Loss of heterozygosity by SCRaMbLEing

Abstract

Genetic variation drives phenotypic evolution within populations. Genetic variation can be divided into different forms according to the size of genomic changes. However, study of large-scale genomic variation such as structural variation and aneuploidy is still limited and mainly based on the static, predetermined feature of individual genomes. Here, using SCRaMbLE, different levels of loss of heterozygosity (LOH) events including short-range LOH, long-range LOH and whole chromosome LOH were detected in evolved strains. By contrast, using rapid adaptive evolution, aneuploidy was detected in the adaptive strains. It was further found that deletion of gene GLN3, long-range LOH in the left arm of synthetic chromosome X, whole chromosome LOH of synthetic chromosome X, and duplication of chromosome VIII (trisomy) lead to increased rapamycin resistance in synthetic yeast. Comparative analysis of genome stability of evolved strains indicates that the aneuploid strain has a higher frequency of degeneration than the SCRaMbLEd strain. These findings enrich our understanding of genetic mechanism of rapamycin resistance in yeast, and provide valuable insights into yeast genome architecture and function.

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References

  1. Andersen, M.P., Nelson, Z.W., Hetrick, E.D., and Gottschling, D.E. (2008). A genetic screen for increased loss of heterozygosity in Saccharomyces cerevisiae. Genetics 179, 1179–1195.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Bennett, R.J., Forche, A., and Berman, J. (2014). Rapid mechanisms for generating genome diversity: whole ploidy shifts, aneuploidy, and loss of heterozygosity. Cold Spring Harbor Perspect Med 4, a019604.

    Google Scholar 

  3. Blount, B.A., Gowers, G.O.F., Ho, J.C.H., Ledesma-Amaro, R., Jovicevic, D., McKiernan, R.M., Xie, Z.X., Li, B.Z., Yuan, Y.J., and Ellis, T. (2018). Rapid host strain improvement by in vivo rearrangement of a synthetic yeast chromosome. Nat Commun 9, 1932.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Brown, E.J., Albers, M.W., Bum Shin, T., Ichikawa, K., Keith, C.T., Lane, W.S., and Schreiber, S.L. (1994). A mammalian protein targeted by G1-arresting rapamycin-receptor complex. Nature 369, 756–758.

    Article  CAS  PubMed  Google Scholar 

  5. Cardenas, M.E., Cutler, N.S., Lorenz, M.C., Di Como, C.J. and Heitman, J. (1999). The TOR signaling cascade regulates gene expression in response to nutrients. Genes Dev 13, 3271–3279.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Chen, G., Bradford, W.D., Seidel, C.W., and Li, R. (2012). Hsp90 stress potentiates rapid cellular adaptation through induction of aneuploidy. Nature 482, 246–250.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Comai, L., Young, K., Till, B.J., Reynolds, S.H., Greene, E.A., Codomo, C. A., Enns, L.C., Johnson, J.E., Burtner, C., Odden, A.R., et al. (2004). Efficient discovery of DNA polymorphisms in natural populations by Ecotilling. Plant J 37, 778–786.

    Article  CAS  PubMed  Google Scholar 

  8. Crespo, J.L., and Hall, M.N. (2002). Elucidating TOR signaling and rapamycin action: lessons from Saccharomyces cerevisiae. Microbiol Mol Biol Rev 66, 579–591.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Dujon, B. (2010). Yeast evolutionary genomics. Nat Rev Genet 11, 512–524.

    Article  CAS  PubMed  Google Scholar 

  10. Dujon, B., Sherman, D., Fischer, G., Durrens, P., Casaregola, S., Lafontaine, I., De Montigny, J., Marck, C., Neuvéglise, C., Talla, E., et al. (2004). Genome evolution in yeasts. Nature 430, 35–44.

