Ty1 Retrotransposition Frequency Assay Using a Chromosomal Ty1his3AI or Ty1kanMXAI Element   

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Original research article

A brief version of this protocol appeared in:
PLOS Genetics
Feb 2018

Abstract

Here I describe a simple genetic assay to determine the frequency of retrotransposition of a single chromosomal Ty1 element that is marked with the retrotransposition indicator gene, his3AI or kanMXAI. The assay is used to determine the effect of mutations or environmental conditions on the frequency of Ty1 retrotransposition in the yeast, Saccharomyces cerevisiae.

Keywords: Ty1, Retrotransposon, Saccharomyces cerevisiae, Retrotransposition Indicator Gene, RIG, his3AI

Background

Ty1 is a long terminal repeat (LTR) retrotransposon that is structurally and evolutionarily related to retroviruses. Retrotransposition of Ty1 occurs when Ty1 RNA is reverse transcribed within cytoplasmic virus-like particles, resulting in synthesis of a cDNA that is transported back to the nucleus and integrated into the genome of its host (Boeke et al., 1985; Garfinkel et al., 1985). The integrated Ty1 element consists two LTRs in the same orientation that flank two open reading frames: GAG, which encodes a structural protein that forms the virus-like particle and binds Ty1 RNA, and POL, which encodes three enzymatic proteins- protease, reverse transcriptase and integrase (Figure 1). Ty1 elements are the most active and abundantly transcribed of all five families of LTR-retrotransposons in S. cerevisiae. Ty1 has been shown to be regulated by hundreds of genes, several different environmental conditions and various DNA damaging agents (Curcio et al., 2015). To measure the relative frequencies of Ty1 retrotransposition in different genetic backgrounds and under different environmental conditions, a chromosomal Ty1 element under the control of the native promoter was marked with a retrotransposition indicator gene (Figure 1). A retrotransposition indicator gene is a selectable marker gene whose coding sequence is interrupted by an intron in the opposite orientation to transcription (Figure 1). When placed within the Ty1 retrotransposon such that Ty1 and the marker gene are in opposite transcriptional orientations, the intron is spliced out of the Ty1 transcript but cannot be spliced out of the marker gene transcript. When the spliced Ty1 transcript undergoes retrotransposition, however, it recreates a functional marker gene in the transposed copy of the element. The fraction of viable cells in which the selectable marker gene is detected phenotypically is the retrotransposition frequency. This assay is a modification of that first described by Curcio and Garfinkel in 1991.

The specific assay conditions provided here are for derivatives of strain BY4741 containing a chromosomal Ty1 element that is marked with the retrotransposition indicator gene, his3AI or kanMXAI (Curcio and Garfinkel, 1991, Bryk et al., 2002). Cells that sustain retrotransposition of Ty1his3AI harbor a Ty1HIS3 element and are His+ prototrophs (i.e., capable of growth on medium lacking histidine; Figure 1). Cells that sustain retrotransposition of Ty1kanMXAI harbor a Ty1kanMX element and are resistant to G418.


Figure 1. Genetic assay for detection of cells that sustain a retrotransposition event of the chromosomal Ty1his3AI element. The Ty1 element consists of long terminal repeats (boxed arrowheads) flanking a GAG and a POL open reading frame. The his3AI indicator gene, which consists of the HIS3 marker gene interrupted by an intron in the antisense orientation, is inserted downstream of POL. Ty1his3AI RNA is transcribed, and the intron within the HIS3 open reading frame is spliced out. The spliced RNA is then subject to reverse transcription during the process of retrotransposition. The resulting Ty1HIS3 cDNA lacks the intron and contains functional HIS3 gene sequences. Integration of the cDNA into the host genome renders the cell His+. (Adapted from Curcio et al., 2015)

