Cloning Tetrahymena Gene THD9 Bridget Tabora & Caitlin Roam Fall 2009

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1 Cloning Tetrahymena Gene THD9 Bridget Tabora & Caitlin Roam Fall 2009 Abstract: HDACs are enzymes that modify histones and regulate gene expression. Sirtuins are a specific class of HDAC, and they relate to gene activation that associates with aging and life span. (Michan and Sinclair, 2007) SIRT6, a mammalian sirtuin, is associated with DNA damage. (Mostoslavsky, et al., 2006) A Tetrahymena homolog found for SIRT6 is THD9. Through the identification of this homolog, we were able to proceed with conducting experiments on this homolog. The DNA was isolated, determined to be pure, Polymerase Chain Reaction (PCR) was carried out on it, and visualization of the PCR product occurred from gel electrophoresis. We found there to only have products in two of the gdna samples and none of the cdna from the conditions we had. We then cloned the gdna and it went through an E. Coli transformation. We constructed a plasmid map and reaction enzyme digest design and found the best reaction enzyme to be NsiI for slicing our gene. We purified the plasmid and did a restriction enzyme digest with NsiI. Lastly, we ran six samples of our restriction enzyme digest on a gel, and the results showed that two of our samples contained the correct plasmid. The confirmation that it was the correct plasmid was evidence that we had successfully cloned our gene THD9. Other scientists can now use those samples for experimentation to better understand the role of SIRT6 in Tetrahymena thermophila, and later in humans. Introduction Chromatin is a template of genetic information. Nucleosomes represent a basic repeating unit of chromatin. In each nucleosome, DNA wraps around proteins called histones, and these histones have tails that can be modified because of specific enzymes that alter their original structure. The modifications in turn alter the nucleosome and therefore the chromatin structure

2 and the genetic information that it holds (Jenuwein et al., 2001). A histone deacetylase, or HDAC, is a particular enzyme that modifies histones and regulates gene expression through the suppression of transcription in certain genes. HDACs are organized into three classes, the third one being sirtuins (De Ruijter, et al., 2003). Sirtuins relate to gene activation that associates with aging and possibly extended life span. Sirtuins within the mammalian category have diverse functions. There are seven mammalian sirtuins, each with its own function (Michan and Sinclair, 2007). Specifically we are concentrating on the SIRT6 that promotes resistances to DNA damage and suppresses genomic instability (Mostoslavsky, et al., 2006). It suppresses genomic instability because it is involved in base excision repair (BER) and thus promotes resistance to DNA damage. SIRT6 maintains glucose homeostasis, which relates to metabolism. (Mostoslavsky, et al., 2006) In general, cellular metabolic rates may regulate aging through DNA repair pathways for genomic stability. This directly relates to SIRT6 s role in DNA repair and genomic stability and thus its relation to metabolism. (Rodgers and Puigserver, 2006) Also, studies have shown that the deficiency of SIRT6 results in genome instability through the DNA base excision repair pathway and leads to aging-associated degenerative phenotypes. (Rodgers and Puigserver, 2006) Through cloning the SIRT6 gene in Tetrahymena, experimentation of that gene may help to define better the role it has in DNA repair in the cell and overall a better understanding of its role in aging and life span. Materials and Methods Bioinformatics This lab was done previously as previously described in Lab 3 of the BMS110 Fall 2009 class (BMS110 Lab 3, Fall 2009). To acquire the information in the lab, the NCBI website, was used to find the amino acid sequence of SIRT6 gene.

3 The Tetrahymena homolog was found from the Tetrahymena Genome Database. The protein and nucleotide sequences of the Tetrahymena homolog, as well as the gene s start, stop and introns prediction were obtained also from the Tetrahymena Genome Database. The Tetrahymena homolog s coding sequence was compared with the genomic sequence at the MGAlignIt homepage: Lastly, again using the NCBI website helped to compare to protein sequences of the organism and the TGD predicted homologs as to find the e-value, identity, and positives. Genomic DNA Isolation using the Urea Lysis Buffer The procedure for this lab is described in Lab 4 of the BMS110 Fall 2009 class (BMS110 Lab 4, Fall 2009). DNA Isolation utilized the following solutions in various ways in order to prepare it to be quantified: Urea Lysis Buffer, phenol:chloroform:isoamyl alcohol, 5M NaCl, isopropyl alcohol, 70% ethanol, Tris-EDTA (TE) Buffer, and RNase A. Once the last reagent was added, we incubated the tube of genomic DNA at 37 C for 15 minutes. To quantify the amount of DNA in solution, spectrophotometric readings were taken at wavelengths of 260 nm and 280 nm. Acquiring the absorbance reading at 260 nm allowed the calculation of the concentration of nucleic acid in the DNA sample. Then we calculated the ratio of A 260 to A 280 by dividing the two. That ratio (values greater than 1.8) determined whether or not the DNA was a pure preparation. Polymerase Chain Reaction (PCR) Done as previously described in Lab 5 of the BMS110 Fall 2009 class (BMS110 Lab 5, Fall 2009). The PCR was carried out using the oligonucleotide primers that were designed using the bioinformatics lab data, the genomic DNA that was isolated using the Urea Lysis

