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1.Which is the most unlikely involved in the intronsplicing of pre-mRNA ? [a. U1 RNP; b. 5' cap structure; c. polyadenylation signal; d. secondary structure of pre-mRNA], give a brief explanation if you are not sure your answer (5%) U1A protein inhibits polyadenylation of its pre-mRNA U1A protein binds U1snRNA: 7-base sequence-U1A binding site U1A protein inhibits polyadenylation of its pre-mRNA U1A binding site 2 copies in its own mRNA , at 3' end near the poly(A) signal prevent polyadenylation, but not the cleavage of pre-mRNA rapidly degraded U1A protein inhibits polyadenylation of its pre-mRNA - prevent polyadenylation, but not the cleavage of pre-mRNA truncated mRNA without poly(A) tail, rapidly degraded RNA editing in mammalian apo-B (apolipoprotein B), serum protein of lipid transporter, CAA UAA, glutamine stop codon, in intestine both liver and intestine forms transport lipid only liver apo-B delivers cholesterol containing LDL RNA editing in glutamate receptor mRNA Na+, Ca+2 ion channel, learning and memory CAG CIG, glutamate arginine, Ca +2 cannot pass Both editing of apo-B and glutamate receptor by RNA deaminases Iron dependent regulation of TfR mRNA stability. when iron ion high, IRE -BP inactive, cannot bind IRE, TfR mRNA is protected from degradation; when iron is low, IRE-BP is active and binds IRE. then TfR mRNA stability , TfR mRNA degraded Iron dependent regulation of translation of ferritin stability. ferritin is an intracellular protein that binds iron ions ferritin, 5' IRE free of BP, translation occurs, more ferritin. The IRE in ferritin mRNA has no AU rich ferritin Using Yeast to study Eukaryotic Gene Function from Recombinant DNA (J Waston et al.) Ch 13 Size of genome in the selected organisms Yeast biosynthetic genes are cloned by complementation of E.coli mutations Cloning yeast biosynthetic genes by complementation in E.coli Shuttle vectors replicate in both E.coli and yeast Classes of yeast vectors Yeast genes can be cloned by simple complementation Cloning a yeast gene by complementation Generation of temperature sensitive mutants of yeast Replica plating Homolgous recombination is a relative frequent event in yeast Gene targeting by homologous recombination Replacing a gene by transplacement Tetrad analysis Cloning genes required for mating reveals a signaling pathway similar to that seen in higher organisms Pheromone signaling pathway This fragment DNA only works in high copies, suggesting it was acting as a suppressor Cloning of the GPA1 gene as a high-copy suppressor of pheromone sensitivity Genetic experiments in yeast can answer precise biochemical question The receptor swap experiment U2 RNA base-pairs with an intron sequence Base pairing between U2 and branch point (in yeast) A genetic assay for protein-protein interactions Genetic analysis in yeast can be exploited to identify and study genes from higher organisms Plasmid shuffle