Materials and Reagents

  1. Pipette tips for P10, P100 and P1000 micropipettes
  2. Sterile flat wood toothpicks for obtaining single colonies (VWR, catalog number: 470146-908)
    Manufacturer: Regional Distributors, catalog number: 159864 .
  3. Sterile 12 x 75 mm polypropylene tubes with caps (4 per strain tested) (Evergreen Scientific, CaplugsTM, catalog number: 222-2367-080 )
  4. Borosilicate glass 20 mm x 150 mm culture tubes (Sigma-Aldrich, catalog number: C1048 ) with closure caps (Sigma-Aldrich, catalog number: C1298 ), sterilized (1 per strain tested)
  5. Sterile 1.5 ml microfuge tubes (4 per strain tested)
  6. Petri dishes (Fisher Scientific, FisherbrandTM, catalog number: FB0875713 )
  7. Sterile Rattler Plating Glass Beads, 4.5 mm (Zymo Research, catalog number: S1001 )
  8. Strains
    This assay is performed using derivatives of strain BY4741 that contain a Ty1his3AI[∆1] element, which has the “∆1” version of the his3AI gene. Ty1his3AI[∆1] must be used in strains such as BY4741 that have a his3∆1 allele rather than a complete deletion of the HIS3 gene. DNA recombination between his3AI[∆1] and his3∆1 cannot yield a functional HIS3 allele, so all His+ prototrophs that arise are due to the presence of a retrotransposed Ty1HIS3 element. Strains containing the chromosomal Ty1his3AI[∆1]-3114 element include JC3212 and JC3787 (Mou et al., 2006). The genotypes are:
    JC3212 MATa his3∆1 leu2∆0 met15∆0 ura3∆0 Ty1his3AI[∆1]-3114
    JC3787 MATα his3∆1 leu2∆0 met15∆0 ura3∆0 Ty1his3AI[∆1]-3114
    A derivative of strain BY4741 containing a single chromosomal Ty1kanMXAI element was described recently (Salinero et al., 2018). This strain has the following genotype:
    JC6464 MATa his3∆1 leu2∆0 met15∆0 ura3∆0 Ty1kanMXAI-6464
  9. YPD agar (Sunrise Science, catalog number: 1876-500 ), sterilized, in Petri dishes (4 plates per strain tested)
  10. SC Complete-His Powder (Sunrise Science, catalog number: 1481-100 )
  11. Agar, Yeast Culture Grade (Sunrise Science, catalog number: 1910-1KG
  12. G418 Sulfate (GeneticinTM Selective Antibiotic) (Thermo Fisher Scientific, catalog number: 11811023 )
  13. Sterile YPD broth (Sunrise Science, catalog number: 1875-019 )
  14. Sterile deionized water (Rockland Immunochemicals, catalog number: MB-009-1000
  15. Ethyl alcohol (ethanol), 190 proof, non-denatured (Sigma-Aldrich, catalog number: E7148 )
  16. SC-HIS agar plates (see Recipe 1), or, Sterile YPD + 200 μg/ml G418 agar plates (see Recipe 3)
    Note: A total of 8 plates (of either type) is needed per strain tested.
  17. 200 mg/ml G418 stock solution (see Recipe 2)

Equipment

  1. P10, P100 and P1000 micropipettes
  2. Pipet-Aid
  3. Tissue Culture Rotator (Fisher Scientific, FisherbrandTM, catalog number: 14-251-250 )
  4. Rotator drum (Fisher Scientific, FisherbrandTM, catalog number: 14-251-251 )
  5. Benchtop centrifuge that accommodates 12 x 75 mm tubes in a swinging bucket rotor (Eppendorf, Centrifuge, model: 5810 with swing out rotor S-4-104, catalog number: 5820740000 )
  6. Microcentrifuge (Fisher Scientific, FisherbrandTM, model: accuSpinTM Micro 17, catalog number: 13-100-675 )
  7. Vortex Mixer (Thermo Fisher Scientific, MaxiMix®, model: M16715Q , catalog number: 12-815-50)
  8. Autoclave

Procedure

  1. Choose a strain harboring a chromosomal Ty1his3Al element (e.g., JC3212, JC3787 or JC6464) and a mutant derivative(s) to compare the Ty1 retrotransposition frequencies of the two strains. The retrotransposition frequencies of a single strain with and without a chemical/environmental treatment can also be compared by this procedure. See Figure 2 for an outline of the whole procedure of this protocol.