4 Buffer, and a working stock of 1:10 dilution of cdna wildtype. The foreword and reverse primers were included in the reactions of both the gdna and cdna, along with reaction components. These reaction mixes were then pipetted into separate tubes of each DNA resulting in a total of six PCR tubes (3 containing DNA and 3 containing cdna). Those tubes were loaded into the thermocycler at the temperatures of 52.6 C, 54.3 C, and 58.0 C and stayed in it overnight. Gel Electrophoresis The method for this lab is described in Lab 6 of the BMS110 Fall 2009 class (BMS110 Lab 6, Fall 2009). In order to see the results of the PCR on the DNA, a prepared 1% agarose gel was poured into a casting tray with a comb and let to solidify. Once the gel was ready, the casting tray was taken out from its pouring position and the comb was removed. The DNA, as well as the kb ladder, was then inserted into the wells once it was mixed with dye. The gel then sat as the bands of DNA migrated down the gel. Once it got about ¾ of the way down, the gel was removed from its chamber, and the UV light box lit up the gel so the camera could capture a picture of the gel. The picture would show the size of the DNA if it showed up, but if it did not show up, the DNA would not have been under the optimal conditions for some reason or another. TOPO Cloning and E. Coli Transformation Lab 7 of the Fall 2009 BMS110 class (BMS110 Lab 7, Fall 2009) illustrates this lab step-by-step. We were able to skip to the third step because we did not have any primer dimers when we visualized our DNA in the gel electrophoresis. We were able to start right in on cloning and transformation and first set up the TOPO cloning reaction. The reaction components included 2 L of PCR product, salt solution, sterile water, and TOPO vector. (The PCR product

5 was diluted from 250 ng/ L to 2.5 ng/ L by taking 1 L of the PCR product and adding 9 L of water.) Once the whole reaction was mixed, it was incubated for 30 minutes at room temperature. The second part consisted of thawing the E. Coli cells, adding 2 L of the TOPO cloning reaction to the cells, incubating that on ice for 10 minutes, heat shocking the cells for 30 seconds at 42 C, and transferring the tube of cells to ice. Once they were in the ice, room temperature SOC medium was added, the tube was shaken for an hour, and finally the transformation was spread onto two separate pre-warmed plates (one containing 50 L and the other 200 L) to be incubated overnight in 37 C. The next day the plates were moved to a cold room with a temperature of 4 C. Construction of Plasmid Map and Restriction Enzyme Digestion Design Done as previously described in Lab 8 of the BMS110 class of Fall 2009 (BMS110 Lab 8, Fall 2009). For constructing the plasmid map, the Gene Construction Kit was utilized through inserting the gene (THD9) into the designated region and highlighting the introns. Once the plasmid map was constructed, restriction enzyme design was then made from it. All of the restriction enzymes were displayed so that one enzyme could be found and used to cut the gene we used NsiI. From the restriction enzyme design, a table and gel picture were printed to compare to the actual gel that will be run to visualize this DNA. Plasmid Purification and Restriction Enzyme Digest The procedure for this lab is described in Lab 10 of the BMS110 Fall 2009 class (BMS110 Lab 10, Fall 2009). The day before the lab we inoculated six 2 L of LB liquid media tubes with six transformant colonies off of the plate from the TOPO cloning and E. Coli Transformation lab. The day of the lab we actually began the plasmid isolation procedure. The first few steps of isolation include the addition of Sucrose Lysis Buffer and lysozyme solution

