Discovery Talk Questions and Answers: Telomeric DNA and Telomerase

Elizabeth Blackburn

Assessments created by Dr. Gabrielle Miller-Messner

Questions

1. Which of these statements define to the ‘end-replication’ problem described by Dr. Blackburn in her talk? (Select all that apply)
   1. Although telomeric sequences are highly conserved, the number of repeats is variable
   2. DNA polymerase is unable to replicate the nucleotides at the very end of chromosomes
   3. The action of telomerase renders cells immortal, thus disturbing the signal to end replication
   4. None of the above
2. During DNA replication, the addition of nucleotides by DNA polymerase requires a DNA template. What acts as a template during the addition of a telomeric repeat?
   1. DNA
   2. An amino acid sequence
   3. RNA
   4. RNA polymerase
   5. None of the above
3. The Central Dogma states that the flow of information goes from _________ to _________.  The addition of telomeres represents an exception to the Central Dogma, because information flows from _________ to _________.
   1. DNA, RNA; proteins, DNA
   2. DNA, proteins; RNA, DNA
   3. DNA, RNA; RNA, DNA
   4. RNA, DNA; proteins, DNA
   5. None of the above
4. In their 1985 paper, Dr. Greider and Dr. Blackburn mixed Tetrahymena cell extracts with synthetic DNA oligonucelotides. What evidence suggested that telomeric repeats were being added to the synthetic oligonucleotides? (Select all that apply)In the paper, the authors suggest that “telomere replication involves a terminal transferase-like activity.” Dr. Blackburn and colleagues hypothesized that telomeric sequences were added de novo to the ends of chromosomes, rather than through a known DNA replication mechanism.
   1. Autoradiograms showed longer reaction products only when a-type DNA polymerase was present
   2. Autoradiograms showed longer reaction products only when both dGTP and dTTP were present.
   3. Autoradiograms showed longer reaction products exhibiting a pattern of 6 base pair repeats
   4. None of the above
5. Name two pieces of evidence from the literature that may have influenced the authors’ thinking about the mechanism of telomere addition.
6. At the beginning of her talk, Dr. Blackburn presents a theoretical representation of the change in chromosome length with successive cell divisions (0:43). The graph depicts what would happen if DNA replication alone controlled the length of chromosomes.In which experiment, described in her talk, did Dr. Blackburn find similar results? Explain what these particular results tells us about the significance of telomeres in the life cycle of a cell.
7. In her talk, Dr. Blackburn describes the evidence that helped her and her team single out telomerase – among other enzymes – as responsible for telomere addition.
   In the paper, Figure 2a, the lanes represent the following mixtures: Lane 3: dTTP + dATP + dCTP + radiolabeled dGTP + Tetrahymena extract
   1. Describe this evidence.
   2. Explain how this evidence implied a mechanism for telomerase function.
8. In the paper, Figure 2a, the lanes represent the following mixtures:
   Lane 3: dTTP + dATP + dCTP + radiolabeled dGTP + Tetrahymena extract
   Lane 4:  dGTP + dATP + dCTP + radiolabeled dTTP + Tetrahymena extract
   Lane 6: dTTP + radiolabeled dGTP + Tetrahymena extract

   If telomere (TTGGGG) replication only requires dTTP and dGTP along with the terminal transferase enzyme, why do we see a 6-base repeat pattern in lanes 3 and 6, but not in lane 4?

9. In the paper, the authors write: “the ability of Oxytricha and Tetrahymena to stabilize linear plasmids in yeast argues that many features of telomere structure and function are highly conserved among lower eukaryotes.”Describe another basic genetic mechanism that is conserved across eukaryotes.
10. Dr. Blackburn concludes her talk with the following message: “What I take home as a message from this is that one really wants to understand how biology works by working at it in the most, sort of, curiosity-driven, question-driven ways, and not necessarily trying to ask the question of, some of application, but just simply trying to understand how things work, because I think we won’t predict necessarily what the ramifications of that would be.” (19:08) Discuss one unforeseen application of Blackburn’s work on the fundamental mechanisms of telomerase. From a personal life experience, provide an example of a technology that is ultimately the result of basic research.

 

Answers

1. b is correct. Answer a is true, but does not define the end-replication problem.In her talk (1:00), Dr. Blackburn explains: “It was also known around the 1970s that the DNA replication machinery of the cell wasn’t able to completely copy all the way out to the very ends of the chromosome. And so the prediction of what might happen every time a DNA replicated in order for a cell to divide would look something like what’s depicted in this slide here, which would be that the chromosomal DNA end region would get shorter and shorter and shorter until eventually the cells would have such short chromosomes that they would be perhaps missing something from their ends and then not able to divide anymore …”
2. c is correct. In the talk (12:14), Dr. Blackburn explains that “One of the things we found out not too long after first finding this activity was that the enzyme actually had within it an essential ribonucleic acid component (…) Within this RNA there was a sequence, shown in blue here, which was the exact complement of the TTGGGG sequence that we found was being synthesized in the test tube. So, with such a very strong hint, we decided this really was the enzyme. And now the challenge was to find out if this really was behaving in cells as it was behaving in the test tube. Because what we were finding in the test tube was that a DNA such as a mimic of a chromosomal end, shown in black here, could be elongated by addition of nucleotides and copying the template- this was our model –  and that would now make the chromosomal end longer. Was that really happening in cells? (…) if this templating mechanism, as it’s called, were really true, then if we changed a particular C into a G then instead of being copied, by templated synthesis, into a G it would now be copied into a C, or, for example, if we changed one of those As into a G it would now be copied into a C instead of into its normal T. And in fact that was what we were able to show…”
3. c is correct. In the talk (17:34), Dr. Blackburn explains that “I think in the back of my head I was always aware that nature tends to be very conserved in many of its most fundamental molecular mechanisms, as had been amply learned from the Central Dogma of DNA information going to RNA going to protein. Although, interestingly, this was a case in which a perfectly normal cellular enzyme was breaking the rule and RNA was being copied into DNA. Something that people before had been thinking was only the kind of thing that certain viruses and retroelements did but they didn’t realize until, we found telomerase, that in fact copying RNA into DNA can be a normal part of a normal cell’s life.”
4.  b and c are correct.Regarding, answer c, in her talk, Dr. Blackburn describes the autoradiogram (11:17) as follows: “[The reaction products] start with just the very short DNA oligonucleotide, we call it a primer, and then the DNAs get longer and longer and longer and you can see that there is this lovely repeating pattern and that pattern is the 6 base repeats of the TTGGGG motif being repeated over and over.”Regarding, answers b and c, on page 407 of the paper, the authors explain: “The autoradiograms in Fig 2 show that an activity in extracts made from cells during macronuclear development elongated the unlabeled input (TTGGGG)4  oligomer by the addition of DNA with a 6 base repeating pattern (…) In Figure 2A each of the four a-32P-dNTPs was added in turn in the presence of the other three dNTPs, or different combinations of only two nucleotides were added at a time. The 6 base repeat pattern was seen only with a-32P-dGTP and three unlabeled dNTPs(lane 3) or a-32P-dGTP plus unlabeled dTTP (lane 8).”

