Project description:The Gram-positive pathogenic bacterium Staphylococcus aureus permanently colonizes the nasal mucosa in about 30% of the healthy population. However, as an opportunistic pathogen, S. aureus can also cause a variety of diseases, the treatment of which is becoming increasingly difficult due to antibiotic resistance. Adaptation to changing environmental conditions during colonization and infection of the host requires precise regulation of the expression of genes for virulence factors and metabolic functions. The host conditions essentially include low iron availability, with the majority of iron in the human organism being present intracellularly in the form of haemoglobin. Therefore, S. aureus has various adaptive mechanisms, in particular iron uptake systems, which are regulated by the global iron uptake regulator Fur (ferric-uptake regulator). Iron deficiency conditions lead to the derepression of Fur-dependent genes. In many bacteria, these genes also include regulatory RNAs (small regulatory RNAs, sRNAs), which mediate an iron-sparing response by inhibiting the synthesis of non-essential iron-containing proteins. A Fur-dependent sRNA (S596) was identified in a transcriptome analysis of S. aureus. In a study by Coronel-Tellez et al. (2022) it was shown that S596/IsrR (“iron sparing response regulator”) is necessary for the virulence of S. aureus. In the presented secretome analysis, the S. aureus HG001 wild type (WT) was compared with an isogenic isrR mutant (ΔisrR) under iron-limiting conditions and a constitutively isrR-expressing strain (HG001 pJLisrR) with an empty vector control (HG001 pJLctrl) under iron-rich conditions as well as corresponding sae mutant (Δsae) strains. Culture supernatants were collected in the exponential and stationary growth phases and extracellular proteins were subsequently analyzed by mass spectrometry. Statistical data analysis was performed to identify proteins with significantly altered amounts between the respective strains. We demonstrate that IsrR positively influences the protein levels of numerous virulence factors which in particular belong to the Sae regulon.
Project description:Microarray technology was used to monitor the level of expression of 7,657 human genes in a set of 35 nodal peripheral T-cell lymphomas.
Project description:Identification of gene expression signatures associated with favourable or unfavourable treatment response and clinical outcome of Hodgkin lymphoma patients.
Project description:We previously described the use of a spotted oligonucleotide array to identify the mir-17 cluster as a direct transcriptional target of Myc. In order to determine whether Myc regulates additional miRNAs, we produced custom microarrays with an expanded set of probes capable of assaying the expression of 313 human miRNAs and 233 mouse miRNAs. P493-6 cells which are Epstein-Barr virus-immortalized human B cells that harbor a tetracycline (tet)-repressible allele of Myc were studied. These cells are tumorigenic in immunocompromised mice and represent a model of human B cell lymphoma. miRNA expression profiles were examined in the high Myc (-tet) and low Myc (+tet) state. Keywords: Dose response P493 cells with high Myc (-tet) and low Myc (+tet) were compared.
Project description:End-to-end chromosome fusions that occur in the context of telomerase deficiency can trigger genomic duplications. These duplications are suggested to arise via Breakage-Fusion-Bridge cycles. To test this hypothesis, we examined end-to-end fusions isolated from C. elegans telomere replication mutants. Genome level rearrangements revealed fused chromosome ends possessing interrupted terminal duplications accompanied by template switching events. These features are very similar to disease-associated duplications of interstitial segments of the human genome. A model termed Fork Stalling and Template Switching has been proposed previously to explain such duplications, where promiscuous replication of large, non-contiguous segments of the genome occurs. Thus, a DNA synthesis-based process can create duplications that seal end-to-end fusions, in the absence of Breakage-Fusion-Bridge cycles. Numerous C. elegans mutant samples were studied with comparative genomic hybridization. There were no replicates or dye-swap hybridizations.