6 after decanting the supernatant, heating for a minute at 99 C, centrifuging for 15 minutes, and removing the pellet. After the discarding the pellet, the isolation continued through precipitating the DNA from the supernatant by adding 3M NaOAc and isopropanol, centrifuging again for 10 minutes, pouring off the supernatant, washing the plasmid pellet in 70% ethanol, centrifuging again for two minutes, drying the pellet, and finally resuspending the pellet in Tris-EDTA buffer. Once the isolation was complete, the confirmation of plasmid by restriction enzyme digest followed. The mixture of a cocktail containing 10X Buffer, NsiI, 100X BSA, and water, separating the cocktail into tubes, adding plasmid to each tube and mixing, and incubating the reactions at 37 C for an hour allowed NsiI to drop out a distinct set of bands different from the starting plasmid. We then took six samples of the restriction enzyme digest and ran them on a gel to visualize whether or not we had the correct plasmid through the band sizes of the particular sections that were dropped out compared to the predicted band sizes in the Construction of Plasmid Map and Restriction Enzyme Digestion Design Lab. Results Identification of the gene SIRT6 Tetrahymena homolog When determining whether or not there was a Tetrahymena homolog for SIRT6, three potential homologs were found: TTHERM_ (e-value of 2.3e-42), TTHERM_ (e-value of 8.7e-30), and TTHERM_ (e-value of 2e-27). The one we chose to proceed with in the lab was the second one: TTHERM_ (e-value of 8.7e- 30). We obtained the genomic and coding sequences of the homolog. For future references, the homolog s name was THD9 for simplification purposes. CACCCTCGAGAAAATTAATAAATTCATTAAAAAAGCGTTCTCTACTATTTAGGAAAG CGAATATAGCAAATACTTTTCTAAAAACTCTGTATACCATTAGAAAGATAAGTTCTT GTTTAATCCTAGACTTAAAGATACTTAAGAGCATTAAGATTCACCAGAATAAATAGA TACCAAAGTAAATCAGCTTATAGAATTACTTTAAAAAAGTAAGAATGCTGTGATATT

7 AACTGGAGCTGGAGTTAGTACAGCTTCAGGTATACCTGATTATAGAAGTGGTGCAA ATACTATTTTAAAAACAGGACCTGGTAAGTGGGAATTAGAGGAAAACAAAAAAAAA TTTTTGGAAGAAAAAGGTAAGCCATAAATAATATTAGCAATAAATGCATTCCCTTCT CCAACTCATATGGCAATTTCAAAACTATATAAAGAGAATTTGATAAAATCAGTAATT ACTTAAAATGTTGATAACTTGCATCATTAAAGTGGTATTCCAAGGAAAGATATTCAT GAGCTGCATGGGAATATTATTTCAGAGAGATGTGAAAAGTGTAATTATGTCCATTAT AGAGATTTTTATACTCGTTTAAAACATTTGAAATGGGGAGATCCACATAACACTGGA AGAATTTGCTAAAAAAATGGATGTGATGGATAATTGCACGACACTTTGGTATTTTTT GGAGAATCAGTATTACAAAATATAAAATAATCAGTAAAATTAATATGAGAATTAGG GATTTAAATATACTTTTATTTAATTAATTAATAAACTCTACTTATAAAGTAGAAAAA AGACTATACCTCCTTTATTTAATTTAATTTTTTTATTAATATTAAAAGGCATAAGAAT AAATTGAAAGTGCAGATTTGTGCATAGTGGTAGGAACGAGCTTGACAGTTTAATCTG CAGCTAGGTTAGTTTGGATTTCTTAGTAAAGAGGAATTCCTATAGTAATAATAAATC TACAAAAAACAAGCTATGATTCGAAGGCACTTAAAATTAATGGTTTATGTGAACCTA TCTTTGATTTGATTCTTAAAAAACTTAATTTCTAGCCCGATAAATTTACAGTTTAAAG GTAAATATTTGAATTTATTTTTTAAAATGTAAAGCTACTTGTGTCAAATTAATATAAT TTCGCTAAAAGAAATATTTAATCTTACTCATTAAATTTTTATTTAAAAAAAAGCTTTT TTACATTTTGCATTAAATTTTTTTAAATACATATTTTTGTTTTTTAATATGAAATTAAG GGATATTATATTGAGATTTTAGAAAACTGGGTTTTGCAGCTTTGATTTGTTTGCTGAC TGTGAAAGTTTTGATGGAAGCCATTTGAGTGCTATTAAATAATTAGAAATTTGGTAT AGAAAAGAAAATGGAAATTAATTATATAAAAGCTTTGAAGGTCATCCATACTATTTT TAAGCTGAAGATTCATTTAACATAGATCAAATTAAATTAAATTTTTTTTCTCACCATA AAGAAAAGAGTCACTTTATTTATGACTAGGAGGTATTATAAAATGGATAGCTATTTA GTATGAGCTCTCCAGGGTCATTAATTTTCCTTACATCTAAATTAACATATGAATTTTA AGATTAAAAAGATGAATGGATTGCTTAACATAAAATTGAAAGCTACAAATGACCTA GGCTCT Figure 1. Genomic Sequence of SIRT6 Tetrahymena thermophila (THD9). The alignment summary found the length of this genomic sequence to be 1647 bp. There are two introns within this gene as highlighted in gray and three exons flanking them. The yellow highlighted portion is the foreword primer, and the blue portion is the reverse primer. Genomic DNA Isolation and Visualization in Agarose Gel Electrophoresis We obtained Tetrehymena genomic DNA (THD9), and put it through the isolation procedure. The product of the genomic DNA isolation was two 100 L of purified DNA one having a dilution ratio of 1:100 and the other 1:200. The A 260 concentration of the 1:100 diluted DNA was g/ml, and its A 260 /A 280 value was 2.8. The A 260 concentration for the 1:200 diluted DNA was 2.13 g/ml, and it s A 260 /A 280 value was Our A 260 /A 280 values were greater than and 2.69 which indicate that the DNA was pure. The confirmation of the

