| Generally it is believed that the ribosomal protein mainly involve in ribosome assembly and composed by ribosomal protein and rRNA. Ribosomal protein not only involved in ribosome assembly and related to the efficiency of mRNA translation. Researchers show that the expression of many ribosomal proteins have related with cancer and some physiological disease. The title of this subject is that the eukaryotic specific ribosomal protein L36 is an important structural component in regulating rRNA processing through modulation of the translational efficiency of genes involved in that pathway. In our lab’s previous work, we have completed a series of constructs about yRPL36B Mutation. Subsequently we transported these constructions into the yeast strains of the deletion of endogenous RPL36A and RPL36B,then we remove ycplac33 plasmid which maintains wild-type yRPL36B by 5-FOA and we use RT-PCR method to verify the yeast that yRPL36B mutation as the only source of RPL36 protein. Next we use the method of spotting assay to observe the influence of yRPL36B mutation on cell growth. In this object,we focus on mutl(5’HA)(there is a mutation in the 88 amino acid of yRPL36B). For one thing,we adopt the general research idea of yeast ribosomal protein,we use anti-yRPL36 and anti-HA to detect the difference of amounts of yRPL36 protein when mutl(5’HA) us yRPL36B(dele.36) or FY251. Also we use the RNA probes for yRPS4b,yRPL36B,yRPL22A,yRPL10 to detect the difference of other ribosomal proteins expression when mutl(5’HA) us yRPL36B(dele.36). We employ green fluorescent protein EGFP to find out whether there is a significant accumulation in the nucleus for the protein yRPL36B or not. Next we adopt the reporting system of translational expression.identification of,reading frame frameshift,internal-mediated translation initiation to check whether mutl(5’HA) lead to changes in translational details. We find mutl(5’HA) is not conducive to the assembly of 60S and that have apparent half-mers. We also try to use some structure prediction and analysis software. Such as:phyre,pymol to predict spatial structure of yRPL36B and possible sites of action. For another, not only do we use polysome analysis combined with high-throughput chip to analysis translational efficiency,but also use high-throughput chip to detect expression levels for mutl(5’HA) and yRPL36B(dele.36). We found that ribosome biogenesis-related genes show significant downward in the level of translation from translational chip. We analysis these ribosomal proteins which have significant changes in the expression microarray by pymol software and find two so-called sub-complex from results of the analysis. We discover energy metabolism-related genes show significant upward in the level of translation and transcription from translational chip and expression microarray. Subsequently,we find translation efficiency of genes which ribosome biogenesis-related and energy metabolism-related have closely related with temperature by polysome analysis and q-PCR. At the same time,we construct the lacZ reporter system of ADE1,HTA2,NOP7,LSG1,ZUO1 in order to find the region which have related with translational efficiency. Next we add 13×myc to zuol and lsgl in order to evaluate amounts of yRPL36 protein in mutl(5’HA) and yRPL36B (dele.36). We also use MeMe software to predict the motif and Weblog software to map sequences surrounding the ATG and RegRNA software to analysis motifs which has been found in order to find structural characteristics which have related with translational efficiency. At the same time, we also want to do translational microarray for RPS14 mutation and RPL11 mutation. |
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