Genome instability of the segmentally aneuploidy progeny
ABSTRACT: Array-based comparative genome hybridization. The aneuploid progeny could return to euploid state. We determined genome-wide gene copy number in the genomes of aneuploid progeny after 14, 24 and 34 days vegetative propagation in dextrose-containing rich medim.
Project description:H. jecorina undergoes a heterothallic reproductive cycle, and the mating yields asci with 16 linearly arranged ascospores. The sixteen ascospores are generated via two rounds of postmeiotic mitosis following the two meiotic divisions. We have discovered that viable aneuploid ascosporesare are frequently (~90%) generated due to a net gain of a conserved ~500 kbp genomic segment. Here we have compared whole genome gene copy number changes in the aneuploid F1 progeny, euploid F1 progeny, parental strains and the F1 progeny of RTU strain backcross with CBS999.97 . We determined genome-wide gene copy number in the genomes of ascospores generated from haploid wild-type strain. We also determined genome-wide gene copy number in the genomes of the F1 progeny of CBS999.97 wild type.
Project description:FGFR1 mediated changes in gene expression at different stages of JOCK1 prostate cancer progression JOCK1 mice which express a prostate localize inducible version of the FGFR1 gene (iFGFR1) were used to determine what are the changes in gene expression at distinct stages of JOCK1 tumor progression. Activation of iFGFR1 is mediated by biweekly treatements of JOCK1 mice with AP20187 (50mg/kg). Wild type mice were treated for 24 hours with CID as a control, while JOCK1 were treated with CID for 4, 12, 24 and 52 weeks. All these treatments began at 12 weeks of age. After treatment mouse prostates (ventral and dorso-lateral prostate lobes), were dissected, placed in RNAlater and incubated at -800C. Tissue was thawed and placed in TRIzol (Invitrogen) for RNA isolation according to the manufacture. Once RNA was removed, a RNA cleaning step was performed using Qiagen RNEasy columns as described by the manufacturer. Time points were performed in triplicate and each array represents a single prostate. Labeling and hybridization to the mouse Affymetrix gene array 430 2.0 was performed by the Baylor College of Medicine microarray core. All hybridizations were performed at the same time.
Project description:Recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) has a poor prognosis with less than 1-year median survival. Platinum-based chemotherapy (cisplatin or carboplatin) remains the first-line treatment for HNSCC. The cancer stem cell (CSC) hypothesis postulates that tumors are maintained by a self-renewing CSC population that is also capable of differentiating into non-self renewing cell populations that constitute the bulk of the tumor. A small population of CSCs exists within HNSCC that are relatively resistant to chemotherapy and clinically predicted to mediate tumor recurrence. These CSCs are identified by high cell-surface expression of CD44 and high intracellular activity of aldehyde dehydrogenase (ALDH) and termed ALDHhighCD44high. We investigated the molecular pathways active in ALDHhighCD44high cells, which remain poorly studied. Additionally, we performed a molecular examination of cisplatin-resistant ALDHhighCD44high cells, which has not been reported. Two HNSCC cell lines, UM-SCC-1 and UM-SCC-22b, were utilized in this study. For microarray analysis, UM-SCC-22b cells were treated for 5 days in vitro with 2uM cisplatin and analyzed by flow cytometry, sorted and submitted for microarray analysis of ALDHhighCD44high and ALDHlowCD44low cells from untreated and cisplatin treated cells. Four separate flow cytometry experiments were performed using Affymetrix Human Gene ST 2.1 microarrays. Microarray data was analyzed using R/Bioconductor. Files were preprocessed by Robust Multiarray Average (RMA) with background substraction, quantile normalization, and median polish (oligo package). Data was fitted with robust probe level linear models to all the probesets (oligo package). Experiment and processing batch differences were accounted for using 'ComBat' within the SVA package. Differentially expressed genes were identified using univariate comparisons after fitting data to a linear model (limma package). Initial statistics were determined using an empirical Bayesian model. Multiple testing comparisons were adjusted using Benjamini and Hochberg (aka FDR). Probes with an adjusted p-value <0.05 were considered statistically significant. Unsupervised hierarchical clustering with complete linkage and Euclidean distance was performed on only statistically significant probes. In four separate experiments, the head and neck squamous cell carcinoma cell line UM-SCC-22b were cultured for 5 days with or without 2uM (micromolar) cisplatin in 6-well plates. Media was replaced every other day. Control and cisplatin treated cells were trypsinized, procesed, and stained for CD44 cell-surface expression and intracellular aldehyde dehydrogenase (ALDH) activity to identify cancer stem cells (ALDH+CD44+). CSCs and non-CSCs (ALDH-CD44-) were collected by flow cytometry from both groups. Total RNA was collected from each fraction (ALDH+CD44+, ALDH-CD44-), treatment (control, cisplatin), and experiment (#1-4). A total of 16 samples were analyzed. One set of 4 (experiment #4) were analyzed on a Human Gene ST 2.1 strip and the rest on a Human Gene ST 2.1 plate. Differential gene expression was determined with R/Bioconductor with Robust Multiarray Average (RMA) and fitting the data to linear models (limma). Experimental and processing batch effects were accounted for using ComBat. Four sets of univariate comparisons were made: 1) Cisplatin ALDH+CD44+ vs Control ALDH+CD44+; 2) Control ALDH+CD44+ vs Control ALDH-CD44-; 3) Cisplatin ALDH+CD44+ vs Cisplatin ALDH-CD44-; 4) Cisplatin ALDH-CD44- vs Control ALDH-CD44-. Multiple testing comparisons were adjusted using Benjamini and Hochberg (aka FDR). Probes with an adjusted p-value <0.05 were considered statistically significant.
