Yeast artificial chromosomes

From OpenWetWare
Jump to navigationJump to search

Yeast artificial chromosomes (YACs) are synthetic double stranded linear constructs containing the elements necessary for replication as independent chromosomes in yeast. These elements are:

  • an autonomous replication sequence (ARS): ARS1, chromosome III ARS, ARSH4
  • a centromere: CEN4, CEN6: centromeres consist of three centromere determining elements, CDE I, CDE II, and CDE III.
  • a telomeric sequence at each end

Typically the chromosome also contains a selection marker such as TRP1, Lys2 or Ura3.

Minimal size for a YAC is between 50kb and 100kb, while maximum sizes are 1Mb to 3Mb.

A common tool for constructing YACs is a shuttle plasmid such as pYAC4 which replicates in E. coli, has a multiple cloning site, and a pair of telomeres which can be cleaved to form a linear fragment. Available as an E.coli plasmid ATCC 67379, sequence at U01086.

There are two common centromere sequences, CEN4 and CEN6. CEN4 is found in most yeast centromere-containiing vectors, such as pYAC4. These vectors typically use ARS1 sequences.

The pRS313- pRS316 plasmids use the CEN6 + ARSH4 cassette (Sikorski89).

Yeast strains

  • AB1380 (MATa ura3-52 trp1 lys2-1 ade2-1 can1-100 his5) (Burke87) ATCC 204682
  • J57D (MATa, ura3-52, trp1 ade2-101 can1-100 leu2-3, 112 his3-6) (Haldi96)
    • claimed to be 2-3x more efficient at large insert cloning than AB1380
  • W303-1a (MATa leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 ybp1-1) Rothstein notes (see Veal03) Open Biosystems: YSC1058
  • YPH500 (MATα ura3-52 lys2-801_amber ade2-101_ochre trp1-Δ63 his1-Δ200 leu2-Δ1) ATCC:204680 (Sikorski89)
  • S288C (MATα SUC2 gal2 mal mel flo1 flo8-1 hap1) ATCC: 204508 (Mortimer86)

YAC Plasmids

  • pYAC4 (Burke87) has Sup4 for ade2 selection, interrupted by the cloning site (EcoRI), ura3, a pair of facing telomeres surrounding a his3 gene (linearized and cut out with BamHI), a pBR322 based E. coli replication and ampR region, a barely functional TRP1 gene, ARS1 and CEN4. Sequence at Genbank U01086
  • pJS97 / pJS98 plasmids (Shero91) are available at ATCC 77191 as a two-plasmid kit. Each plasmid supplies one chromosome end.
    • pJS97 has CEN4 and ARSH4, URA3 gene, Sup11 tRNA for ade2 selection, a pUC19 derived E. coli replication region, including ampR, and a telomere.
    • pJS98 has ARSH4, a functional TRP1 gene, a pUC19 derived E. coli replication region including ampR, and a telomere.
  • pCGS966 (Smith90, Smith92, Moir93) has ARS1 on both arms, Gal inducible extra copy production, NeoR for mammalian expression and a functional promoter for the Trp gene, unlike pYAC4.