    Article  PubMed  Google Scholar 

  11. Dymond, J.S., Richardson, S.M., Coombes, C.E., Babatz, T., Muller, H., Annaluru, N., Blake, W.J., Schwerzmann, J.W., Dai, J., Lindstrom, D. L., et al. (2011). Synthetic chromosome arms function in yeast and generate phenotypic diversity by design. Nature 477, 471–476.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Gietz, R.D., and Woods, R.A. (2002). Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol 350, 87–96.

    Article  CAS  PubMed  Google Scholar 

  13. Gorringe, K.L. (2016). Loss of heterozygosity. In eLS. pp. 1–8, doi: 10.1002/9780470015902.a0026643.

    Google Scholar 

  14. Guo, F., Gopaul, D.N., and van Duyne, G.D. (1997). Structure of Cre recombinase complexed with DNA in a site-specific recombination synapse. Nature 389, 40–46.

    Article  CAS  PubMed  Google Scholar 

  15. Hassall, C. (2014). The ecology and biodiversity of urban ponds. WIREs Water 1, 187–206.

    Article  Google Scholar 

  16. Hidalgo, M., and Rowinsky, E.K. (2000). The rapamycin-sensitive signal transduction pathway as a target for cancer therapy. Oncogene 19, 6680–6686.

    Article  CAS  PubMed  Google Scholar 

  17. Hochrein, L., Mitchell, L.A., Schulz, K., Messerschmidt, K., and Mueller-Roeber, B. (2018). L-SCRaMbLE as a tool for light-controlled Cremediated recombination in yeast. Nat Commun 9, 1931.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Huber, A., Bodenmiller, B., Uotila, A., Stahl, M., Wanka, S., Gerrits, B., Aebersold, R., and Loewith, R. (2009). Characterization of the rapamycin-sensitive phosphoproteome reveals that Sch9 is a central coordinator of protein synthesis. Genes Dev 23, 1929–1943.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Jain, M., Koren, S., Miga, K.H., Quick, J., Rand, A.C., Sasani, T.A., Tyson, J.R., Beggs, A.D., Dilthey, A.T., Fiddes, I.T., et al. (2018). Nanopore sequencing and assembly of a human genome with ultra-long reads. Nat Biotechnol 36, 338–345.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Jeffares, D.C., Jolly, C., Hoti, M., Speed, D., Shaw, L., Rallis, C., Balloux, F., Dessimoz, C., Bähler, J., and Sedlazeck, F.J. (2017). Transient structural variations have strong effects on quantitative traits and reproductive isolation in fission yeast. Nat Commun 8, 14061.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Jia, B., Wu, Y., Li, B.Z., Mitchell, L.A., Liu, H., Pan, S., Wang, J., Zhang, H.R., Jia, N., Li, B., et al. (2018). Precise control of SCRaMbLE in synthetic haploid and diploid yeast. Nat Commun 9, 1933.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Kidd, J.M., Cooper, G.M., Donahue, W.F., Hayden, H.S., Sampas, N., Graves, T., Hansen, N., Teague, B., Alkan, C., Antonacci, F., et al. (2008). Mapping and sequencing of structural variation from eight human genomes. Nature 453, 56–64.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Lander, E., and Kruglyak, L. (1995). Genetic dissection of complex traits: guidelines for interpreting and reporting linkage results. Nat Genet 11, 241–247.

    Article  CAS  PubMed  Google Scholar 

  24. Lander, E.S., and Schork, N.J. (1994). Genetic dissection of complex traits. Science 265, 2037–2048.

    Article  CAS  PubMed  Google Scholar 

  25. Lau, T.K., Jiang, F.M., Stevenson, R.J., Lo, T.K., Chan, L.W., Chan, M.K., Lo, P.S.S., Wang, W., Zhang, H.Y., Chen, F., et al. (2013). Secondary findings from non-invasive prenatal testing for common fetal aneuploidies by whole genome sequencing as a clinical service. Prenat Diagn 33, 602–608.