8 purity allowed us to go through with the amplification of our THD9 gene through the use of PCR. The PCR was then visualized through performing electrophoresis and taking a picture of the gel. Figure 2. 1% Agarose Gel of PCR results. The gdna was run with annealing temperatures of 52.6 C (lane 3), 54.3 C (lane 4), and 58.0 C (lane 5). The cdna also ran at those same temperatures in lanes 7-9. The gdna produced product at temperatures 52.6 C and 58.0 C, whereas the cdna did not produce any product when the gel was run. The cdna that was used was a wildtype and had a 1:10 dilution. In lane 1, the kb ladder indicates that the size of the DNA is approximately 1.6 kb. Referring back to the size predicted for the genomic DNA in the bioinformatics lab (1647 bp) confirms that the prediction was accurate. TOPO Cloning and E. Coli Transformation We chose to clone the PCR product of gdna from lane 5 (Fig. 1). The PCR product was then colonized onto the TOPO vector. The plate with 200 L of transformation showed to have 141 colonies on it. The other plate with 50 L had 21 colonies dispersed on it.

9 Construction of Plasmid Map and Restriction Enzyme Digestion Design Once we observed our success of the growth of colonies from the TOPO cloning and E. Coli transformation, we constructed a map of all of the restriction enzymes in THD9. We had to add in both the foreword and reverse primers (Fig. 1) to the gene sequence so that we could construct the map. After inserting the sequence into the plasmid, we added each of the restriction enzymes. Once the restriction enzymes were found, we determined NsiI to be used to slice the THD9 gene because it was found the most throughout the plasmid. Figure 3. Plasmid map with the insertion of the THD9 gene with all of the restriction enzymes labeled. The green section of the map is the THD9 sequence, pink is the antibiotic resistance (KAN r ), yellow is the origin of replication, and the brown dashed is the section of the plasmid that is used to transfer the gene to other plasmids.

10 Figures 4. This figure represents how the restriction enzyme NsiI should slice the plasmid and thus THD9 gene. Both pictures show the band sizes that will result from the slicing. The gel picture is on the left, and the table is on the right. Plasmid Purification and Restriction Enzyme Digest After we found the predicted band sizes, we were able to actually purify the plasmid and put it through the restriction enzyme digest with NsiI. We put the samples of the mix into the gel and ran it. Through visualizing the gel, we confirmed that the 5 th and 6 th samples of the restriction enzyme digest in lanes 6 and 7 (Fig. 5) were the correct plasmid because the band sizes on the gel top band just over the 2.0 kb, the second band right 2.0 kb, and the third band way at the bottom were close to the predicted band sizes of the sections of the plasmid: 2137 bp, 1519 bp, and 266 bp (Fig. 4).

11 Figure % Agarose Gel of Restriction Enzyme Digest of pentr::thd9 done with NsiI. Lane 1 has 5 ml of kb ladder and lanes 2-7 contain the six samples of restriction enzyme digest. Lanes 6 and 7 contained the correct plasmid. Discussion The outcome of this project has resulted in acquiring the specific homolog of THD9 to use it in experimentation to better understand the SIRT6 gene in Tetrahymena thermophila. Once my partner and I isolated the THD9 gene, we were able to quantify the amount of DNA and determine its purity. The pureness of the DNA allowed us to carry out Polymerase Chain Reaction and put the mix into a thermocycler. The thermocycler prepared the gdna and cdna to go through agarose gel electrophoresis. We visualized only the gdna and found that the predicted size from the bioinformatics was similar to the size indicated in the gel about 1600 bp. We did not visualize the cdna because we might not have started it at the right time or because the DNA was not on at that particular time. We also did not visualize gdna in lane 4 (Fig. 2). Also visualized in the gel was the fact that no primer dimers were seen. This allowed us to be able to go straight into the cloning of the PCR product and the transformation of it into E. Coli. We used the gdna 3 sample for moving into cloning. It did not matter whether we used