Project description:Phenotype-driven forward genetic experiments are among the most powerful approaches for linking biology and disease to genomic elements. Although widely used in a range of model organisms, positional cloning of causal variants is still a very laborious process. Here, we describe a novel universal approach, named fast forward genetics that combines traditional bulk segregant techniques with next-generation sequencing technology and targeted genomic enrichment, to dramatically improve the process of mapping and cloning multiple mutants in a single experiment. In a two-step procedure the mutation is first roughly mapped by ‘light’ sequencing of the bulk segregant pool, followed by genomic enrichment and deep-sequencing of the mutant pool for the linked genomic region. The latter step allows for simultaneous fine-mapping and mutation discovery. We successfully applied this approach to three Arabidopsis mutants, but the method can in principle be applied to any model organism of interest and is largely independent of the genome size. Moreover, we show that both steps can be performed in multiplex using barcoded samples, thereby increasing efficiency enormously. Inducible overexpression of the RETINOBLASTOMA-RELATED (RBR-OE) gene in Arabidopsis roots causes the complete differentiation of stem cells and premature differentiation of daughter cells, leading to a full exhaustion of the primary root meristem. In order to identify regulators of RBR function in cell differentiation, RBR-OE plants in the Columbia background (Col0) were treated with EMS mutagenesis and a set of genetic suppressors of RBR-OE, which restores root growth capacity, were isolated. In this study, we used one the identified suppressor lines, which segregated as a recessive mutation. Mapping populations were generated by outcrossing to Ler ecotype. Seedlings from the F2 population were grown for 15 days post germination (dpg). A pool of 60 seedlings each with a clear suppressor phenotype (homozygous for suppressor mutation) and of 60 seedlings showing RBOE phenotype (Heterozygous for the suppressor mutation) were prepared and genomic DNA was isolated with the RNeasy Plant Mini Kit from QIAGEN according to manufacturer's protocol. The other two, mutants 136 and 193 were obtained in fluorescence based mutant screen and a QCmarker based mutagenesis, respectively. Mutants were generated by chemical mutagenesis (EMS) in Colombia (Col) genetic background. Mutants were subsequently crossed to the Landsberg (Ler) ecotype to create the mapping populations. Bulk-segregant pools of about 200 mutant as well as wild-type plants were generated for every mutant line.
Project description:Transposon insertion site sequencing (TIS) is a powerful tool that has significantly advanced our knowledge of functional genomics. While providing valuable insights, these applications of TIS focus on (conditional) gene essentiality and neglect possibly interesting but subtle differences in the importance of genes for fitness. Notably, data from TIS experiments can be used for fitness quantification and constructing genetic interaction maps, though this potential is only sporadically exploited. We aimed to develop a method to quantify the fitness of gene disruption mutants using data obtained from the TIS screen SATAY. This dataset was used to determine the reproducibility of the fitness estimates across biological and technical replicates of the same strain of S. cerevisiae. In addition, a mutant bem3∆ strain was utilized to compare the genetic interactions inferred from these fitness estimates with those documented in published databases. The dataset for the wild-type strain was created by transforming strain yWT01 with plasmid pBK549 and picking 4 different colonies from the transformation plate. These 4 biological replicates were then renamed to FD7, FD9, FD11 and FD12.
Project description:Comparison at two different timepoints of expression profiles of wild_type Bacillus and a deletion mutant of the PlcR regulator. Additional processed data can be found in the FTP directory for this experiment <a href="ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/MEXP/E-MEXP-1472/">ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/MEXP/E-MEXP-1472/</a>
Project description:BACKGROUND: Microarray comparative genomic hybridization (CGH) is currently one of the most powerful techniques to measure DNA copy number in large genomes. In humans, microarray CGH is widely used to assess copy number variants in healthy individuals and copy number aberrations associated with various diseases, syndromes and disease susceptibility. In model organisms such as Caenorhabditis elegans (C. elegans) the technique has been applied to detect mutations, primarily deletions, in strains of interest. Although various constraints on oligonucleotide properties have been suggested to minimize non-specific hybridization and improve the data quality, there have been few experimental validations for CGH experiments. For genomic regions where strict design filters would limit the coverage it would also be useful to quantify the expected loss in data quality associated with relaxed design criteria. RESULTS: We have quantified the effects of filtering various oligonucleotide properties by measuring the resolving power for detecting deletions in the human and C. elegans genomes using NimbleGen microarrays. Approximately twice as many oligonucleotides are typically required to be affected by a deletion in human DNA samples in order to achieve the same statistical confidence as one would observe for a deletion in C. elegans. Surprisingly, the ability to detect deletions strongly depends on the oligonucleotide 15-mer count, which is defined as the sum of the genomic frequency of all the constituent 15-mers within the oligonucleotide. A similarity level above 80% to non-target sequences over the length of the probe produces significant cross-hybridization. We recommend the use of a fairly large melting temperature window of up to 10 C, the elimination of repeat sequences, the elimination of homopolymers longer than 5 nucleotides, and a threshold of -1 kcal/mol on the oligonucleotide self-folding energy. We observed very little difference in data quality when varying the oligonucleotide length between 50 and 70, and even when using an isothermal design strategy. CONCLUSIONS: We have determined experimentally the effects of varying several key oligonucleotide microarray design criteria for detection of deletions in C. elegans and humans with NimbleGen's CGH technology. Our oligonucleotide design recommendations should be applicable for CGH analysis in most species. One-copy deletions were studied in both the C. elegans (2 samples) and human (1 sample) genomes. There were no replicate or dye-swap hybridizations.