    Article  CAS  PubMed  Google Scholar 

  26. Lauer, S., Avecilla, G., Spealman, P., Sethia, G., Brandt, N., Levy, S.F., and Gresham, D. (2018). Single-cell copy number variant detection reveals the dynamics and diversity of adaptation. PLoS Biol 16, e3000069.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Lengauer, C., Kinzler, K.W., and Vogelstein, B. (1997). Genetic instability in colorectal cancers. Nature 386, 623–627.

    Article  CAS  PubMed  Google Scholar 

  28. Lewandoski, M., and Martin, G.R. (1997). Cre-mediated chromosome loss in mice. Nat Genet 17, 223–225.

    Article  CAS  PubMed  Google Scholar 

  29. Lindstrom, D.L., and Gottschling, D.E. (2009). The Mother Enrichment Program: A genetic system for facile replicative life span analysis in Saccharomyces cerevisiae. Genetics 183, 413–422.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Liti, G., Carter, D.M., Moses, A.M., Warringer, J., Parts, L., James, S.A., Davey, R.P., Roberts, I.N., Burt, A., Koufopanou, V., et al. (2009). Population genomics of domestic and wild yeasts. Nature 458, 337–341.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Liu, W., Luo, Z., Wang, Y., Pham, N.T., Tuck, L., Pérez-Pi, I., Liu, L., Shen, Y., French, C., Auer, M., et al. (2018). Rapid pathway prototyping and engineering using in vitro and in vivo synthetic genome SCRaMbLE-in methods. Nat Commun 9, 1936.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Lu, S., Zong, C., Fan, W., Yang, M., Li, J., Chapman, A.R., Zhu, P., Hu, X., Xu, L., Yan, L., et al. (2012). Probing meiotic recombination and aneuploidy of single sperm cells by whole-genome sequencing. Science 338, 1627–1630.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Luo, Z., Wang, L., Wang, Y., Zhang, W., Guo, Y., Shen, Y., Jiang, L., Wu, Q., Zhang, C., Cai, Y., et al. (2018a). Identifying and characterizing SCRaMbLEd synthetic yeast using ReSCuES. Nat Commun 9, 1930.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Luo, Z., Yang, Q., Geng, B., Jiang, S., Yang, S., Li, X., Cai, Y., and Dai, J. (2018b). Whole genome engineering by synthesis. Sci China Life Sci 61, 1515–1527.

    Article  PubMed  Google Scholar 

  35. Mercy, G., Mozziconacci, J., Scolari, V.F., Yang, K., Zhao, G., Thierry, A., Luo, Y., Mitchell, L.A., Shen, M., Shen, Y., et al. (2017). 3D organization of synthetic and scrambled chromosomes. Science 355, eaaf4597.

    Google Scholar 

  36. Mitchell, L.A., Wang, A., Stracquadanio, G., Kuang, Z., Wang, X., Yang, K., Richardson, S., Martin, J.A., Zhao, Y., Walker, R., et al. (2017). Synthesis, debugging, and effects of synthetic chromosome consolidation: synVI and beyond. Science 355, eaaf4831.

    Google Scholar 

  37. O’Donnell, A.F., Apffel, A., Gardner, R.G., and Cyert, M.S. (2010). α-Arrestins Aly1 and Aly2 regulate intracellular trafficking in response to nutrient signaling. Mol Biol Cell 21, 3552–3566.

    PubMed  Google Scholar 

  38. Peter, J., De Chiara, M., Friedrich, A., Yue, J.X., Pflieger, D., Bergström, A., Sigwalt, A., Barre, B., Freel, K., Llored, A., et al. (2018). Genome evolution across 1,011 Saccharomyces cerevisiae isolates. Nature 556, 339–344.

    Article  CAS  PubMed  Google Scholar 

  39. Philipp, O., Otipoby, K.L., Mitsuo, M., and Klaus, R. (2003). Unidirectional Cre-mediated genetic inversion in mice using the mutant loxP pair lox66/lox71. Nucleic Acids Res 31, e140.

