspective of potato biodiversity enhancement.     

5-Azacytidine treatment of a murine cytotoxic T cell line alters interferon-gamma gene induction by interleukin 2

Genetic susceptibility to multiple sclerosis (MS) in Caucasians was previously shown to be correlated to the presence of given alleles at the HLA-DR and Gm loci. We now demonstrate that the humoral immune response in MS central nervous system (CNS) is modulated by both loci: the levels of IgG1 subclass and IgG1 allotypes in cerebrospinal fluid of MS patients depend on both their Gm genotype and their HLA-DR2 or HLA-DR7 phenotype. That HLA-DR molecules may either participate in a preferential recruitment of IgG1 allotype-producing B cells in MS CNS or act after such a selective homing is discussed. These results demonstrate that both HLA and Gm loci are synergistically involved in the modulation of the humoral immune response.