Suppose you have purified a novel protein from the Golgi membranes of a poorly s

Question

Suppose you have purified a novel protein from the Golgi membranes of a poorly studied but pathogenic species of amoeba, and you perform Edman degradation and determine its N-terminal five amino acids as T-Y-W-I-N. You decide to construct single-stranded, radioactive DNA probes to screen a genomic library produced from this amoeba, in hopes of cloning the gene that encodes this protein. How many different DNA probes should you synthesize? (show your math).

You decide to name this new amoeba protein Lannisterase, and you estimate its size as 468 amino acids. Soon, predictably, your hated arch-rival (who is also studying these amoeba) shows up at a scientific meeting with a poster in which he hypothesizes that Lannisterase is integral to the Golgi membrane due to a single start-transfer sequence that encompasses amino acids 48-66, and that no other membrane-spanning regions exist in Lannisterase. You agree that the Lannisterase protein purified from amoeba has its only membrane-spanning region between amino acids 48-66, but you claim that this region is a stop-transfer sequence. Explain your rationale and evidence for this alteration of your rival's hypothesis.

Explanation / Answer

               Thr          Tyr          Try           Ile        Asn

Codons   ACT        TAT        TGG       ATT        AAT

               ACC        TAC                       ATC        AAC

               ACA                                      ATA                      

               ACG

Now select the 15 base region to prepare 15mer degenerate probe to screen the genomic library

ACTTACTGGATTAAT   - this will be the complementary oligonuceotide in the degenerate probe

TGAATGACCTAATTA   -this is the coding sequence.

48 (= 4×2×1×3×2) different 15 base DNA sequences could encode this one sequence of amino acids. The 48 possible DNA sequences from these 15 bases will have a probe which perfectly complementary to the actual coding sequence from the genomic library.

For the second question,

The start transfer sequence is involved in the initiation of transfer, where as the stop transfer signal will stop the translocation of the protein. When start transfer signal directs the protein to the Endoplasmic membrane, the pores will be opened. Signal peptidase will cleave this start transfer peptide and the protein will be allowed to pass through the membrane. When translocated protein is released in the ER lumen, the start signal peptide will be released from the pore and will be degraded to amino acids by the proteases present in the ER. So your prediction is correct that the peptide of 48-66 amino acids is a stop transfer sequence. It is not a start transfer sequence, as it is degraded soon after translocation.

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