C60 Fullerene Effects on Diphenyl-N-(trichloroacetyl)-amidophosphate Interaction with DNA In Silico and Its Cytotoxic Activity Against Human Leukemic Cell Line In Vitro.

New representative of carbacylamidophosphates - diphenyl-N-(trichloroacetyl)-amidophosphate (HL), which contains two phenoxy substituents near the phosphoryl group, was synthesized, identified by elemental analysis and IR and NMR spectroscopy, and tested as a cytotoxic agent itself and in combination with C60 fullerene.According to molecular simulation results, C60 fullerene and HL could interact with DNA and form a rigid complex stabilized by stacking interactions of HL phenyl groups with C60 fullerene and DNA G nucleotide, as well as by interactions of HL CCl3 group by ion-π bonds with C60 molecule and by electrostatic bonds with DNA G nucleotide.With the use of MTT test, the cytotoxic activity of HL against human leukemic CCRF-CM cells with IC50 value detected at 10 µM concentration at 72 h of cells treatment was shown. Under combined action of 16 µM C60 fullerene and HL, the value of IC50 was detected at lower 5 µM HL concentration and at earlier 48 h period of incubation, besides the cytotoxic effect of HL was observed at a low 2.5 µM concentration at which HL by itself had no influence on cell viability. Binding of C60 fullerene and HL with minor DNA groove with formation of a stable complex is assumed to be one of the possible reasons of their synergistic inhibition of CCRF-CЕM cells proliferation.Application of C60 fullerene in combination with 2.5 µM HL was shown to have no harmful effect on structural stability of blood erythrocytes membrane. Thus, combined action of C60 fullerene and HL in a low concentration potentiated HL cytotoxic effect against human leukemic cells and was not followed by hemolytic effect.

Cytotoxic Effects of Dimorfolido-N-Trichloroacetylphosphorylamide and Dimorfolido-N-Benzoylphosphorylamide in Combination with C60 Fullerene on Leukemic Cells and Docking Study of Their Interaction with DNA.

Dimorfolido-N-trichloroacetylphosphorylamide (HL1) and dimorfolido-N-benzoylphosphorylamide (HL2) as representatives of carbacylamidophosphates were synthesized and identified by the methods of IR, 1H, and 31P NMR spectroscopy. In vitro HL1 and HL2 at 1 mM concentration caused cell specific and time-dependent decrease of leukemic cell viability. Compounds caused the similar gradual decrease of Jurkat cells viability at 72 h (by 35%). HL1 had earlier and more profound toxic effect as compared to HL2 regardless on leukemic cell line. Viability of Molt-16 and CCRF-CEM cells under the action of HL1 was decreased at 24 h (by 32 and 45%, respectively) with no substantial further reducing up to 72 h. Toxic effect of HL2 was detected only at 72 h of incubation of Jurkat and Molt-16 cells (cell viability was decreased by 40 and 45%, respectively).It was shown that C60 fullerene enhanced the toxic effect of HL2 on leukemic cells. Viability of Jurkat and CCRF-CEM cells at combined action of C60 fullerene and HL2 was decreased at 72 h (by 20 and 24%, respectively) in comparison with the effect of HL2 taken separately.In silico study showed that HL1 and HL2 can interact with DNA and form complexes with DNA both separately and in combination with C60 fullerene. More stable complexes are formed when DNA interacts with HL1 or C60 + HL2 structure. Strong stacking interactions can be formed between HL2 and C60 fullerene. Differences in the types of identified bonds and ways of binding can determine distinction in cytotoxic effects of studied compounds.

Directed gap closure in large-scale sequencing projects.

A problem in many sequencing projects is the final closure of gaps left in the clone libraries, which serve as templates for sequencing, because of uncloned or unclonable genomic areas. By use of the Xylella fastidiosa genome as a test system, we present here an approach to generate, in a directed manner, sequence information from those gaps. We suggest using the complete clone library as a competitor against the genomic DNA of interest in a subtractive hybridization procedure similar to representational difference analysis (RDA). The resulting sequence information can be used to screen selectively other clone resources or serve directly for gap closure.

Contig selection in physical mapping.

In physical mapping, one orders a set of genetic landmarks or a library of cloned fragments of DNA according to their position in the genome. Our approach to physical mapping divides the problem into smaller and easier subproblems by partitioning the probe set into independent parts (probe contigs). For this purpose we introduce a new distance function between probes, the averaged rank distance (ARD) derived from bootstrap resampling of the raw data. The ARD measures the pairwise distances of probes within a contig and smoothes the distances of probes across different contigs. It shows distinct jumps at contig borders. This makes it appropriate for contig selection by clustering. We have designed a physical mapping algorithm that makes use of these observations and seems to be particularly well suited to the delineation of reliable contigs. We evaluated our method on data sets from two physical mapping projects. On data from the recently sequenced bacterium Xylella fastidiosa, the probe contig set produced by the new method was evaluated using the probe order derived from the sequence information. Our approach yielded a basically correct contig set. On this data we also compared our method to an approach which uses the number of supporting clones to determine contigs. Our map is much more accurate. In comparison to a physical map of Pasteurella haemolytica that was computed using simulated annealing, the newly computed map is considerably cleaner. The results of our method have already proven helpful for the design of experiments aimed at further improving the quality of a map.

