Use the information below for the next four 4 questions T.pdf
- 1. Use the information below for the next four (4) questions: T-cell receptor spectratype analysis is used to examine the diversity of T-cell receptor beta chain sequences in an individuals T cells. For this technique, T cells are isolated from a sample of thymocytes (developing T cells) or mature peripheral T cells from an individual. The mRNA, indicating the expressed proteins, is isolated from these cells and cDNA is generated by reverse transcription. This pool of cDNA is mixed with PCR primers that are used to amplify part of the rearranged T-cell receptor beta chain sequence containing the complete CDR3 loop. The position of these primers relative to the rearranged T-cell receptor b chain gene in the DNA locus is shown in the figure below: Following PCR amplification, the mixture of different DNA molecules is then size-separated by electrophoresis on an apparatus that can separate molecules that differ by a single nucleotide! At the end, the quantity of material deposited in each band of a given nucleotide sequence length is quantified by densitometry, and the spectratype trace is produced. The x-axis of the spectratype depicts the number of nucleotides in each PCR product from the beginning of the forward primer to the end of the reverse primer while the y-xis depicts the quantity of each PCR product. Select all that apply This is what a spectratype trace would like for mature peripheral T cells from a healthy individual. What is explanation for the separation of the heterogeneous population of T-cell receptor beta chain sequences into multiple sharp peaks of size lengths (rather than have one continuous curve)? In other words, why aren't all lengths possible? Select all that apply. When V(D)J recombination occurs, enzymatic activity makes possible to only create sequences of certain lengths but not others In order for the T-cell receptor beta chain locus to encode a functional protein, the V, D, and J sequences, along with any P or N nucleotides, must be 'in-frame.' There are fewer TCRs with very short and very long lengths because these receptors are less effective at binding epitopes Each TCR sequence can only differ from other sequences by a multiple of three nucleotides, as each three nucleotides encodes one amino acidThe spectratype below shows T cells from an individual that is missing an important enzyme that contributes to T-cell receptor beta chain diversity during the recombination process. Which enzyme is most likely absent in this individual? RAG-1 or RAG-2 Artemis The Ku70:Ku80 complex TdT DNA ligaseThe figure below shows the spectratype analysis of T-cell receptor beta chain sequences in developing T cells (thymocytes) that have just completed the V-D-J recombination process. Which of the following statements explain why this spectratype looks different from the one shown in question 13 of mature peripheral T cells. Select all that apply. During V(D)J recombination, all lengths of nucleotide sequences are created Developing T cells need to have more sequence lenghts in their TCRs to protect important primary lymphoid tissue from becoming infected Mature peripheral T cells express a functional beta chain protein, which mandates all TCRS can only have nucleotide lengths that are in-frame (every 3 nucleotides) Prior to testing the beta chain for funcationality, all possible sequence lengths generated by recombination would still be detected TCRs on developing T-cells are less specific than TCRs on mature T cellsThe figure below shows a more restricted example of spectratype analysis, where the forward primer used only binds to one specific V-beta sequence. In this example, the primer is specific for the Vb17 segment. When such a Vbetaspecific primer is used the spectratype analysis only shows the junctional sequence
- 2. lengths for all the T cells whose beta chain uses that specific Vb17 segment in their receptor. In a healthy individual, the Vb17 spectratype should look just like the one shown in question 13. In other words, it should show a random distribution of Vb17+T-cell receptor beta chains with a normal distribution of junctional lengths. However, in this case, the individual being studied has been infected with influenza virus, and is in the midst of a robust T cell response against the virus. Which of the following statements most likely explain the non-random pattern of peaks on the Vb17 spectratype from this individual at this timepoint? SelectThe flu virus has killed the other T cells so they are no longer detectable The flu infection prevented TCRs of different lengths from being generated during T cell development The peaks represent selective expansion of T cells responding to the virus infection Only T cells with receptors using this Vb17 segment are capable of responding to the flu virus The prominent expansions a few viral-specific T cell clones makes it difficult to detect the background of random Vb17+T-cell receptors in the rest of the T cell population in this individual