Genomic imbalances associated with methotrexate resistance in human osteosarcoma cell lines detected by comparative genomic hybridization-based techniques

Methotrexate (MTX) is one of the most important drugs for osteosarcoma (OS) treatment. To identify genetic aberrations associated with the development of MTX resistance in OS cells, in addition to the previously reported expression changes of dihydrofolate reductase (DHFR) and reduced folate carrier (RFC) genes, comparative genomic hybridization (CGH)-based techniques were used. The direct comparison between MTX-resistant variants of U-2OS or Saos-2 human OS cell lines with their respective parental cell lines by CGH on chromosomes revealed that development of MTX resistance was associated with gain of the chromosomal regions 5q12-q15 and 11q14-qter in U-2OS variants, and with gain of 8q22-qter in Saos-2 variants. Further analyses by CGH on microarrays demonstrated a progressively increasing gain of mixed lineage leukemia (MLL) gene (11q23) in U-2OS MTX-resistant variants, which was also confirmed by fluorescence in situ hybridization (FISH), in addition to gain of FGR (1p36), amplification/overexpression of DHFR, and slight decrease of RFC expression. In Saos-2 MTX-resistant variants, gain of MYC (8q24.12-q24.13) was detected, together with a remarkable decrease of RFC expression. Further analyses of DHFR, MLL, MYC, and RFC gene status in four additional human OS cell lines revealed that only gain of DHFR and MLL were associated with an inherent lower sensitivity to MTX. These data demonstrate that genetic analyses with complementary techniques are helpful for the identification of new candidate genes, which might be considered for an early identification of MTX unresponsive tumors.     

Carcinogenic potential and genomic instability of beryllium sulphate in BALB/c‐3T3 cells

Occupational exposure to beryllium (Be) and Be compounds occurs in a wide range of industrial processes. A large number of workers are potentially exposed to this metal during manufacturing and processing, so there is a concern regarding the potential carcinogenic hazard of Be. Studies were performed to determine the carcinogenic potential of beryllium sulfate (BeSO₄) in cultured mammalian cells. BALB/c3T3 cells were treated with varying concentrations of BeSO₄ for 72 h and the transformation frequency was determined after 4 weeks of culturing. Concentrations from 50–200 g BeSO₄/ml, caused a concentrationdependent increase (9–41 fold) in transformation frequency. Nontransformed BALB/c3T3 cells and cells from transformed foci induced by BeSO₄ were injected into both axillary regions of nude mice. All ten Beinduced transformed cell lines injected into nude mice produced fibrosarcomas within 50 days after cell injection. No tumors were found in nude mice receiving nontransformed BALB/c3T3 cells 90 days postinjection. Gene amplification was investigated in Kras, cmyc, cfos, cjun, csis, erbB2 and p53 using differential PCR while random amplified polymorphic DNA fingerprinting was employed to detect genomic instability. Gene amplification was found in Kras and cjun, however no change in gene expression or protein level was observed in any of the genes by Western blotting. Five of the 10 transformed cell lines showed genetic instability using different random primers. In conclusion, these results indicate that BeSO₄ is capable of inducing morphological cell transformation in mammalian cells and that transformed cells induced by BeSO₄ are potentially tumorigenic. Also, cell transformation induced by BeSO₄ may be attributed, in part, to the gene amplification of Kras and cjun and some BeSO₄induced transformed cells possess neoplastic potential resulting from genomic instability.     

Determination of the copy number of the nuclear rDNA and beta-tubulin genes of Pneumocystis carinii f. sp. hominis using PCR multicompetitors

Multiple copies of a gene may lead to difficulty in the interpretation of typing results because polymorphism of the copies may wrongly lead to the conclusion that different types are present in a specimen. To determine the copy number per genome of the nuclear rDNA and beta-tubulin genes analyzed for the typing of Pneumocystis carinii f. sp. hominis, we developed a strategy based on the use of the same multicompetitor molecule in two different quantitative-competitive PCRs, one for the gene under study and the other for a reference single copy gene, allowing direct comparison of the results of both PCRs. Control experiments showed that the strategy was sensitive enough to detect duplication of a gene. The copy number of the nuclear rDNA operon was determined by amplification of the intron of the 26S rDNA gene and that of the beta-tubulin by amplification of the region surrounding the intron no. 6. The method was first tested on P. c. carinii, the special form commonly infecting rats. Pneumocystis c. carinii was found to contain a single copy of the rDNA operon. The method was then applied to P. c. hominis. The results confirmed that P. c. hominis genome contains a single copy of the nuclear rDNA and beta-tubulin genes.     