    Article  CAS  Google Scholar 

  40. Proud, C.G. (1996). p70 S6 kinase: an enigma with variations. Trends Biochem Sci 21, 181–185.

    CAS  PubMed  Google Scholar 

  41. Rafalski, A. (2002). Applications of single nucleotide polymorphisms in crop genetics. Curr Opin Plant Biol 5, 94–100.

    Article  CAS  PubMed  Google Scholar 

  42. Redon, R., Ishikawa, S., Fitch, K.R., Feuk, L., Perry, G.H., Andrews, T.D., Fiegler, H., Shapero, M.H., Carson, A.R., Chen, W., et al. (2006). Global variation in copy number in the human genome. Nature 444, 444–454.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Reid, R.J., Sunjevaric, I., Voth, W.P., Ciccone, S., Du, W., Olsen, A.E., Stillman, D.J., and Rothstein, R. (2008). Chromosome-scale genetic mapping using a set of 16 conditionally stable Saccharomyces cerevisiae chromosomes. Genetics 180, 1799–1808.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Ren, H., and Zhao, H. (2017). SynV and SynX: A story more than DNA synthesis. Sci China Life Sci 60, 558–560.

    Article  CAS  PubMed  Google Scholar 

  45. Richardson, S.M., Mitchell, L.A., Stracquadanio, G., Yang, K., Dymond, J. S., DiCarlo, J.E., Lee, D., Huang, C.L.V., Chandrasegaran, S., Cai, Y., et al. (2017). Design of a synthetic yeast genome. Science 355, 1040–1044.

    Article  CAS  PubMed  Google Scholar 

  46. Sabatini, D.M. (2006). mTOR and cancer: insights into a complex relationship. Nat Rev Cancer 6, 729–734.

    Article  CAS  PubMed  Google Scholar 

  47. Schmittgen, T.D., and Livak, K.J. (2008). Analyzing real-time PCR data by the comparative CT method. Nat Protoc 3, 1101–1108.

    Article  CAS  PubMed  Google Scholar 

  48. Schwartz, D.C., and Cantor, C.R. (1984). Separation of yeast chromosomesized DNAs by pulsed field gradient gel electrophoresis. Cell 37, 67–75.

    Article  CAS  PubMed  Google Scholar 

  49. Shen, M.J., Wu, Y., Yang, K., Li, Y., Xu, H., Zhang, H., Li, B.Z., Li, X., Xiao, W.H., Zhou, X., et al. (2018). Heterozygous diploid and interspecies SCRaMbLEing. Nat Commun 9, 1934.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Shen, Y., Stracquadanio, G., Wang, Y., Yang, K., Mitchell, L.A., Xue, Y., Cai, Y., Chen, T., Dymond, J.S., Kang, K., et al. (2016). SCRaMbLE generates designed combinatorial stochastic diversity in synthetic chromosomes. Genome Res 26, 36–49.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Shen, Y., Wang, Y., Chen, T., Gao, F., Gong, J., Abramczyk, D., Walker, R., Zhao, H., Chen, S., Liu, W., et al. (2017). Deep functional analysis of synII, a 770-kilobase synthetic yeast chromosome. Science 355, eaaf4791.

    Google Scholar 

  52. Stankiewicz, P., and Lupski, J.R. (2010). Structural variation in the human genome and its role in disease. Annu Rev Med 61, 437–455.

    Article  CAS  Google Scholar 

  53. Torres, E.M., Sokolsky, T., Tucker, C.M., Chan, L.Y., Boselli, M., Dunham, M.J., and Amon, A. (2007). Effects of aneuploidy on cellular physiology and cell division in haploid yeast. Science 317, 916–924.

    Article  CAS  PubMed  Google Scholar 

  54. Väli, U., Brandström, M., Johansson, M., and Ellegren, H. (2008). Insertion-deletion polymorphisms (indels) as genetic markers in natural populations. BMC Genet 9, 8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Wang, J., Xie, Z.X., Ma, Y., Chen, X.R., Huang, Y.Q., He, B., Bin Jia, B., Li, B.Z., and Yuan, Y.J. (2018). Ring synthetic chromosome V SCRaMbLE. Nat Commun 9, 3783.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Weaver, B.A.A., and Cleveland, D.W. (2006). Does aneuploidy cause cancer? Curr Opin Cell Biol 18, 658–667.

