UBC13 Cloning from Tetrahymena Thermophila. Jessie Meyer. Fall 2009

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1 UBC13 Cloning from Tetrahymena Thermophila Jessie Meyer Fall 2009 Abstract There have been times that DNA damage has occurred during cell replication. However, To counter this threat and maintain genome integrity, eukaryotic cells employ three main strategies: DNA repair pathways which directly reverse DNA damage, cell cycle checkpoints which allow time to repair the damage prior to replication, and DNA damage tolerance (DDT), which is a method of bypassing DNA damage lesions during the DNA replication phase of the cell cycle (Carlile et al., 2009.) UBC13 is a gene needed for some of these processes, especially seeing as how subsequent polyubiquitination of PCNA at the same K164 residue by Mms2- Ubc13-Rad5 promotes error-free lesion bypass (Anderson et al., 2008). Because of this, we have taken interest in UBC13 in the hopes that through research, we can find a method where we can repair DNA after it has been damaged, such as in cases of cancer. In order to promote the study of this gene, we will clone UBC13 from the ciliate Tetrahymena thermophila into a plasmid that can then be used by other researchers in their study of topics such as DNA repair and cancer. During this procedure, we will first find a UBC13 Tetrahymena homolog to work with. We will then isolate the DNA, design reactions to amplify the gene (PCR), and after determining the purity, clone the gene using a TOPO vector and transform it into E. coli in order to propagate and store the gene for further research. Introduction

2 Ubiquitin-conjugating enzymes (Ubc) are essential in a process called ubiquitination. "Ubiquitination is a post-translational modification carried out by a set of three enzymes, E1, E2 and E3. Ubiquitin is first activated by ubiquitin-activating enzyme E1, before being transferred to its active site, the amino acid cystein The ubiquitin molecule is then passed on to the second enzyme of the complex, E2 (ubiquitin-conjugating enzyme), before reaching the final enzyme, E3, the ubiquitin protein ligase, which recognizes, binds the target substrate and labels it with the ubiquitin (Combet, 2008). In most cases, the protein targeted is taken to the proteasome, where it gets degraded or dissembled, which is an important part of the human immune system. However, UBC13 is different in that it is heavily involved in DNA damage repair, as opposed to protein degradation. Though UBC13 is crucial to DNA repair, it cannot act alone. Ubc13 requires the presence of a Ubc variant (Uev) for polyubiquitination (Anderson et al., 2005). Because of this, UBC13 can have many different roles in DNA repair, depending on the Ubc variant it interacts with. More often than not, UBC13 s partners in DNA damage tolerance (DDT) are the genes Mms2, a member of the Uev gene family, and Rad5. These enzymes, along with Rad6-Rad18, work together to perform DDT, which are significant in maintaining genomic stability (Anderson, 2008). Methods and Procedures Bioinformatics (Refer to Lab 3 Handout) Before we got started cloning any DNA, we first had to pick a homo-sapien gene, and to find a Tetrahymena homolog of that gene. (Tetrahymena is a simpler organism than homosapiens, so is easier to study and operate on.) To do this, we used bioinformatics and molecular computational tools such as those found at

3 and First, we went to the NCBI homepage to find a human protein (UBC13), and then went to the Tetrayhymena Genome Database (TGD) to look for a tetrahymena homolog, using TBLASTN. After gathering information on the homolog, we compare the genomic and the coding sequence to that of the human gene. Tetrahymena Genomic DNA Isolation (Refer to Lab 4 Handout) After gathering the cells into a pellet by centrifugation and removing supernatant, we added 700µL of Urea Lysis Buffer and 600µL of Phenol:Chloroform:Isoamyl alcohol to phenolextract the lysate. Then we removed the phenol-chloroform mix from the top buffer layer, and we repeated the procedure starting from the phenol extraction. Afterwards, we added 150µL of 5M NaCl to help reduce the carbohydrate content. In order to precipitate the DNA we then added 700µL of Isopropyl alcohol, and let the solution incubate for 10 minutes at room temperature, centrifuging it for 10 minutes at max speed after incubation. After removing supernatant, we added 500µL of 70% ethanol. Once again, we centrifuged the tubes, this time for 3 minutes and removed supernatant, letting the left over pellet air dry. When it s dry, 50µL of TE Buffer is added and mixed, followed by the RNase A, and then it is incubated at 37ºC for 15 minutes. Once incubated, we created dilutions of DNA with a 1:100 and 1:200 ratio, and used a spectrophomtometer to measure the quantification of genomic DNA. Polymerase Chain Reaction (Refer to Lab Handout 5) First, we needed to rehydrate the lyophilized primers given to us. To do this, we calculated how much water to put in to create our stock with a final concentration of 200µM.