Structural Analysis ofArabidopsis thaliana Chromosome 5. VI. Sequence Features of the Regions of 1,367,185 bp Covered by 19 Physically Assigned P1 and TAC Clones

Nineteen Pl and TAC clones, which have been mapped on the finephysical map of the Arabidopsis thaliana chromosome 5, weresequenced according to the shotgun-based strategy, and theirstructural features were analysed. The total length of the regionssequenced in this study was 1,367,185 bp. Combining this withthe regions covered by 90 P1 and TAC clones proviously reported,the total length of chromosome 5 sequenced to date becomes 8,058,855bp. On the basis of similarity search against protein and ESTdatabases and gene modeling with computer programs, a totalof 330 potential protein-coding regions were identified, bringingan average density of the genes to approximately one gene per4.1 kb. Introns were identified in 81.0% of the potential proteingenes for which the entire gene structure was predicted, withan average number per gene of 4.2 and an average length of theintrons of 180 bp. The RNA-coding genes identified were 9 tRNAgenes corresponding to 8 amino acid species and 2 genes forU2 nuclear RNA. These sequence features are essentially identicalto those in the previously reported sequences. The sequencedata and gene information are available on the World Wide Webdatabase KAOS (Kazusa Arabidopsis data Opening Site) at http://www.kazusa.or.jp/arabi/.     

Taxonomic relationships in East AsianVicia species with unijugate leaves based on random amplified polymorphic DNA markers

Random amplified polymorphic DNA(RAPD) markers were investigated to clarify the taxonomic positions ofVicia linearifolia andV. bifolia, and to assess the genomic diversity among the 9 populations ofV. unijuga, each of which represents a geographical variation or infraspecific taxa in southern Korea. These species are characterized by unijugate leaves in East Asia and have been controversial as to infra-or interspecific classification. The polymorphic markers among the populations examined were observed for fifteen decamer primers. The degree of band sharing was used to calculate genetic similarity between populations, and a phenogram using UPGMA cluster analysis was generated based on the Dice similarity coefficient. The taxa studied were divided into two main groups and the populations ofV. unijuga were all grouped together in the phenogram. The genetic similarities ofV. unijuga were very high among the populations and did not show distinctions between the infraspecific taxa, although the populations of Mt. Odae and adjacent areas in eastern Korea were different from others of the species.V. linearifolia fell within the range of the genomic variation among the populations ofV. unijuga, whileV. bifolia was grouped withV. venosa var.cuspidata having multijugate leaves rather thanV. unijuga. The result from studying RAPD markers suggested thatV. linearifolia should be integrated intoV. unijuga and that species with unijugate leaves ofV. bifolia andV. unijuga are polyphyletic.     

A genome map of Salmonella enterica serovar Agona: numerous insertions and deletions reflecting the evolutionary history of a human pathogen

Salmonella enterica serovar Agona is an important zoonotic pathogen, causing serious human illness worldwide, but knowledge about its genetics and evolution, especially regarding the genomic events that might have contributed to the formation of S . Agona as an important pathogen, is lacking. As a first step toward understanding this pathogen and characterizing its genomic differences with other salmonellae, we constructed a physical map of S . Agona in strain SARB1 using I-CeuI, XbaI, AvrII and Tn 10 insertions with pulsed-field gel electrophoresis techniques. On the 4815-kb genomic map, we located 82 genes, revealed one inversion of about 1000 kb and resolved seven deletions and seven insertions ranging from 10 to 67 kb relative to the genome of Salmonella typhimurium LT2. These genomic features clearly distinguish S . Agona from other previously analyzed salmonellae and provide clues to the molecular basis for its genomic divergence. Additionally, these kinds of physical maps, combined with emerging high-speed sequencing technologies, such as the Solexa or SOLiD techniques, which require a pre-existing high-resolution physical map such as the S . Agona map reported here, will play important roles in genomic comparative studies of bacteria involving large numbers of strains.     

