The following is the DNA sequence of a hypothetical gene for the TINY protein. I
ID: 89838 • Letter: T
Question
The following is the DNA sequence of a hypothetical gene for the TINY protein. It is called the TINY gene. 1 atgatcatca actgtcacga ccaattcatt gtatgaaaag 41 acacagcaag caggatatag Imagine you are doing a research project in a laboratory and your supervisor asks you to clone the TINY gene into a plasmid vector so you can express the gene in an E. coli bacterial host. The process you use is shown below. The direction of TINY gene transcription is shown by the dotted arrow. Other sequences that are necessary in the plasmid are shown with an X, and with circles labelled Sequence 1' and 'Sequence 2. A) Name the two (2) sequence elements that must be in the plasmid at position X for the gene to be transcribed and translated. Briefly describe the function of each sequence element. B) The host cells also need two additional sequence elements to maintain and copy the plasmid. These are shown as 'Sequence 1 and 'Sequence 2. What are the correct names of these two (2) sequence elements? Briefly describe the function of each sequence element. C) Design a forward PCR primer that you could use as part of a forward and reverse primer pair to amplify the entire TINY gene. Write the primer in your answer book, indicating the 5 and 3 ends of the sequence. You may show your working for the primer design. D) Assume the first three bases of the TINY gene sequence shown are the start codon (ATG) and the last three bases are a stop codon (TAG). How many amino acid residues are in the encoded protein? Explain your answer briefly. E) What is the sequence of the first ten bases of the mRNA produced from the TINY coding sequence?Explanation / Answer
A) Ans-
X corresponds to promoter region. Promoter region consists of following sequence elements-
1) core promoter
it contains binding site for RNA polymerase -RNA pol I-transcribes genes encodind 18s 5.8s 28s ribosomal RNA
RNA pol II-transcribes genes encoding messenger RNA and certain small nuclear RNAs and microRNA
RNA pol III-transcribes genes encoding transfer RNA, 5s ribosomal RNAs and other small RNAs
2) Pribnow box (bacteria)- it is the sequence of six nucleotides base i.e.TATAAT which is recognized by RNA polymerase during transcription. it is the essential part of DNA for trancription (bacteria)
Ans-B
1) ori -origin of replication is the particular sequence in the plasmid at which replication is initiated.
2) antibiotic resistant gene- this gene is required for the selection of transformed plasmid. In the presence of particular antibiotic only tranformed plasmid will grow.
Ans-C
Forward primer- 5’ ATGATCATCAACTG 3’
Reverse primer-5’ CTATATCCTGCTTG 3’
Ans-D
19 amino acid long polypeptide.
Total number of codons in the sequence is 20, but last codon corresponds to stop codon it will not code for any amino acid.
Ans-E
DNA- ATG ATC ATC AAC TGT CAC GAC CAA TTC ATT
TRANSLATION - Met I I N C H D Q F I
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