    Article  CAS  PubMed  Google Scholar 

  57. Weber, T.S., Dukes, M., Miles, D.C., Glaser, S.P., Naik, S.H., and Duffy, K. R. (2016). Site-specific recombinatorics: in situ cellular barcoding with the Cre Lox system. BMC Syst Biol 10, 1–13.

    Article  CAS  Google Scholar 

  58. Wu, Y., Li, B.Z., Zhao, M., Mitchell, L.A., Xie, Z.X., Lin, Q.H., Wang, X., Xiao, W.H., Wang, Y., and Zhou, X. (2017). Bug mapping and fitness testing of chemically synthesized chromosome X. Science 355.

    Google Scholar 

  59. Wu, Y., Zhu, R.Y., Mitchell, L.A., Ma, L., Liu, R., Zhao, M., Jia, B., Xu, H., Li, Y.X., and Yang, Z.M. (2018). In vitro DNA SCRaMbLE. Nat Commun 9.

    Google Scholar 

  60. Wullschleger, S., Loewith, R., and Hall, M.N. (2006). TOR signaling in growth and metabolism. Cell 124, 471–484.

    Article  CAS  Google Scholar 

  61. Xie, Z.X., Li, B.Z., Mitchell, L.A., Wu, Y., Qi, X., Jin, Z., Jia, B., Wang, X., Zeng, B.X., and Liu, H.M. (2017). “Perfect” designer chromosome V and behavior of a ring derivative. Science 355.

    Google Scholar 

  62. Shen, Y., Betzendahl, I., Tinneberg, H.R., and Eichenlaub-Ritter, U. (2008). Enhanced polarizing microscopy as a new tool in aneuploidy research in oocytes. Mutat Res/Genet Toxicol Environ Mutagenesis 651, 131–140.

    Article  CAS  Google Scholar 

  63. Yona, A.H., Manor, Y.S., Herbst, R.H., Romano, G.H., Mitchell, A., Kupiec, M., Pilpel, Y., and Dahan, O. (2012). Chromosomal duplication is a transient evolutionary solution to stress. Proc Natl Acad Sci USA 109, 21010–21015.

    Article  PubMed  Google Scholar 

  64. Yue, J.X., Li, J., Aigrain, L., Hallin, J., Persson, K., Oliver, K., Bergström, A., Coupland, P., Warringer, J., Lagomarsino, M.C., et al. (2017). Contrasting evolutionary genome dynamics between domesticated and wild yeasts. Nat Genet 49, 913–924.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Zhang, F., and Voytas, D.F. (2018). Synthetic genomes engineered by SCRaMbLEing. Sci China Life Sci 61, 975–977.

    Article  PubMed  Google Scholar 

  66. Zhang, W., Zhao, G., Luo, Z., Lin, Y., Wang, L., Guo, Y., Wang, A., Jiang, S., Jiang, Q., Gong, J., et al. (2017). Engineering the ribosomal DNA in a megabase synthetic chromosome. Science 355, eaaf3981.

    Google Scholar 

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (21621004, 21750001 and 21676192) and Young Elite Scientist Sponsorship Program by CAST (YESS) (2018QNRC001).

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Correspondence to Yingjin Yuan.

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Li, Y., Wu, Y., Ma, L. et al. Loss of heterozygosity by SCRaMbLEing. Sci. China Life Sci. 62, 381–393 (2019). https://doi.org/10.1007/s11427-019-9504-5

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Keywords

  • SCRaMbLE
  • loss of heterozygosity (LOH)
  • rapid adaptive evolution
  • aneuploidy
  • structural variation
  • synthetic yeast genome