Project description:100mM EMS was added to mid-log phase Halobacterium NRC-1 cultures. After constant stress of EMS for 30 minutes, cultures were spun down and pellets were re-suspended in same volume of GN101 media. Cultures were then harvested by centrifugation and pellets were snap frozen on a dry-ice ethanol bath. Samples for RNA preparation were collected during recovery time points at 0, 10, 20, 30, 40, 60 and 120 minutes. Keywords: stress response, dose response
Project description:100mM EMS was added to mid-log phase Halobacterium NRC-1 cultures. After constant stress of EMS for 30 minutes, cultures were spun down and pellets were re-suspended in same volume of GN101 media. Cultures were then harvested by centrifugation and pellets were snap frozen on a dry-ice ethanol bath. Samples for RNA preparation were collected during recovery time points at 0, 10, 20, 30, 40, 60 and 120 minutes. 16 samples (8 samples from EMS perturbed and 8 non-perturbed controls) were analyzed on replicate arrays (dye-flips) all against the same standard reference sample.
Project description:Granulosa cells are essential to the growth, development, and maturation of oocytes and follicles. In patients with EMs-related infertility, follicle maturation disorders, poor oocyte quality may be closely related to GCs in the follicle. However, the molecular biological mechanism of GCs in EMs-related infertility has not been reported. Therefore, we tried to explore the possible causes of EMs-related infertility through the study of ovarian GCs in patients with EMs-related infertility and tubal factor infertility.
Project description:The TET proteins TET1, TET2 and TET3 constitute a new family of dioxygenases that utilize molecular oxygen and the cofactors Fe(II) and 2-oxoglutarate to convert 5-methylcytosine (5mC) to 5-hydroxy-methylcytosine (5hmC) and further oxidation products in DNA1-5. Here we show that Tet1 and Tet2 have distinct roles in regulating 5hmC deposition and gene expression in mouse embryonic stem cells (mESC). Tet1 depletion in mESC primarily diminishes 5hmC levels at transcription start sites (TSS), whereas Tet2 depletion is mostly associated with decreased 5hmC in gene bodies relative to TSS. 5hmC is enriched at exon start and end sites, especially in exons that are highly expressed, and is significantly decreased upon Tet2 knockdown at the boundaries of high-expressed exons that are selectively regulated by Tet2. In differentiating murine B cells, Tet2 deficiency is associated with selective exon exclusion in the gene encoding the transmembrane phosphatase CD45. Tet2 depletion is associated with increased 5hmC and decreased 5mC at promoters/ TSS regions, possibly because of the redundant activity of Tet1. Together, these data indicate a complex interplay between Tet1 and Tet2 in mESC, and show that loss-of-function of a single TET protein does not necessarily lead to loss of 5hmC and a corresponding gain of 5mC, as generally assumed. The relation between Tet2 loss-of-function and selective changes in exon expression could potentially explain the frequent occurrence of both TET2 loss-of-function mutations and mutations in proteins involved in pre-mRNA splicing in myeloid malignancies in humans. Gene and exon expression analysis in mESC, Tet1 knockdown mESC, and Tet2 knockdown mESC by RNA-sequencing. Mapping of 5-hydroxymethylcytosine in mESC, Tet1 knockdown mESC, and Tet2 knockdown mESC by anti-CMS-seq. Mapping of methylcytosine in mESC, and Tet2 kd mESC by MeDIP-seq.
Project description:Goal:identify the genes regulated by RON2 involved in the delay of floral transitional and those associated with leaf development. The gene expression profile of mature leaf 6 of ron2-1 EMS mutant and the wild-type Ler are compared. The plant material for this experiment was grown in LEPSE-INRA Montpellier, and the microarrays at the MAF-VIB Leuven with PSB-VIB Ghent.