To study the splicing of mRNA X you generate a 32 P labeled in vitro RNA

To study the splicing of mrna x you generate a 32 p

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16. To study the splicing of mRNA X, you generate a 32P-labeled in vitro containing the 140 nucleotide (n) pre-mRNA X intron 1, flanked by the 100 n exon 1 and the 60 n exon 2, as shown below (‘A’ refers to the branch point A). You incubate your pre-mRNA X with a nuclear extract from a pancreatic cell line for 0, 5, 15 or 60 minutes, and subject the resulting RNA products to denaturing gel electrophoresis, followed by autoradiography as shown on the right (‘M’ refers to a marker). (a) (2 pts)Is the band migrating around 450 n (marked by ‘*’) a precursor, intermediate, or product of the splicing reaction? nuclear extractRNA Intermediate (b) (2 pts)Please draw how the RNA that migrates around 450 n looks, clearly indicating the elements present (i.e exon 1, intron and/or exon 2). (c)(2 pts) You create an AG to GG mutation at the 3’ splice site of pre-mRNA X. This mutation allows 17. (4 pts) You are interested in identifying important transcription factors for a gene and therefore perform two experiments. Using a reporter assay, you test the effects of mutating three elements within the promoter and get the results shown below. M 0 5 15 60Pancreas nuclear extract--Time (mins)--500 n -*5 6
Points____________ Name: ____________________________ 8You also perform a DNase footprinting assay for the wild-type promoter, 32P-labeled at the +20 position (see below), and incubated with the proteins A, B, C and D, respectively. (‘-’: no protein) Based on these experiments, for each of proteins A-D, indicate which position in the promoter is their binding site, and whether they are activators or repressors of transcription. (If a protein does not bind and/or affect transcription, write ‘None’ in the respective column). Activator/Repressor? 18. To identify important control regions for gene Xexpression, you create 9-base pair (bp) deletions in each of the places in gene Xindicated by arrows below. B_

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