Direct and inverted DNA repeats associated with P-glycoprotein gene amplification in drug resistant Leishmania.

The H circle of Leishmania species contains a 30 kb inverted duplication separated by two unique DNA segments, a and b. The corresponding H region of chromosomal DNA has only one copy of the duplicated DNA. We show here that the chromosomal segments a and b are flanked by inverted repeats (198 and 1241 bp) and we discuss how these repeats could lead to formation of H circles from chromosomal DNA. Selection of Leishmania tarentolae for methotrexate resistance indeed resulted in the de novo formation of circles with long inverted duplication, but two mutants selected for arsenite resistance contained new H region plasmids without such duplications. One of these plasmids appears due to a homologous recombination between two P-glycoprotein genes with a high degree of sequence homology. Our results show how the same DNA region in Leishmania may be amplified to give plasmids with or without long inverted duplications and apparently by different mechanisms.     

Monitoring gene expression in mixed microbial communities by using DNA microarrays

A DNA microarray to monitor the expression of bacterial metabolic genes within mixed microbial communities was designed and tested. Total RNA was extracted from pure and mixed cultures containing the 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterium Ralstonia eutropha JMP134, and the inducing agent 2,4-D. Induction of the 2,4-D catabolic genes present in this organism was readily detected 4, 7, and 24 h after the addition of 2,4-D. This strain was diluted into a constructed mixed microbial community derived from a laboratory scale sequencing batch reactor. Induction of two of five 2,4-D catabolic genes (tfdA and tfdC) from populations of JMP134 as low as 10(5) cells/ml was clearly detected against a background of 10(8) cells/ml. Induction of two others (tfdB and tfdE) was detected from populations of 10(6) cells/ml in the same background; however, the last gene, tfdF, showed no significant induction due to high variability. In another experiment, the induction of resin acid degradative genes was statistically detectable in sludge-fed pulp mill effluent exposed to dehydroabietic acid in batch experiments. We conclude that microarrays will be useful tools for the detection of bacterial gene expression in wastewaters and other complex systems.     

The application of DNA microarrays in gene expression analysis

DNA microarray technology is a new and powerful technology that will substantially increase the speed of molecular biological research. This paper gives a survey of DNA microarray technology and its use in gene expression studies. The technical aspects and their potential improvements are discussed. These comprise array manufacturing and design, array hybridisation, scanning, and data handling. Furthermore, it is discussed how DNA microarrays can be applied in the working fields of: safety, functionality and health of food and gene discovery and pathway engineering in plants.     

Genomic DNA macroarrays as a tool for analysis of gene expression in Leishmania

Gene-array technologies have been applied in a wide number of organisms to study gene expression profiling under several physiological and experimental conditions. Gene-array implementations combined with the information arising from emerging genome sequencing projects are expected to be in the near future a major tool to characterize genes involved in different processes. So far, gene expression profile studies in trypanosomatids have been performed in microarrays that use a glass support to immobilize fragments of genomic DNA followed by fluorescent detection. Here, we wanted to test the potential of genomic DNA macroarrays of Leishmania infantum using nylon membranes and radioactive detection. Nylon macroarrays present a number of advantages since the processing of the membranes is based on standard Southern blotting protocols familiar to molecular biologists, and the data acquisition equipment is available to most research institutions. Nylon macroarrays were employed to search for genes showing increased mRNA abundance during an axenic differentiation of L. infantum promastigotes to amastigotes. Several clones were rescued and, after validation by Northern blot assays, these L. infantum sequences were used to screen the Leishmania major gene database. The L. major contigs with high homology to the L. infantum sequences allowed a consistent identification of the regulated genes.     

Simultaneous measurement of gene expression for hepatotoxicity in thioacetamide-administered rats by DNA microarrays

DNA microarray technology was developed as a tool for simultaneously measuring a number of gene expression changes, and has been applied for investigations of toxicity assessments of chemicals. In this study, we used a typical hepatotoxicant, thioacetamide (TA), to find correlations between the extent of hepatotoxicity and certain gene expression patterns or specific gene expression profiles. TA was intraperitoneally administered at high (400 mg/kg), medium (150 mg/kg) or low (50 mg/kg) dose (four rats per group) and then the serum and liver were collected at the indicated time (6, 12, 24, 36 and 48 h). Serum biochemical markers were measured and hepatic mRNA expression profiles were analyzed by a DNA microarray. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were increased by TA-administration in a dose-dependent manner and reached the maximum at 24h. Hierarchical clustering analysis of all dosage groups revealed in 2 major clusters, distinguished by an early (6 and 12h) and a late (24, 36 and 48 h) phase. The early and late phase clusters were sorted in time- and dose-dependent manners. The major gene expression profile obtained by quality-threshold (QT) clustering analysis showed the same maximal toxic time as that estimated by the serum biochemical markers. The individual expression profiles of the candidate genes selected in our previous studies and the simultaneous gene expression patterns measured by five typical hepatotoxicants including TA also reflected the hepatotoxicity of TA. These findings suggest that the potential toxic effects appearing as gene expression changes are independent of the dosage of TA. This study suggested that the major gene expression profile estimated by QT clustering would be a sensitive marker of hepatotoxicity.     