    Figure 2. Flow chart of the Ty1his3AI retrotransposition frequency assay

  2. Obtain single colonies of JC3212, JC3787 or JC6464 and a mutant derivative(s) by growing cells on YPD agar at 30 °C, a non-permissive temperature for retrotransposition. Inoculate 10 ml of sterile YPD broth in a capped glass culture tube with a single colony and grow for 18-24 h on a tissue culture roller at 30 °C. Saturated cultures of JC3212, JC3787 or JC6464 will be at a density of approximately 1 x 108 viable cells per ml.
  3. For each strain being tested, label four 12 x 75 mm sterile polypropylene culture tubes with caps removed and placed gently on top. Vortex each culture at a low to moderate speed for 3 to 5 sec, making sure that the cells are well suspended. Remove 8.5 μl of culture and add to 8.5 ml of sterile YPD in a capped tube. (This is a 1:1,000 dilution.) Vortex to suspend well. Immediately pipet 2,000 μl of cells into each of four 12 x 75 mm polypropylene tubes and replace cap. (Caps should be pushed down but not tightly, so that they rotate freely and are not airtight. If they are pushed down completely, they will pop off during incubation.) Discard the remaining ~0.5 ml of diluted culture. 
  4. Grow cultures on a tissue culture roller at 20 °C, a permissive temperature for retrotransposition, for 64-72 h. The tissue culture roller should continue rotating for the entire incubation.
  5. The evening before the incubation is finished, label four YPD agar plates and eight SC-HIS agar plates (if using Ty1his3AI) or eight YPD + 200 μg/μl G418 agar plates (if using Ty1kanMXAI). Leave these plates unbagged on the bench at room temperature (or 4 °C for YPD + 200 μg/ml G418) to get rid of excess moisture. The agar should be slightly dry but not cracked for best results.
  6. Label four sterile 1.5 ml microfuge tubes per strain analyzed. Add 999 μl of sterile deionized water to each tube. Cap and label the tubes.
  7. Following incubation at 20 °C, vortex one culture until cells are well suspended, remove 1 μl and add to 999 μl of sterile water in the microfuge tube. This is a 1:1,000 dilution. Cap the original culture and diluted culture. Repeat with the other three cultures. 
  8. To one of the YPD plates, add 200 μl of sterile water to the center of the agar, and then add four to eight sterile glass beads on top of the water. Glass beads can be autoclaved in culture tubes, so that it is easy to pour out a limited number of beads. Vortex a 1:1,000 diluted culture, remove 1 μl and add it to the water on the YPD plate. Cover the plate and gently shake the plate (agar side down) from side to side, so that liquid spreads as evenly as possible over the surface of the YPD agar. Avoid shaking up and down. When liquid is absorbed into the agar, pour the beads into a beaker of 95% ethanol. Cover and let the plate sit for 5-10 min, agar side down, on the bench. Repeat with the other three 1:1,000 diluted cultures. Turn plates agar side up and incubate at 30 °C for three days.
  9. Centrifuge the undiluted cultures (1,999 μl) in 12 x 75 mm tubes in the swinging bucket rotor of a benchtop centrifuge at 2,000 x g for 10 min at room temperature.
  10. Carefully remove medium by slowly inverting each tube so that the cell pellet is not disrupted. Add 400 μl of sterile water to the tube. Resuspend cells by vortexing at low to moderate speed for 3 to 5 sec. Repeat the vortexing if necessary to ensure that the cell pellet is fully resuspended.
  11. Add 4 to 8 glass beads to the surface of the agar of each of two SC-HIS plates (or two YPD + 200 μg/ml G418 plates). Using a large pipet tip, remove all of the cell suspension from one 12 x 75 mm tube. The volume will be about 450 μl. Distribute equivalent amounts between the two SC-HIS plates (or YPD + 200 μg/ml G418 plates). It is critical that the cells are fully resuspended and all the cell suspension is plated, but the amounts of cell suspension per plate do not need to be exactly equal.
  12. Cover the plates, stack them, and gently shake the plates to distribute cell suspension as evenly as possible. When liquid is absorbed into the agar, transfer beads to a beaker of 95% ethanol, and let the plates sit for 5-10 min, agar side down, on the bench. Repeat with the other three cell suspensions. Turn all plates agar side up and incubate at 30 °C for three days. 
  13. After three days, count the number of colonies on each YPD plate. Count the total number of colonies on each set of two SC-HIS (or YPD + 200 μg/ml G418) plates. Repeat for the other three sets of plates. Representative data for strains JC3212 and JC5298 (an spt3∆ derivative of JC3212 are provided in Supplement file.