Mapping analysis of the Xylella fastidiosa genome.

A cosmid library was made of the 2.7 Mb genome of the Gram-negative plant pathogenic bacterium Xylella fastidiosa and analysed by hybridisation mapping. Clones taken from the library as well as genomic restriction fragments of rarely cutting enzymes were used as probes. The latter served as a backbone for ordering the initial map contigs and thus facilitated gap closure. Also, the co-linearity of the cosmid map, and thus the eventual sequence, could be confirmed by this process. A subset of the eventual clone coverage was distributed to the Brazilian X.FASTIDIOSA: sequencing network. Data from this effort confirmed more quantitatively initial results from the hybridisation mapping that the redundancy of clone coverage ranged between 0 and 45-fold across the genome, while the average was 15-fold by experimental design. Reasons for this not unexpected fluctuation and the actual gaps are being discussed, as is the use of this effect for functional studies.

The genome sequence of the plant pathogen Xylella fastidiosa. The Xylella fastidiosa Consortium of the Organization for Nucleotide Sequencing and Analysis.

Xylella fastidiosa is a fastidious, xylem-limited bacterium that causes a range of economically important plant diseases. Here we report the complete genome sequence of X. fastidiosa clone 9a5c, which causes citrus variegated chlorosis--a serious disease of orange trees. The genome comprises a 52.7% GC-rich 2,679,305-base-pair (bp) circular chromosome and two plasmids of 51,158 bp and 1,285 bp. We can assign putative functions to 47% of the 2,904 predicted coding regions. Efficient metabolic functions are predicted, with sugars as the principal energy and carbon source, supporting existence in the nutrient-poor xylem sap. The mechanisms associated with pathogenicity and virulence involve toxins, antibiotics and ion sequestration systems, as well as bacterium-bacterium and bacterium-host interactions mediated by a range of proteins. Orthologues of some of these proteins have only been identified in animal and human pathogens; their presence in X. fastidiosa indicates that the molecular basis for bacterial pathogenicity is both conserved and independent of host. At least 83 genes are bacteriophage-derived and include virulence-associated genes from other bacteria, providing direct evidence of phage-mediated horizontal gene transfer.

Use of representational difference analysis and cDNA arrays for transcriptional profiling of tumor tissue.

Representational difference analysis of cDNA (cDNA-RDA) was used for a comparison of the global transcript level of tumor of the larynx and the corresponding normal epithelial tissue toward the end of detecting differentially expressed genes. Overall, some 130 gene fragments were identified. By sequence analysis and homology comparison, they could be put into several groups related to (potential) functions. Apart from genes whose overexpression was most likely a result of tumor growth or dedifferentiation of epithelial tissue, a lot of genes were isolated that play major roles in signal transduction pathways or apoptosis or act as oncogenes or tumor suppressor genes, in addition to new, entirely unknown genes. Moreover, some cDNAs of known genes were identified that derived from unconventional splicing activity or other transcript modifications. All identified fragments were arrayed on solid support and used for reverse Northern blot analyses. The use of preselected RDA fragments as targets in array-based profiling experiments circumvents many of the problems encountered when dealing with large clone libraries.

Novel technology for detection of genomic and transcriptional alterations in pancreatic cancer.

AIM: The present review summarizes our strategies aimed at identifying and characterizing genetic alterations occurring at the transcriptional and chromosomal level in pancreatic cancer. METHODS: To study transcriptional alterations we have used a number of techniques including modified versions of differential hybridizations and cDNA-RDA (representational difference analysis). Comparative genomic hybridization (CGH) was used to study chromosomal aberrations occurring in pancreatic cancer tissues. RESULTS: The study of transcriptional alterations led to the identification of more than 500 genes with differential expression in pancreatic cancer. The sum of these alterations represented the first expression profile characteristic for pancreatic tumors. The CGH analysis allowed the identification of a number of chromosomal regions containing putative tumor suppressor genes or oncogenes. These regions are presently being characterized at the molecular level. In a first approach the myb-oncogene was identified as the relevant oncogene of an amplification on 6q occurring in up to 10% of pancreatic cancer patients. CONCLUSIONS: Genes isolated in both approaches represent potential new disease genes for pancreatic cancer and are at present being characterized by individual or serial analysis.

Strategies for the detection of disease genes in pancreatic cancer.

