Yeast artificial chromosomes

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.

• pRML1 / pRML2 (Spencer93) are similar to pJS97/8 (Haldi96)
  • see sequence information here
  • see US Patent 5776745, Ketner et al. 1998
  • pRML1 vector NTI genbank format file: here
  • pRML2 vector NTI genbank format file: here
  • Knight:pRML1/2-sequencing

References

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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)

32. US Patent 5776745 Ketner et al. 1998