Data analysis

  1. Calculate the retrotransposition frequency for each of your original cultures:
    Total number of His+ (or G418R) colonies on both plates/[1999 x (number of colonies on YPD plate x 1000)]
    (The total number of His+ [or G418R] colonies on both plates/1999 = the number of His+ [or G418R] colonies per μl. The number of colonies on YPD plate x 1000 = the number of colony forming units per μl).
    See Supplement file for an example of calculating the retrotransposition frequency. 
  2. Average the four retrotransposition frequencies for each of your biological replicates (individual cultures). Determine the standard deviation.
    1. If one to three of the cultures assayed yield 0 His+ (or G418R) colonies on both plates, obtain the sum of the His+ colonies on all eight plates, and obtain the sum of the colonies on all four YPD plates. Determine the maximum retrotransposition frequency using the following formula:
      Total number of His+ (or G418R) colonies on all 8 plates/[4 x 1999 x (number of colonies on all four YPD plates x 1000)]
      This formula gives you a single number–the maximum retrotransposition frequency for all four biological replicates. This number represents the upper limit of retrotransposition when one or more cultures assayed yielded zero His+ colonies. 
    2. If all four of your cultures yield 0 His+ (or G418R) colonies, obtain the sum of the colonies on all four YPD plates and use the following formula to calculate the maximum retrotransposition frequency:
      1/[4 x 1999 x (number of colonies on all four YPD plates x 1000)]

Notes

If you are analyzing a mutant derivative of one of the strains listed above, or are treating the strain with a chemical that alters growth or other aspects of cell physiology, the assay conditions may need to be altered. See below for specific issues.

  1. Always compare the wild-type strain to the mutant (or chemically treated wild-type strain) in the same experiment. Variations in growth media, temperature, and length of incubation can alter the retrotransposition frequency from one experiment to the next. 
  2. Chemical treatments should be added during the 20 °C incubation, as this is when retrotransposition is occurring. 
  3. If the standard deviations are unacceptably large, or if there is more than a 10-fold difference in the retrotransposition frequencies among biological replicates, repeat the experiment using more replicates. Up to 12 cultures per strain or condition is standard. Large standard deviations can also arise from pipetting inaccurate volumes or not completely resuspending the cell pellet.
  4. The average number of colonies on YPD will be 50 to 250. If you obtain an average of fewer than 25 colonies/YPD plate for the wild-type strains (listed above), extend the 20 °C incubation period up to 6 hours. If cultures of the wild-type strain yield an average of ≥ 25 colonies/YPD plate, but cultures of the mutant (or chemically treated) strain that you are comparing to the wild-type strain yield an average of < 25 colonies/YPD plate, do the following: plate the wild-type cultures after the standard incubation at 20 °C, and continue to incubate cultures of the mutant (or chemically treated) strain at 20 °C for an additional 6 to 24 h, depending on how severely the plating efficiency is reduced. After the extended incubation period, plate cells using the standard protocol.
  5. If using a mutant strain that grows more slowly than the wild-type strain, plates with the mutant strain can be incubated beyond three days until the mutant colonies are similar in size to those of the wild-type strain. Use caution when incubating YPD + 200 μg/μl G418 plates, as small spontaneous G418R colonies will appear after an extended incubation period. These are not a result of retrotransposition and should not be counted. 
  6. For optimal reproducibility, do not plate more than 2,000 μl of culture onto two SC-HIS or two YPD + 200 μg/ml G418 plates. 
  7. If using a mutant strain with an increased retrotransposition frequency, there may be too many cells on the SC-HIS or YPD + 200 μg/μl G418 plates to count accurately. In this case, estimate the number of His+ or G418R colonies per plate (by counting colonies on one quarter or one-eighth of the plate) and use these estimates to calculate an estimated retrotransposition frequency. Then repeat the experiment with the following change: plate an aliquot of each 1,999 μl culture on to SC-HIS or YPD + 200 μg/μl G418 agar plates. For example, if a mutant has a retrotransposition frequency that is about 10-fold higher than the wild-type strain, remove 200 μl of the vortexed culture (about 1/10 of the total volume), and pellet the cells and resuspend them in sterile water as described in detail in Step 9 of the Procedure section above. Remember to change “1999” to “200” in the formula provided in Step 1 of the Data Analysis section above. 
  8. This protocol can be used to measure Ty1his3AI or Ty1kanMXAI retrotransposition in other S. cerevisiae or S. paradoxus strains that support Ty1 retrotransposition (S. cerevisiae strain W303 does not.). In this case, the retrotransposition frequency, which is between 1 and 10 x 10-7 His+ or G418R cells per total viable cells plated on selective medium, may be different, and the amounts plated may have to be adjusted.