12 gdna 1 or 3 (lanes 3 and 5, Fig. 2) because both had the same brightness of bands and thus the same concentration. After we transformed gdna 3 (THD9) and put the mix onto the plates, we checked on the plates the next day to see if they had grown any colonies. The success of colony growth indicated that we inserted the THD9 gene into the plasmid. We constructed a plasmid map and found the restriction enzyme NsiI to be the one to use for slicing the gene. We used NsiI and performed the plasmid purification and restriction enzyme digest and ran a gel on six samples of it. The gel showed that two of our six samples contained the correct plasmid because the band sizes were close to what was predicted in the restriction enzyme digest design. The only problems that occurred during these labs came from a lack of precision. When we visualized the gdna in the first Agarose Gel Electrophoresis, we realized how high the concentration was of the samples, and we had to dilute it to move on to the next step. This high concentration occurred probably because of pipetting over the correct amount needed. Also reasons we did not visualize the gdna in lane 4 (Fig. 2) could have been because we messed up the sampling that entered the well, either through incorrect pipetting or a contamination occurring. Also, we did not visualize in the restriction enzyme digest gel all six of the samples containing the correct plasmid. Reasons for this occurring include inaccurate pipetting, contamination, or simply a failure at fully digesting every sample. Ways to fix these problems would to be more careful when pipetting and transferring certain tubes between places and solutions to avoid contamination. The successful results of these consecutive labs show that the final results may lead to other scientists further experimentation. Future experiments may be done on the THD9 gene as to further knowledge of THD9 specifically in Tetrahymena, and to generally understand SIRT6 better. Specifically, a lot can be done with the plasmid to further understand THD9. Scientists

13 can exchange the plasmid region with our gene THD9 with other plasmids for studying it. They can take the plasmid and put it under certain conditions to see how it reacts and functions. The gene could be studied as to how it reacts with different agents that are placed in the plasmid. Also to better understand THD9 s role within the cell, a highlighting mechanism could be used when the gene is inserted into a cell to see where it goes within the cell. An over expression of the gene could help the understanding of what happens to the cell. These various experiments can lead to a better understanding of SIRT6 s role in the cell and in the body of Tetrahymena and similarly humans. Understanding SIRT6 in humans will lead to the understanding of the process of aging and the determination of life span. References BMS110-Fa09-999: Concepts in Biomedical Sciences. (Fall 2009). Lab 3: Bioinformatics & Molecular Computational Tools. BMS110-Fa09-999: Concepts in Biomedical Sciences. (Fall 2009). Lab 4: Genomic DNA Isolation. BMS110-Fa09-999: Concepts in Biomedical Sciences. (Fall 2009). Lab 5: Polymerase Chain Reaction. BMS110-Fa09-999: Concepts in Biomedical Sciences. (Fall 2009). Lab 6: Agarose Gel Electrophoresis. BMS110-Fa09-999: Concepts in Biomedical Sciences. (Fall 2009). Lab 7: Cloning PCR Product. BMS110-Fa09-999: Concepts in Biomedical Sciences. (Fall 2009). Lab 8: Plasmid Map Construction & Reaction Enzyme Design. BMS110-Fa09-999: Concepts in Biomedical Sciences. (Fall 2009). Lab 10: Plasmid Purification and Restriction Enzyme Digest. De Ruijter, A. J. M., Van Gennip, A. H., Caron, H. N., Kemp, Stephan, and Van Kuilenburg, Andre B. P. (2003) Histone deacetylases (HDACs): characterization of the classical HDAC family. Biochem J. 370, Jenuwein, Thomas, and Allis, D. C. (2001) Translating the Histone Code. Science. 293, Michan, Shaday and Sinclair, David. (2007) Sirtuins in mammals: insights into their biological function. Biochem J. 404, Mostoslavsky, Raul, Chua, K. F., Lombard, D. B. Alt, F. W. (2006) Genomic Instability and Aging-like Phenotype in the Absence of Mammalian SIRT6. Cell. 124, Rodgers, Joseph T. and Puigserver, Pere. (2006) Certainly can t live without this: SIRT6. Cell Metabolism. 3,