Project description:These are the ribosomal subunit fractions from the polysome gradients. investigating effect of heat shock on procyclic-form trypanosomes.
Project description:JN54 (wild-type) cells were incubated in YPD medium at 30 degrees C to a logarithmic phase (OD660=1), followed by treatment with mild heat-shock at 43 degrees C for 30 min in pre-warmed (43 degrees C) 100ml of YPD medium using 500 ml Erlenmeyer flask. JN54 (wild-type) cells were incubated in YPD medium at 30 degrees C to a logarithmic phase (OD660=1), followed by treatment with mild heat-shock at 43 degrees C for 60 min in pre-warmed (43 degrees C) 100ml of YPD medium using 500 ml Erlenmeyer flask. Keywords: Stress response
Project description:hCINAP is essential for human 18S rRNA processing and ribosome assembly. hCINAP is highly expressed in human cancers and promotes cancer cell growth. To connect the role of hCINAP in ribosome biogenesis and tumor growth, genome-wide polysome profiling was performed to detect the regulation of hCINAP on ribosome assembly and translation control. The results showed taht hihgly expressed hCINAP promotes ribosome biogenesis and selectively modulates cancer-associated translatome to promote malignancy. This result provides important insights into the molecular mechanism underlying the role of hCINAP in tumorigenesis. For the transcriptional profile, MCF7 cells transfected with control vector or GFP-hCINAP were collected and total RNA was extracted. For the polysoem profile, MCF7 cells with stably expressed GFP or GFP-hCINAP were subjected to surcose gradient fractionation. Ribosome profiles were obtained by measuring the absorbance of the sucrose gradient at a wavelength of 254 nm using a BioComp Gradient Fractionator. The fractions corresponding to subpolysome and polysome were collected and RNA was extracted. The cDNA library was generated and sequenced via Illumina HiSeqTM 2000. The clean reads were mapped to the UCSC hg19 reference genome and FPKM was assigned to calculate expression levels. The recruitment to polysome of each mRNA was calculated according to their FPKM values in the monsome and polysome.
Project description:Procyclic trypanosomes (strain 427 lister) were grown in culture under standard conditions at 27ºC in SDM79 medium with 10% foetal bovine serume (Brun and Schnenberger, 1979), in a gazed incubator (5% CO2). Logarithmically growing procyclic cells (at about 5*10^6 cells/ml, at 27°C) were added to one volume medium that had been heated to 53°C and incubated at 41ºC for 60 minutes in a waterbath in a closed tube (41ºC sample). The control cells were added to one volume medium at 27ºC and also incubated for 60 minutes in a closed tube at 27°C. Cells were harvested and washed once in PBS. The harvesting was done within 8 minutes.