Field of research: Biology
Lab: Diego Loayza
School: Manhattan Hunter Science High School
Year: 10th/ 11th Grade
Probing telomere regulation and elongation
Research in Professor Loayza's lab focused on the replication/elongation, and regulation of the length of both G, and C-strand portions of the telomere.
The telomeres are TTAGGG repeats at the end portion of chromosomes, they do not code for any protiens. Their purpose is to protect the integrity of the DNA by keeping it from being "lost" during , so that the DNA itself would stay intact. (its the nature of to only replicate the middle portion of linear strands of DNA, which keeps the end portions from being replicated, and will be lost.) The telomeres are located at the end portion for this purpose, so that they themsleves will be "lost" instead of the DNA. In normal cells where division isn't constant, the length of the telomeres will decrease over time until all of it has been "lost", at this point the cell will stop dividing. In cells where division is constant (like , or immune cells) there is an enzyme which adds on to the telemere after replication so that it will never diminish in length over time. And a protein complex which inhibits this enzyme to keep the length of the telemere constant. This enzyme is called Telomerase, and this complex, Shelterin. We know for this to occur only on the top G-strand of the telemere.
The G strand being the top strand consists of the TTAGGG repeats (make up the telemeres) and consists of more of the nucleotide Guanine or G than the lower strand. The lower strand is rich in C repeats so it is called the C-strand. Researchers know much about how the G-strand in cancer cells (or consistently dividing immune cells) replicate and are regulated: the enzyme telomerase adds TTAGGG repeats to the G-strand replenishing amounts that were lost during cell division, and the Shelterin Complex, a group of proteins (TRF1, Tin1, TRF2, and Pot1) all work to gether to attach themselves directly to the telomere to inhibit telomerase from adding to the G-strand. This will keep the G-strand at a constant length with in these cells. The question the professor and the graduate students working in this lab decided to address was how the C-strand was replicated.
We started our research with the knowledge that the two proteins found in yeast, Fen1 and PolA2, were involved with the replication of the C-strand in yeast cells. Being so that yeast proteins are homologous to humans, this may be the same in human cells. In order to find out if this was true, we conducted several procedures to determine if this was true. The final procedure, a Chromatin IP would allow us to see first ahnd if these proteins were involved in the replication of the C-strand of the telomeres.
We performed four procedures to clone the PolA2 and Fen1. A ligation to create plasmids with the inserts PolA2, and Fen, then a transformation to incorporate these plasmids with PolA2 and Fen1 inserts into E.coli bacteria. Then an innoculation where we picked 6 colonies in culture tubes. Then performed a Miniprep to purify the plasmids.After these procedures were done, (clone the inserts PolA2 and Fen1 and prurify them) we performed a digestion to check to make sure the plasmids contained the correct inserts, and then performed a to visualize these results. Then we did a western blot see if our proteins PolA 2 and Fen1 were expressed in our human cells. Then once we saw this was true, we performed the Chromatim IP to be able to see if these proteins PolA2 and FEn1 would interact directly at the C-strand of the telemeres. Since we didn't receive our equiptment (Dotblotter) in time to finish our Chromatin IP and get our results, we will continue in the fall where we will be able to conclude whether PolA2 and Fen1 are involved in C-strand replication of the telomeres.
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