DNA microarrays and papillary thyroid carcinoma gene expression profile

The paper presents gene expression profile analysis with DNA microarrays and compares two core technological platforms used for this purpose - high density oligonucleotide microarrays and cDNA microarrays. With this background recent results of papillary thyroid carcinoma analysis with DNA microarrays are presented.     

Differential gene expression analysis by DNA microarrays technology and its application in molecular oncology

Accumulation of genetic and epigenetic aberrations leads to malignant transformation of normal cells. Functional studies of cancer using genomic and proteomic tools will help to reveal the true complexity of the processes leading to cancer development in humans. Until recently, diagnosis and prognosis of cancer was based on conventional pathologic criteria and epidemiological evidence. Certain tumors were divided only into relatively broad histological and morphological subcategories. Rapidly developing methods of differential gene expression analysis promote the search for clinically relevant genes changing their expression levels during malignant transformation. DNA microarrays offer a unique possibility to rapidly assess the global expression picture of thousands genes in any given time point and compare the detailed combinatory analysis results of global expression profiles for normal and malignant cells at various functional stages or separate experimental conditions. Acquisition of such "genetic portraits" allows searching for regularity and difference in expression patterns of certain genes, understanding their function and pathological importance, and ultimately developing the "molecular nosology" of cancer. This review describes the basis of DNA microarray technology and methodology, and focuses on their applications in molecular classification of tumors, drug sensitivity and resistance studies, and identification of biological markers of cancer.     

Using DNA microarrays to study gene expression in closely related species

MOTIVATION: Comparisons of gene expression levels within and between species have become a central tool in the study of the genetic basis for phenotypic variation, as well as in the study of the evolution of gene regulation. DNA microarrays are a key technology that enables these studies. Currently, however, microarrays are only available for a small number of species. Thus, in order to study gene expression levels in species for which microarrays are not available, researchers face three sets of choices: (i) use a microarray designed for another species, but only compare gene expression levels within species, (ii) construct a new microarray for every species whose gene expression profiles will be compared or (iii) build a multi-species microarray with probes from each species of interest. Here, we use data collected using a multi-primate cDNA array to evaluate the reliability of each approach. RESULTS: We find that, for inter-species comparisons, estimates of expression differences based on multi-species microarrays are more accurate than those based on multiple species-specific arrays. We also demonstrate that within-species expression differences can be estimated using a microarray for a closely related species, without discernible loss of information. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

Glycosylation and post-translational modification gene expression analysis by DNA microarrays for cultured mammalian cells

DNA microarray analysis of gene expression has become a valuable tool for bioprocessing research aimed at improving therapeutic protein yields. The highly parallel nature of DNA microarray technology allows researchers to assess hundreds of gene simultaneously, essentially enabling genome-wide snapshots. The quality and amount of therapeutic proteins produced by cultured mammalian cells rely heavily on the culture environment. In order to implement beneficial changes to the culture environment, a better understanding of the relationship between the product quality and culture environment must be developed. By analyzing gene expression levels under various environmental conditions, light can be shed on the underlying mechanisms. This paper describes a method for evaluating gene expression changes for cultured NS0 cells, a mouse-derived myeloma cell line, under culture environment conditions, such as ammonia buildup, known to affect product quality. These procedures can be easily adapted to other environmental conditions and any mammalian cell lines cultured in suspension, so long as a sufficient number of gene sequences are publicly available.     

Identification of differential gene expression profiles in placentas from preeclamptic pregnancies versus normal pregnancies by DNA microarrays

The purpose of this study was to perform a comprehensive analysis of gene expression profiles in placentas from preeclamptic pregnancies versus normal placentas. Placental tissues were obtained immediately after delivery from women with normal pregnancies (n=6) and patients with preeclampsia (n=6). The gene expression profile was assessed by oligonucleotide-based DNA microarrays and validated by quantitative real-time RT-PCR. Functional relationships and canonical pathways/networks of differentially-expressed genes were evaluated by GeneSpring? GX 11.0 software, and ingenuity pathways analysis (IPA). A total of 939 genes were identified that differed significantly in expression: 483 genes were upregulated and 456 genes were downregulated in preeclamptic placentas compared with normal placentas (fold change ≥ 2 and p<0.05 by unpaired t-test corrected with Bonferroni multiple testing). The IPA revealed that the primary molecular functions of these genes are involved in cellular function and maintenance, cellular development, cell signaling, and lipid metabolism. Pathway analysis provided evidence that a number of biological pathways, including Notch, Wnt, NF-κB, and transforming growth factor-β (TGF-β) signaling pathways, were aberrantly regulated in preeclampsia. In conclusion, our microarray analysis represents a comprehensive list of placental gene expression profiles and various dysregulated signaling pathways that are altered in preeclampsia. These observations may provide the basis for developing novel predictive, diagnostic, and prognostic biomarkers of preeclampsia to improve reproductive outcomes and reduce the risk for subsequent cardiovascular disease.