4 The TF primer for UBC13 required 164µL of water, while the TR primer required 184µL of water. Once rehydrated, we made a dilution of the primers using 180µL of water and 20µL of primer solution for each primer. Then we set up the PCR reactions by mixing 3µL of gdna, 1.5µL of Phusion polymerase, 30µL of GC Buffter, 30µL of Betaine, 3µL of dntp s, 3µL of TF primer, 3µL of TR primer, and 76.5µLwater (making sure to put the polymerase in last), repeating the procedure for cdna. Reactions were kept on ice until taken to thermocycler, which was set to heat reactions for 1 minute at 98ºC, run through 34 cycles of denaturation, annealing, and polymerase extension, 10 minutes at 72ºC, and then hold at 4ºC. The annealing temperatures used for UBC13 were 55.5ºC, 58ºC, and 60ºC. Agarose Gel Electrophoresis (Refer to Lab 6 Handout) The next step in the lab is to separate, identify, and purify the DNA fragments, but first, we must prepare the agarose gel. To create the gel, we mixed.75 grams of agarose with 50µL of 1X TAE, heating it in the microwave for 1 minute, and then for 4-5 sets of 10 second increments. When all agarose was dissolved, we let the flask cool until safe to touch. Once the solution was cooled, we added ethidium bromide, swirling to mix, and filled the casting tray with the mix until the comb teeth here submerged halfway. Next, we wait for the gel to solidify, and then we removed the comb. Next we prepare the samples by mixing all samples (six total; three of gdna at annealing temperatures, three of cdna at annealing temperatures) with dye, and then inserting into the gel. After the gel has been fully prepared, we set the electrophoresis power supply to run at a constant 120 volts for 90 minutes. Results shown below.

5 TOPO Cloning and E. Coli Transformation (Refer to Lab 7 Handout) After seeing the result of the electrophoresis, it was determined that the UBC13 did not need further purification. However, the DNA was in need of diluting, so we made a solution using 9µL of water and 1µL of DNA to end up with a solution with a 5ng concentration. After mixing 1µL of the PCR product, 1µL of salt solution, 3µL of water, and 1 µl of TOPO Vector, we let the solution incubate for 10 minutes at room temperature. Then we mixed 2µL of it with E. coli and let it incubate again for 10 minutes on ice, then heat shock the cells for 30 sec. at 42ºC. Once transferred back to ice, 250µL of SOC Medium was added, then put it shaking incubator at 200rpm for 1 hour at 37ºC. It was then spread on a pre-warmed plate of Kanamycin using glass beads. It was left to incubate overnight at 37ºC, then put in cold room the next day. Plasmid Map and Restriction Enzyme Digestion Design (Refer to Lab 8 Handout) In this lab, we used a Gene Construction Kit program to mark specific regions of a plasmid that interests us and search the DNA sequence for specific markers like restriction enzyme sites. See results below. Plasmid Purification and Restriction Enzyme Digest (Refer to Lab 10 Handout) The next step was to isolate the plasmid. Before that, however, we needed to inoculate six cultures of LB liquid media tubes containing 50µg/mL Kanamycin with six transformant colonies from the TOPO cloning lab. Once completed, the tubes were placed in a shaking incubator set at 37 C. The next day, the cultures were transferred into microcentrifuge tubes and centrifuged for 2 minutes on maximum speed. After removing supernatant, 350µL of Sucrose Lysis Buffer was added, and the pellets were resuspended. Then we added 25µL of lysozyme