The present review summarizes our strategies aimed at identifying and characterizing genetic alterations occurring at the transcriptional and chromosomal level in pancreatic cancer. To study transcriptional alterations we have used a number of techniques including modified versions of differential hybridizations and cDNA RDA (representational difference analysis). These approaches have led to the identification of more than 500 genes with differential expression in pancreatic cancer. To study chromosomal aberrations occurring in pancreatic cancer tissues we used comparative genomic hybridization (CGH). This allowed the identification of a number of chromosomal regions containing putative tumor suppressor genes or oncogenes. Genes isolated in both approaches represent potential new disease genes for pancreatic cancer and are at present being characterized by individual or serial analysis.

Isolation of differentially expressed genes by combining representational difference analysis (RDA) and cDNA library arrays.

The difference products (DP) of representational difference analyses (RDA) were used as hybridization probes on cDNA arrays. The effectivity of RDA products obtained with increasing driver/tester ratios (DP 1 = 100:1, DP 2 = 800:1 and DP 3 = 400,000:1) to isolate differentially expressed genes was compared with the effectivity of conventional differential hybridizations. Pacreatic cancer and control tissues were used as a test system to isolate differentially expressed genes. The use of RDA products as hybridization probes showed two major advantages: (i) a reliable identification of true differential signals; and (ii) only one autoradiograph had to be analyzed, which eliminated the need for a laborious subtraction of signal intensities obtained with different cDNA probes. Increasing driver/tester ratios in iterative rounds of RDA delivered more specific results, though the total yield of differential clones was gradually reduced. In this situation, the intermediate RDA product DP 2 provided the best compromise.

Selective generation of chromosomal cosmid libraries within the Trypanosoma cruzi genome project.

From a total genomic cosmid library of the pathogen Trypanosoma cruzi, specific sublibraries of the smallest four chromosomes were isolated by hybridization of the respective chromosomal bands obtained from pulsed-field gels. These libraries form the basis for initial mapping analyses that should provide information useful for both the ongoing physical mapping of the entire genome and eventual sequence analyses. Selectivity of the procedure was high with 75% to 92%, although cross-hybridization had to be expected from ubiquitous DNA features, such as centromeric and telomeric sequences, and other regions homologous between individual chromosomes. Overall, the number of identified clones was slightly higher than expected but well within the intrinsic experimental variation considering the uncertainty about the exact genome size, the variability in clonability and the higher frequency of repeat sequences in larger chromosomes. Chromosome III- and IV-specific cosmids were analyzed on Southern blots of chromosomal separations. For strain CL Brener, all clones tested exhibited cross-hybridization to a homologous chromosome larger than 1 Mbp, supporting the assumption of the respective chromosome couple being diploid pairs.

Hybridization mapping of Trypanosoma cruzi chromosomes III and IV.

As part of the Trypanosoma Genome Initiative launched by the World Health Organization (WHO), a physical clone map of Trypanosoma cruzi chromosomes III and IV was generated to facilitate both DNA sequence analysis of the parasite's genome and the investigation of chromosome organization. Apart from a few genetic markers, anonymous cosmids were taken from chromosomal sublibraries and individually hybridized to filter arrays of the relevant cosmid library. The probe order was determined from the hybridization fingerprint results and used to define a fitting clone order, with few gaps remaining. The results were independently verified by hybridizations to a bacterial artificial chromosome (BAC) library and, in case of chromosome III, restriction mapping. For gap closure, additional experiments on a total cosmid library were carried out. The possible tiling paths consist of 26 clones for chromosome III (610 kbp) and 28 clones for chromosome IV (680 kbp).

Identification of genes with specific expression in pancreatic cancer by cDNA representational difference analysis.

cDNA representational difference analysis (cDNA-RDA) is a polymerase-chain-reaction-coupled subtractive and kinetic enrichment procedure for the isolation of differentially expressed genes. In this study, the technique was used to isolate novel genes specifically expressed in pancreatic cancer. cDNA-RDA was done on cDNA reverse transcribed from a poly(A)+ mRNA pool made from 10 cancer tissues (tester) by using as a driver a cDNA from a poly(A)+ mRNA pool made from a combination of 10 tissues of chronic pancreatitis and 10 healthy pancreatic tissues. The use of chronic pancreatitis in addition to healthy pancreas mRNA in the driver preparation eliminated the influence of stromal tissue components present as contamination in the cancer-specific preparations. Such cDNA-RDA led to the isolation of 16 distinct, cancer-specific gene fragments. These were confirmed to be overexpressed in pancreatic cancer tissues by Northern blot analysis. Sequence analysis revealed homologies to five genes previously implicated in the carcinogenesis of the pancreas or other tissues. Eleven fragments had no significant homology to any known gene and thus represent novel candidate disease genes. The experiments demonstrate that cDNA-RDA is a reproducible and highly efficient method for the identification of novel genes with cancer-specific expression.