  1. Hieter P, Pridmore D, Hegemann JH, Thomas M, Davis RW, and Philippsen P. Functional selection and analysis of yeast centromeric DNA. Cell. 1985 Oct;42(3):913-21. DOI:10.1016/0092-8674(85)90287-9 | PubMed ID:2996783 | HubMed [Hieter85]
  2. Mann C and Davis RW. Structure and sequence of the centromeric DNA of chromosome 4 in Saccharomyces cerevisiae. Mol Cell Biol. 1986 Jan;6(1):241-5. DOI:10.1128/mcb.6.1.241-245.1986 | PubMed ID:3537685 | HubMed [Mann86]
  3. Mortimer RK and Johnston JR. Genealogy of principal strains of the yeast genetic stock center. Genetics. 1986 May;113(1):35-43. DOI:10.1093/genetics/113.1.35 | PubMed ID:3519363 | HubMed [Moritmer86]
  4. Burke DT, Carle GF, and Olson MV. Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science. 1987 May 15;236(4803):806-12. DOI:10.1126/science.3033825 | PubMed ID:3033825 | HubMed [Burke87]
  5. Cottarel G, Shero JH, Hieter P, and Hegemann JH. A 125-base-pair CEN6 DNA fragment is sufficient for complete meiotic and mitotic centromere functions in Saccharomyces cerevisiae. Mol Cell Biol. 1989 Aug;9(8):3342-9. DOI:10.1128/mcb.9.8.3342-3349.1989 | PubMed ID:2552293 | HubMed [Cottarel89]
  6. Anand R, Villasante A, and Tyler-Smith C. Construction of yeast artificial chromosome libraries with large inserts using fractionation by pulsed-field gel electrophoresis. Nucleic Acids Res. 1989 May 11;17(9):3425-33. DOI:10.1093/nar/17.9.3425 | PubMed ID:2542900 | HubMed [Anand89]
  7. Sikorski RS and Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19-27. DOI:10.1093/genetics/122.1.19 | PubMed ID:2659436 | HubMed [Sikorski89]
  8. Sgaramella V, Ferretti L, Damiani G, and Sora S. A procedure for cloning restriction fragments of DNA as single inserts in yeast artificial chromosomes. Biochem Int. 1990;20(3):503-10. PubMed ID:2189412 | HubMed [Sgaramella90]
  9. Smith DR, Smyth AP, and Moir DT. Amplification of large artificial chromosomes. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8242-6. DOI:10.1073/pnas.87.21.8242 | PubMed ID:2236036 | HubMed [Smith90]
  10. Wells RA, Germino GG, Krishna S, Buckle VJ, and Reeders ST. Telomere-related sequences at interstitial sites in the human genome. Genomics. 1990 Dec;8(4):699-704. DOI:10.1016/0888-7543(90)90257-u | PubMed ID:2276741 | HubMed [Wells90]
  11. Van Houten JV and Newlon CS. Mutational analysis of the consensus sequence of a replication origin from yeast chromosome III. Mol Cell Biol. 1990 Aug;10(8):3917-25. DOI:10.1128/mcb.10.8.3917-3925.1990 | PubMed ID:2196439 | HubMed [VanHouten90]
  12. McCormick MK, Antonarakis SE, and Hieter P. YAC cloning of DNA embedded in an agarose matrix. Genet Anal Tech Appl. 1990 Sep;7(5):114-8. DOI:10.1016/0735-0651(90)90016-9 | PubMed ID:2091693 | HubMed [McCormick90]
  13. Burke DT. YAC cloning: options and problems. Genet Anal Tech Appl. 1990 Sep;7(5):94-9. DOI:10.1016/0735-0651(90)90013-6 | PubMed ID:2091697 | HubMed [Burke90]
  14. Shero JH, McCormick MK, Antonarakis SE, and Hieter P. Yeast artificial chromosome vectors for efficient clone manipulation and mapping. Genomics. 1991 Jun;10(2):505-8. DOI:10.1016/0888-7543(91)90343-d | PubMed ID:2071158 | HubMed [Shero92]
  15. Burke DT and Olson MV. Preparation of clone libraries in yeast artificial-chromosome vectors. Methods Enzymol. 1991;194:251-70. DOI:10.1016/0076-6879(91)94020-d | PubMed ID:2005791 | HubMed [Burke91]
  16. Connelly C, McCormick MK, Shero J, and Hieter P. Polyamines eliminate an extreme size bias against transformation of large yeast artificial chromosome DNA. Genomics. 1991 May;10(1):10-6. DOI:10.1016/0888-7543(91)90477-v | PubMed ID:2045094 | HubMed [Connelly91]
  17. Smith DR, Smyth AP, and Moir DT. Copy number amplification of yeast artificial chromosomes. Methods Enzymol. 1992;216:603-14. DOI:10.1016/0076-6879(92)16052-l | PubMed ID:1336106 | HubMed [Smith92]
  18. Ragoussis J, Trowsdale J, and Markie D. Mitotic recombination of yeast artificial chromosomes. Nucleic Acids Res. 1992 Jun 25;20(12):3135-8. DOI:10.1093/nar/20.12.3135 | PubMed ID:1620611 | HubMed [Ragoussis92]
  19. de Bruin D, Lanzer M, and Ravetch JV. Characterization of yeast artificial chromosomes from Plasmodium falciparum: construction of a stable, representative library and cloning of telomeric DNA fragments. Genomics. 1992 Oct;14(2):332-9. DOI:10.1016/s0888-7543(05)80223-x | PubMed ID:1427849 | HubMed [deBruin92]
  20. Deshpande AM and Newlon CS. The ARS consensus sequence is required for chromosomal origin function in Saccharomyces cerevisiae. Mol Cell Biol. 1992 Oct;12(10):4305-13. DOI:10.1128/mcb.12.10.4305-4313.1992 | PubMed ID:1406623 | HubMed [Deshpande92]
  21. Marahrens Y and Stillman B. A yeast chromosomal origin of DNA replication defined by multiple functional elements. Science. 1992 Feb 14;255(5046):817-23. DOI:10.1126/science.1536007 | PubMed ID:1536007 | HubMed [Marahrens92]
  22. Christianson TW, Sikorski RS, Dante M, Shero JH, and Hieter P. Multifunctional yeast high-copy-number shuttle vectors. Gene. 1992 Jan 2;110(1):119-22. DOI:10.1016/0378-1119(92)90454-w | PubMed ID:1544568 | HubMed [Christianson92]
  23. Moir DT, Dorman TE, Smyth AP, and Smith DR. A human genome YAC library in a selectable high-copy-number vector. Gene. 1993 Mar 30;125(2):229-32. DOI:10.1016/0378-1119(93)90334-y | PubMed ID:8462878 | HubMed [Moir93]
  24. Spencer F, Ketner G, Connelly C, and Hieter P. Targeted recombination-based cloning and manipulation of large DNA segments in yeast. Methods: A companion to methods in enzymology 1993; 5:161-175 (no pubmed entry)

