You are working to characterize a novel protein in mice. Analysis shows that high levels of the primary transcript that codes for this protein are found in tissue from the brain, muscle, liver, and pancreas. However, an antibody that recognizes the C-terminal portion of the protein indicates that the protein is present in brain, muscle, and liver, but not in the pancreas. What is the most likely explanation for this result?

Question

A.  The gene that codes for this protein is not transcribed in the pancreas.
B.  The transcript is immediately degraded in the pancreas.
C.  The transcript receives a 5’ cap in the pancreas.
D.  Alternative splicing in the pancreas creates a protein missing the domain that the antibody recognizes.
E.  The transcript receives a 3’ poly-A tail in the pancreas.

Clarify Question
· What is the key concept addressed by the question?
· What type of thinking is required?
· What key words does the question contain and what do they mean?

Gather Content
· What do you already know about post-transcriptional regulatory mechanisms?

Consider Possibilities
· Consider the different answer options. Which can you rule out?

Choose Answer · Given what you now know, what information and/or problem solving approach is most likely to produce the correct answer?

Reflect on Process
· Did your problem-solving process lead you to the correct answer? If not, where did the process break down or lead you astray? How can you revise your approach to produce a more desirable result?

Answer 1

D.  Alternative splicing in the pancreas creates a protein missing the domain that the antibody recognizes.

Clarify Question
· What is the key concept addressed by the question?
        o This question addresses post-transcriptional regulatory mechanisms.
· What type of thinking is required?
        o This question is asking you to weigh and judge evidence, or evaluate, to choose the best of the possible answers.
· What key words does the question contain and what do they mean?
        o A novel protein is one that has just recently been discovered, and isn’t well understood by scientists.
        o The primary transcript is the initial pre-mRNA transcript before splicing takes place.
        o The C-terminal portion of a protein is the end near the final –COOH.

Gather Content
· What do you already know about post-transcriptional regulatory mechanisms?
        o Cells use several kinds of post-transcriptional regulatory mechanisms.
        o A gene can be transcribed or not in different cells, or the transcript can be degraded.
        o Alternative splicing creates proteins with different domains.
        o Most mRNAs receive a 5’ cap and poly-A tail to improve stability.

Consider Possibilities
· Consider the different answer options. Which can you rule out?
        o Could the gene not be transcribed in the pancreas, or the transcript degraded? No, because we are told that the primary transcript was found in the pancreas.
        o Could the addition of a 5’ cap or 3’ poly-A tail in the pancreas explain the data? No. Most transcripts get those anyway, and they only improve stability and the likelihood of translation into protein.

Choose Answer · Given what you now know, what information and/or problem solving approach is most likely to produce the correct answer?
        o What could explain why the antibody reacts with the protein in some cells but not others? Either the protein is not present in the pancreas cells, or the protein is different and not recognizable by the antibody.
        o What types of regulation lead to different sets of proteins in different cells? In addition to transcriptional regulation, alternative splicing is a mechanism that ensures that different cell types produce specialized sets of proteins.
        o The best explanation of the answer options is that alternative splicing produced a different form of the protein in pancreas cells, that could not be recognized by the antibody.

Reflect on Process
· Did your problem-solving process lead you to the correct answer? If not, where did the process break down or lead you astray? How can you revise your approach to produce a more desirable result?
        o This question asked for the best explanation for the presence of transcript but no apparent protein in one cell type.
        o The question required you to weigh and judge evidence, or evaluate, to choose the best of the possible answers.
        o Did you recognize that the regulation must be post-transcriptional?
        o Did you realize that antibodies may recognize only a portion of a protein?