   Answer a is incorrect, as demonstrated on page 408 of the paper: “We have shown that 10-20 ug/mL of aphidicolin strongly inhibits a-type DNA polymerase activity in our crude Tetrahymena extracts … The repeat synthesis was completely insensitive to 20 ug/mL aphidicolin (Figure 2B, lanes 1,3, and 5). These experiments indicate that the addition activity is independent of both endogenous DNA and endogenous DNA polymerase a.”

5. Below are two pieces of evidence from the literature that may have influenced the authors’ thinking about the mechanism of telomere addition:1) Trypanosomes and Tetrahymena telomeres exhibit increases in length.  Such increases in length would not be expected from DNA polymerase, because DNA polymerase requires a template and a primer, leading to shortening of chromosomes, not lengthening. On page 405 of the paper, the authors explain: “Together, these findings show that telomeres are dynamic structures capable of a net increase in length. However, DNA polymerases function in the 5’ to 3’ direction and require a template and primer for DNA synthesis, which means that the ends of chromosomal DNA should become progressively shortened over the course of many rounds of DNA replication (Cavalier-Smith, 1974). The fact that chromosome length is maintained, and even increased, strongly suggests that the replication of telomeric ends is not accomplished solely by the action of conventional DNA replication enzymes.”2) When Tetrahymena telomeres are appended to the end of linear DNA molecules in yeast, yeast telomeres are subsequently added to the Tetrahymena telomeres. This suggests that the addition of the yeast telomeres is untemplated. On page 405 of the paper, the authors explain: “Yeast repeats are added onto the ends of Tetra- hymena telomeres after maintenance and replication of a linear plasmid in yeast”
6.  In her talk (14:44), Dr. Blackburn describes the manipulation of the RNA template component of telomerase, and the fact that, in some cases, it resulted in telomerase inactivation. Upon telomerase inactivation “the telomeres started getting shorter and shorter and then over the course of about 20-25 cell divisions they progressively got shorter and shorter, and then the cells ceased to divide, altogether.” The results of telomerase inactivation imply that telomerase plays an important role in preventing cell senescence.
7. a. The two “hints” are:(1)   The enzyme’s sensitivity to ribonuclease; and(2)   The fact that it contained a complementary sequence to the TTGGGG telomere sequence.

   In her talk (12:14), Dr. Blackburn explains that: “One of the things we found out, not too long after first finding this activity, was that the enzyme actually had within it an essential ribonucleic acid component. And what this does is depicted here. So it had a ribonucleic acid component in it, which made the enzyme very sensitive to the enzyme ribonuclease, not what you’d expect if it were just a protein enzyme. And within this RNA there was a sequence, shown in blue here, which was the exact complement of the TTGGGG sequence that we found was being synthesized in the test tube. So, with such a very strong hint, we decided this really was the enzyme.”

   b. (1)   Sensitivity to ribonuclease enabled the authors to rule out other enzymes in the Tetrahymena cell extracts, such as DNA polymerase.

   (2)   The probability of an enzyme carrying the exact RNA complement of the TTGGGG sequence merely by chance is low.

   Thus the RNA component sequence suggested a templating mechanism.

8. The results in figure 3B suggest that dATP and dCTP compete with dTTP.On page 409 of the paper, the authors explain: “The minimum concentration of dGTP needed to achieve elongation is less than 0.1 uM (data not shown), whereas dTTP must be present at concentrations above 1uM before significant elongation is seen (Figure 3B).”
9. Sample Answer: The genetic code is a fundamental aspect of genetics that is conserved across all life.
10. Sample Answer:- Continued work on telomerase indicates that a high percentage of cancer cell lines show increased telomerase activity. Notably, some telomerase variants in humans are associated with increased risk of certain cancers. [1]- I had an MRI of my brain, because I get migraines with aura. The MRI was developed with a basic theoretical understanding of nuclear magnetic resonance (NMR), the subject of several basic physics research programs and several Nobel Prizes in the first half of the 20th century.[2]

    [1] “The 2009 Nobel Prize in Physiology or Medicine – Advanced Information”. Nobelprize.org. 2 May 2013 http://www.nobelprize.org/nobel_prizes/medicine/laureates/2009/advanced.html

    [2] http://en.wikipedia.org/wiki/Nuclear_magnetic_resonance#History

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