Recipes

  1. SC-HIS agar plates (ca. 40)
    28.62 g SC Complete-His powder
    20 g of agar
    1,000 ml deionized water
    Add deionized water to 28.62 g of SC Complete-His powder up to 500 ml. Add a stir bar and mix by stirring for 15 min. Add deionized water to 20 g agar up to 500 ml in a separate container. Autoclave both mixtures at 121 °C for 15 min. Cool each solution to ~65 °C. Slowly add agar to the SC Complete-His solution while stirring. Continue stirring for 2 min and then pour ~25 ml into each plastic Petri dish on a flat surface. Ensure that the agar remains sterile throughout the procedure. Once the agar is completely solidified, turn the Petri dishes upside-down and store in an airtight container at room temperature or 4 °C. 
  2. 200 mg/ml G418 stock solution
    2 g G418 sulfate
    10 ml sterile deionized water
    Dissolve 2 g G418 sulfate in 10 ml deionized water. Aliquot 1 ml into 10 tubes and store at -20 °C.
  3. YPD + 200 μg/ml G418 plates (ca. 40)
    70 g YPD agar powder
    1 ml 200 mg/ml G418 sulfate stock solution
    1,000 ml deionized water
    Add deionized water to 70 g of YPD agar powder up to 1000 ml. Add a stir bar and mix by stirring for 15 min. Autoclave at 121 °C for 15 min. Cool to ~65 °C. Add 1 ml of 200 mg/ml G418 and stir for several minutes. Pour ~25 ml into each plastic Petri dish on a flat surface. Ensure that the agar remains sterile throughout the procedure. Once the agar is completely solidified, turn the Petri dishes upside-down and store in an airtight container at 4 °C.

Acknowledgments

This protocol was adapted from Curcio and Garfinkel (1991). The author has no conflict of interest or competing interests.

References

  1. Boeke, J. D., Garfinkel, D. J., Styles, C. A. and Fink, G. R. (1985). Ty elements transpose through an RNA intermediate. Cell 40(3): 491-500.
  2. Bryk, M., Briggs, S. D., Strahl, B. D., Curcio, M. J., Allis, C. D. and Winston, F. (2002). Evidence that Set1, a factor required for methylation of histone H3, regulates rDNA silencing in S. cerevisiae by a Sir2-independent mechanism. Curr Biol 12(2): 165-170.
  3. Curcio, M. J. and Garfinkel, D. J. (1991). Single-step selection for Ty1 element retrotransposition. Proc Natl Acad Sci U S A 88(3): 936-940.
  4. Curcio, M. J., Lutz, S. and Lesage, P. (2015). The Ty1 LTR-retrotransposon of budding yeast, Saccharomyces cerevisiae. Microbiol Spectr 3(2): 1-35.
  5. Garfinkel, D. J., Boeke, J. D. and Fink, G. R. (1985). Ty element transposition: reverse transcriptase and virus-like particles. Cell 42(2): 507-517.
  6. Mou, Z., Kenny, A. E. and Curcio, M. J. (2006). Hos2 and Set3 promote integration of Ty1 retrotransposons at tRNA genes in Saccharomyces cerevisiae. Genetics 172(4): 2157-2167.
  7. Salinero, A. C., Knoll, E. R., Zhu, Z. I., Landsman, D., Curcio, M. J. and Morse, R. H. (2018). The Mediator co-activator complex regulates Ty1 retromobility by controlling the balance between Ty1i and Ty1 promoters. PLoS Genet 14(2): e1007232.
Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Curcio, M. J. (2018). Ty1 Retrotransposition Frequency Assay Using a Chromosomal Ty1his3AI or Ty1kanMXAI Element. Bio-protocol Bio101: e3004. DOI: 10.21769/BioProtoc.3004.
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