An approach for post‐market monitoring of potential environmental effects of Bt‐maize expressing Cry1Ab on natural enemies

Post‐market monitoring (PMM) consistent with Swiss and European Union legislation should ensure the detection and prevention of adverse effects on the environment possibly deriving from commercial cultivation of genetically modified (GM) crops. Insect‐resistant GM crops (such as Bt‐maize) raise particular questions regarding disturbances of biological control functions provided by beneficial insects such as predators and parasitoids (so‐called natural enemies). Consensus among regulators, scientists and the agricultural biotech industry on appropriate PMM plans allowing the detection and possibly prevention of such adverse effects is still lacking. The aims of this study were to identify the necessity for PMM of Bt‐maize expressing Cry1Ab on natural enemies and to develop an appropriate PMM plan. The approach chosen consisted in determining what type of monitoring is most appropriate to address potential effects of Bt‐maize on natural enemies during commercial cultivation. This included identifying whether there remain substantial scientific uncertainties that would support case‐specific monitoring. Existing pre‐market risk assessment data indicate that Bt‐maize (Cry1Ab) comprises a negligible risk for disturbances in biological control functions of natural enemies. As a consequence, a faunistic monitoring of specific groups of natural enemies is not considered an appropriate approach to detect failures in biological control functions. Alternatively, an approach is proposed that consists in indirectly analysing biological control functions by surveying outbreaks of maize herbivores. Unusual herbivore outbreaks could indicate failures in biological control functions of natural enemies. Data could be collected via questionnaires addressed to farmers growing Bt‐maize. Significant correlations between unusual occurrences of specific maize herbivores and the cultivation of Bt‐maize would subsequently need specific studies to determine possible causalities in more detail. The here proposed approach has the advantage of covering different natural enemy groups. It represents a cost‐effective strategy to obtain scientifically sound data as a basis for regulatory decision‐making.     

Coadaptation and conflict,misconception and muddle,in the evolution of genomic imprinting

Common misconceptions of the ‘parental conflict'' theory of genomic imprinting are addressed. Contrary to widespread belief, the theory defines conditions for cooperation as well as conflict in mother–offspring relations. Moreover, conflict between genes of maternal and paternal origin is not the same as conflict between mothers and fathers. In theory, imprinting can evolve either because genes of maternal and paternal origin have divergent interests or because offspring benefit from a phenotypic match, or mismatch, to one or other parent. The latter class of models usually require maintenance of polymorphism at imprinted loci for the maintenance of imprinted expression. The conflict hypothesis does not require maintenance of polymorphism and is therefore a more plausible explanation of evolutionarily conserved imprinting.     

Genomic prediction in CIMMYT maize and wheat breeding programs

Genomic selection (GS) has been implemented in animal and plant species, and is regarded as a useful tool for accelerating genetic gains. Varying levels of genomic prediction accuracy have been obtained in plants, depending on the prediction problem assessed and on several other factors, such as trait heritability, the relationship between the individuals to be predicted and those used to train the models for prediction, number of markers, sample size and genotype × environment interaction (GE). The main objective of this article is to describe the results of genomic prediction in International Maize and Wheat Improvement Center''s (CIMMYT''s) maize and wheat breeding programs, from the initial assessment of the predictive ability of different models using pedigree and marker information to the present, when methods for implementing GS in practical global maize and wheat breeding programs are being studied and investigated. Results show that pedigree (population structure) accounts for a sizeable proportion of the prediction accuracy when a global population is the prediction problem to be assessed. However, when the prediction uses unrelated populations to train the prediction equations, prediction accuracy becomes negligible. When genomic prediction includes modeling GE, an increase in prediction accuracy can be achieved by borrowing information from correlated environments. Several questions on how to incorporate GS into CIMMYT''s maize and wheat programs remain unanswered and subject to further investigation, for example, prediction within and between related bi-parental crosses. Further research on the quantification of breeding value components for GS in plant breeding populations is required.