6 solution, thoroughly mixing it, and incubated the solutions at room temperature for 5 minutes. When incubation was complete, the microcentrifuge tubes were heated for 1 minute at 99 C and then centrifuged for 15 minutes at maximum speed. Then the pellets were removed, and 40µL of 3M NaOAc and 220µL of isopropanol were added and mixed with the supernatant. They were again left to incubate at room temperature for 5 minutes, and then centrifuged for 10 minutes at maximum speed. After that, the supernatant was poured off, and the remaining plasmid pellets were washed in 1000µL of 70% ethanol, and centrifuged once more for 2 minutes at maximum speed. The ethanol was then discarded, and the pellets air dried before we resuspended them in 50µL Tris-EDTA Buffer. In order to confirm that the plasmid was indeed isolated, we mixed 3µL of each plasmid with a restriction enzyme cocktail, which, for UBC13, consisted of 2µL of 10 X Buffer 2,.5µL of AvrII,.5µL of NheI,.2µL of 100X BSA, and 13.8µL of water. The solutions were then incubated at 37 C for at least 1 hour. When finished incubating, each sample was mixed with 10X sample dye and ran on an agarose gel (see Agarose Gel Electorphoresis [lab 6]). Results are shown below. Results The Tetrahymena homolog chosen to clone was TTHERM_ , because that gene was the most similar to UBC13. In Figure 1a, the protein sequence for both the UBC13 and the Tetrahymena homolog of UBC13 are listed, and one can easily compare the two genes. However, there are some parts in the Tetrahymena homolog that were untranslatable (introns). The placement of the introns in the gene can be seen in Figure 1b. Although there are some

7 differences between the homolog and the original UBC13 gene, the homolog was found fit to clone to be used for further research. Once that was determined, the DNA of the homolog was isolated, and once the procedure was complete, the DNA was measured to see how pure it was, which ended up being (This is a very high purity number, considering that a purity of 1.8 would suffice.) Once the DNA was deemed pure enough, we were ready to go to the next step, which was to stimulate a Polymerase Chain Reaction. Polymerase Chain Reaction is typically used to amplify a portion of DNA. The portion of the gene we wanted to amplify was dictated by the oligo-nucleotide forward and reverse primers we received. Once the PCR reactions were set up for both the genomic DNA (gdna) and the coding DNA (cdna), the reactions were all put in the thermocycler, which was programmed to denature the DNA, anneal the primer to the DNA, and extend the primer (DNA synthesis). When that was complete, we ran an Agarose Gel Electrophoresis, which is shown in Figure 2, so that we could see if the DNA really had been isolated or not. As can be seen from the figure, the cdna was full of primer dymers, and didn t match its predicted gene size (450), which was determined during the PCR lab. The gdna, however, did match its predicted gene sized of 942, and had no primer dymers, although there was some by-product showing up below the gdna. Because the bands that show the gdna were so bright, it was determined that the DNA needed no further purification and was ready to clone into E. coli. On the first attempt, only four colonies grew on the 200µL plate, and none on the other. The second attempt was more

8 successful, and there were at least 100 colonies of cells on the plate, and they were then incubated at 4 C until we were ready for the next step. Before moving on, however, we created a plasmid map and a restriction enzyme digestion design, which is shown in Figure 3. From that, we determined that restriction enzymes that should be used were AvrII and NheI, and that in the next agarose gel, there should be three bands showing for each plasmid sample, if done correctly: one at 266, one at 637, and one at Now that we knew what our next results were supposed to look like, it was time to see what they actually were. After isolating the plasmid, and confirming the isolation using restriction enzyme digestion, we ran another agarose gel electrophoresis, the results of which can be seen in Figure 4. As can be seen from the picture, although the band at 637 is missing in all the plasmid samples, and there is some by-product, the other two bands that are supposed to be there are where they need to be, meaning that the plasmid was successfully cloned into the E. coli, so that now the UBC13 homolog is ready to be used for further research. Conclusion There have been a few instances during the lab where mistakes have been made, or so I believe. Specifically during lab 4, regarding the ratio of the dilutions. The results of the electrophoresis show that the concentration of DNA was very strong, so perhaps the DNA was not diluted correctly, but that can t be known, because some of the data, such as the spectrophotometer readings and the purity calculations, was lost when my lab partner dropped the class. And after the TOPO cloning, I only had 4 colonies of DNA at first. Because of the small number, the procedure was redone and the following result significantly improved; there