  25. Kuhn RM and Ludwig RA. Complete sequence of the yeast artificial chromosome cloning vector pYAC4. Gene. 1994 Apr 8;141(1):125-7. DOI:10.1016/0378-1119(94)90139-2 | PubMed ID:8163163 | HubMed [Kuhn94]
  26. Hugerat Y, Spencer F, Zenvirth D, and Simchen G. A versatile method for efficient YAC transfer between any two strains. Genomics. 1994 Jul 1;22(1):108-17. DOI:10.1006/geno.1994.1351 | PubMed ID:7959756 | HubMed [Hugerat94]
  27. Spencer F, Hugerat Y, Simchen G, Hurko O, Connelly C, and Hieter P. Yeast kar1 mutants provide an effective method for YAC transfer to new hosts. Genomics. 1994 Jul 1;22(1):118-26. DOI:10.1006/geno.1994.1352 | PubMed ID:7959757 | HubMed [Spencer94]
  28. Hamer L, Johnston M, and Green ED. Isolation of yeast artificial chromosomes free of endogenous yeast chromosomes: construction of alternate hosts with defined karyotypic alterations. Proc Natl Acad Sci U S A. 1995 Dec 5;92(25):11706-10. DOI:10.1073/pnas.92.25.11706 | PubMed ID:8524833 | HubMed [Hamer95]
  29. Mahmood A, Kimura T, Takenaka M, and Yoshida K. The construction of mobilizable YACs and their direct conjugative transfer from E. coli to yeasts. Nucleic Acids Symp Ser. 1995(34):45-6. PubMed ID:8841544 | HubMed [Mahmood95]
  30. Mahmood A, Kimura T, Takenaka M, and Yoshida K. The construction of novel mobilizable YAC plasmids and their behavior during trans-kingdom conjugation between bacteria and yeasts. Genet Anal. 1996 Jul;13(2):25-31. DOI:10.1016/1050-3862(95)00146-8 | PubMed ID:8880145 | HubMed [Mahmood96]
  31. Haldi ML, Strickland C, Lim P, VanBerkel V, Chen X, Noya D, Korenberg JR, Husain Z, Miller J, and Lander ES. A comprehensive large-insert yeast artificial chromosome library for physical mapping of the mouse genome. Mamm Genome. 1996 Oct;7(10):767-9. DOI:10.1007/s003359900228 | PubMed ID:8854865 | HubMed [Haldi96]
  32. [Ketner98]
  33. Kouprina N, Nikolaishvili N, Graves J, Koriabine M, Resnick MA, and Larionov V. Integrity of human YACs during propagation in recombination-deficient yeast strains. Genomics. 1999 Mar 15;56(3):262-73. DOI:10.1006/geno.1998.5727 | PubMed ID:10087193 | HubMed [Kooprina99]
  34. Cocchia M, Kouprina N, Kim SJ, Larionov V, Schlessinger D, and Nagaraja R. Recovery and potential utility of YACs as circular YACs/BACs. Nucleic Acids Res. 2000 Sep 1;28(17):E81. DOI:10.1093/nar/28.17.e81 | PubMed ID:10954614 | HubMed [Cocchia00]
  35. Veal EA, Ross SJ, Malakasi P, Peacock E, and Morgan BA. Ybp1 is required for the hydrogen peroxide-induced oxidation of the Yap1 transcription factor. J Biol Chem. 2003 Aug 15;278(33):30896-904. DOI:10.1074/jbc.M303542200 | PubMed ID:12743123 | HubMed [Veal03]

All Medline abstracts: PubMed | HubMed