Project description:To identify the genes required to sustain aneuploid viability, we screened a deletion library of non-essential genes in the fission yeast Schizosaccharomyces pombe, in which most types of aneuploidy are eventually lethal to the cell. Aneuploids remain viable for a period time and can form colonies by reducing the extent of the aneuploidy. We hypothesized that a reduction in colony formation efficiency could be used to screen for gene deletions that compromise aneuploid viability. Deletion mutants were used to measure the effects on the viability of spores derived from triploid meiosis and from a chromosome instability mutant. We found that the CCR4-NOT complex, an evolutionarily conserved general regulator of mRNA turnover, and other related factors, including poly(A)-specific nuclease for mRNA decay, are involved in aneuploid viability. Defective mutations in CCR4-NOT complex components in the distantly related yeast Saccharomyces cerevisiae also affected the viability of spores produced from triploid cells, suggesting that this complex has a conserved role in aneuploids. In addition, our findings suggest that the genes required for homologous recombination repair are important for aneuploid viability. Gene expression profile at exponentially-growing phase.in the fission yeast deletion mutants of the CCR4-NOT complex protein genes.
Project description:BACKGROUND: The ability to adapt to environments with fluctuating nutrient availability is vital for bacterial survival. Although essential for growth, few nitrogen metabolism genes have been identified or fully characterised in mycobacteria and nitrogen stress survival mechanisms are unknown. RESULTS: A global transcriptional analysis of the mycobacterial response to nitrogen stress, showed a significant change in the differential expression of 16% of the Mycobacterium smegmatis genome. Gene expression changes were mapped onto the metabolic network using Active Modules for Bipartite Networks (AMBIENT) to identify metabolic pathways showing coordinated transcriptional responses to the stress. AMBIENT revealed several key features of the metabolic response not identified by KEGG enrichment alone. Down regulated reactions were associated with the general reduction in cellular metabolism as a consequence of reduced growth rate. Up-regulated modules highlighted metabolic changes in nitrogen assimilation and scavenging, as well as reactions involved in hydrogen peroxide metabolism, carbon scavenging and energy generation. CONCLUSIONS: Application of an Active Modules algorithm to transcriptomic data identified key metabolic reactions and pathways altered in response to nitrogen stress, which are central to survival under nitrogen limiting environments. [Data is also available from http://bugs.sgul.ac.uk/E-BUGS-140]
Project description:Background. The Beijing family of Mycobacterium tuberculosis is dominant in countries in East Asia. Genomic polymorphisms are a source of diversity within the M.tuberculosis genome and may account for the variation of virulence among M.tuberculosis isolates. To date there are no studies that have examined the genomic composition of M.tuberculosis isolates from the high TB-burden country, Myanmar. Methodology/Principle findings. Twenty-two M.tuberculosis isolates from Myanmar were screened on whole-genome arrays containing genes from M.tuberculosis H37Rv, M.tuberculosis CDC1551 and M.bovis AF22197. Screening identified 198 deletions or extra regions in the clinical isolates compared to H37Rv. Twenty-two regions differentiated between Beijing and non-Beijing isolates and were verified by PCR on an additional 40 isolates. Six regions (Rv0071-0074 [RD105], Rv1572-1576c [RD149], Rv1585c-1587c[RD149], MT1798-Rv1755c [RD152], Rv1761c [RD152] and Rv0279c) were deleted in Beijing isolates, of which 4 (Rv1572-1576c, Rv1585c-1587c, MT1798-Rv1755c and Rv1761c) were variably deleted among ST42 isolates, indicating a closer relationship between the Beijing and ST42 lineages. The TbD1 region, Mb1582-Mb1583 was deleted in Beijing and ST42 isolates. One M.bovis gene of unknown function, Mb3184c was present in all isolates, except 11 of 13 ST42 isolates. The CDC1551 gene, MT1360 coding for a putative adenylate cyclase, was present in all Beijing and ST42 isolates (except 1). The pks15/1 gene, coding for a putative virulence factor, was intact in all Beijing and non-Beijing isolates, except in ST42 and ST53 isolates. Conclusion. This study describes previously unreported deletions/extra regions in Beijing and non-Beijing M.tuberculosis isolates. The modern and highly frequent ST42 lineage showed a closer relationship to the hypervirulent Beijing lineage than to the ancient non-Beijing lineages. The pks15/1 gene was disrupted only in modern non-Beijing isolates. This is the first report of an in-depth analysis on the genomic diversity of M.tuberculosis isolates from Myanmar. Data is also available from http://bugs.sgul.ac.uk/E-BUGS-66