9 were now over 100 colonies. It should also be noted that during the cloning portion of the lab, I did have to restart a couple times because of mis-measuring or using reagents before they were to be used. However, at this point, everything seems to be going smooth. This procedure has definitely expanded my knowledge of genes, how they work, how they are affected by reagents, and how to use technology in genetic research. I think that this lab could ve been done much smoother if I had more background in doing similar labs, more experience using the materials, and if I had a broader scientific vocabulary before starting, rather than learning it as we go. However, now that this lab procedure is coming to a close, I feel more confident and knowledgeable, and look forward to doing similar labs sometime in the future. References Carlile, Candice M., Cecile M. Pickart, Michael J. Matunis, and Robert E. Cohen. "Synthesis of Free and PCNA-Bound Polyumbiquitin Chains by the Ring E3 Ubiquitin Ligase RAD5." (2009). Anderson, Parker L., Fang Xu, and Wei Xiao. "Eukaryotic DNA Damage Tolerance and Translesion Synthesis through Covalent Modifications if PCNA." (2008). Andersen, P. L., H. Zhou, L. Pastushok, T. Moraes, S. McKenna, B. Ziola, M. J. Ellison, V. M. Dixit, W. Xiao Distinct regulation of Ubc13 functions by the two ubiquitin-conjugating enzyme variants MMS2 and Uev1A. The Journal of Cell Biology

10 Combet, Emilie. "Ubiquitination: labelling the proteins." Society for Experimental Biology Ed. Sigmer Technologies. N.p., Mar Web. 1 Dec < Figures

11 Figure 1a Bioinformatics Figure 1a lists the protein sequence for UBC13 gene, followed by the protein sequence of the UBC13 homolog found in Tetrahymena thermophila. Beneath that are the coding and genome sequences for the homolog.

12 Figure 1b Figure 2 shows the location of the introns and exons. Introns are the parts of the Tetrahymena homolog that are either non-translatable or not found in the original gene, while exons are the parts of the gene that match up with UBC13. Figure 2 Agarose Gel: Polymerase Chain Reaction

13 Figure 2 is a picture of an agarose gel. This one shows the results of the Polymerase Chain Reaction lab, as well as the purity of the DNA (indicated by the bright bands). The temperatures at the top of columns 3-5 and 7-9 are the different annealing temperatures for the UBC13 primers. Columns 7-8 contain samples of cdna, 3-5 contain gdna, and the first column is the kb Ladder, or the kilobase ladder, which measures the size of the gene based on the number of nucleotides. The numbers on the far left of the figure label the sizes. (.5=500 nucleotides, 1=1000 nucleotides, etc.)

14 Figure 3 Plasmid Map & Restriction Enzyme Digestion Design A B C Figure 3a is a picture of an electronic gel, showing what the next gel is supposed to look like. According to the gel, the result of the next gel should have a band between.5 and 1, and between 2 and 3 on the kb ladder. 3b tells where exactly the bands are supposed to be: 266, 637, and c is a plasmid map, showing different sections of the

15 plasmid. The green section is the gene being studied (UBC13), and the pink shows the Kanamycin. The enzymes chosen to use as the digestion enzymes were AvrII and NheI, because they fully envelope the gene. Figure 4 Agarose Gel: Plasmid Purification & Restriction Enzyme Digest Figure 4 is another picture of an agarose gel, but this time it is showing the purity of the plasmid. Once again, the kb Ladder is in column 1, and the samples are in columns 3-5 and 7-9, while columns 2 and 6 are empty. If compared with Figure 3, one will see that the 266 bands and the 2140 bands are there for each sample, though none of the plasmids have the 637 band, all of them have a band above 10 on the kb Ladder, and a couple have an extra band between 1 and 1.5.