3A. After successfully cloning the gene responsible for megacholesterosis, you sequenced the clone and obtained the following 100bp DNA sequence. Answer the questions regarding the following DNA sequence: ATATAATCAG GCAGGAGTGA TGGTCATCCG TTGACTTTAG CCAATTAGTC 50 GCCGCATTTG AATGCCACCC TTGCTGACCA TGAGGGAATT TTCGTAGGGC 100 GIVEN: i) RNA polymerase binds at a T-A-T-A sequence and begins transcription at the 1ST base from the binding site. ii) RNA polymerase terminates immediately AFTER a C-A-T-G-A-G-G sequence. iii) The intron ends have the sequences U-U-G and C-U-U iv) The RNA is cut at the 3' end of a A-U-U-U-U sequence by the POLY-A enzyme complex and a poly A tail is added to the cut end. v) The ribosomal P site aligns with bases 7-9 from the methyl-G cap. vi) All eukaryotic proteins START with the amino acid, methionine (met). (1) What is the sequence of the first 10 nucleotides of the primary RNA? (2) What is the entire sequence of the mature mRNA? (3) What is the sequence of the protein? (4) Based on the structure of the mature mRNA, what cellular compartment would you expect to find the mature transcript? (5) If there is a TRANSITION mutation in base 20: a. what is the effect on the mRNA: b. what is the effect on the protein? (6) If there are 2 TRANSVERSION mutations in bases 80-81 to their complementary bases: a. what is the effect on the primary transcript? b. what is the effect on the mRNA? c. what is the effect on the protein?
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Use the complementary base pairs (A-U, T-A, G-C, C-G) to transcribe the DNA sequence into RNA. Show more…
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A sequence of a eukaryotic gene (coding strand) is shown below, RNA polymerase recognizes the sequence ‘TATAAT’ and initiates transcription six nucleotides downstream of the sequence. The intron splice sites are CUU (5’ splice site) and AAG (3’ splice site), poly-A tails are added following the sequence AGUUGG. The poly-A tails are 20 nucleotides. a. Predict the sequence of mature mRNA and denote 5’ and 3’ ends. (2 points) b. If this is an oncogene that is elevated in cancer cells, design a siRNA (RNA duplex) to knock down the mRNA, list the sequences of both strands of the siRNA and highlight the targeted regions on the mRNA in yellow. (2 points) c. If you plan to study whether the gene is regulated by miRNA(s), you will search for potential miRNA target sequences on the mRNA, highlight the region for miRNA targeting in green. (1 point) GATGAGTTAT AATATTTCTC TCCAGGCATG GAGTATTCCG GTGTGCGATC GCCGTTATCG ATCGATCGAT ATCGATCGAA TCCCCCTTTG GACCACCCTG GGTTGCCCTC TAAGCATAAT TCGTCGTCGT ACAACGCCAT GTTTCTGT AATTAAAATT TGTTTGCCTC AGTTGGATGT What is alternative splicing? (0.5 point) Draw out all possible mature mRNAs of Gene A. ( 1 point) Compare and contrast the mechanisms and function of siRNA and miRNA. (2 points) Describe at least three differences between chromatin regions that are transcriptionally active and those in which genes are transcriptionally silent. (1.5 points)
Sri K.
DNA: template strand 3' T A C T A G G C T A T T 5'
Adi S.
Analysis of Gene Structure and Function Donor Site - GT Acceptor Site - AG Find an interrupted ORF in the sequence below: 5'-ATTACATGGGCCAAGCCCGATTCAGGTAAGTATCAGCCTGGTTTGGTAATTACT-3' Step 1 - Locate potential start codons in the sequence. Step 2 - Locate the first Donor splice site 3' of the start codon. Step 3 - Locate the first Acceptor splice site 3' of the Donor splice site. Step 4 - Draw a box around the potential intron (including the donor and acceptor sites). Step 5 - Ignoring bases in the potential intron and staying in the same translational frame, locate the first stop codon 3' of the acceptor site. Step 6 - Determine whether the potential intron interrupts a codon or falls between two adjacent codons. Step 7 - Use the genetic code to translate the full polypeptide. What are the lengths of the exonic regions? What is the percent GC content of the exons? What is the length of the intron? What is the percent GC content of the non-coding regions? Does the intron interrupt a codon? Assuming that the potential intron is successfully spliced, what would the sequence of the encoded protein be? In the sequence of a human chromosome, how likely is it to encounter potential start codons, stop codons, and splice sites that are non-functional? What "wet lab" procedure(s) could be used to check whether this potential intron is actually spliced? What computational procedure(s) could be used to assess whether this potential gene is a real gene? What computational procedures could be used to identify a possible function for this potential gene?
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