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Construction of YAC Libraries with Large Inserts

Simon Foote1,  Christopher Denny2

1Whitehead Institute, Cambridge, Massachusetts
2University of California at Los Angeles, School of Medicine, Los Angeles, California


Publication Name: 
Current Protocols in Human Genetics
Unit Number: 
Unit 5.2
DOI: 
10.1002/0471142905.hg0502s31
Online Posting Date: 
February, 2002
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Abstract

The yeast artificial chromosome (YAC) cloning system makes it possible to clone large pieces of genomic DNA into yeast. Libraries have been made containing clones with inserts in the megabase-pair range. The basic protocol in this unit describes preparation of YAC vectors and transformation of ligated DNA into yeast spheroplasts. A support protocol describes titration of Lyticase to make spheroplasts. Additional support protocols detail two methods for partial digestion of genomic DNA: EcoRI restriction endonuclease-EcoRI methylase competition and the partial digestion of genomic DNA by use of limiting amounts of Mg2+, respectively.

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol: Construction of a Yeast Artificial Chromosome Library
  • Support Protocol 1: Titration of Lyticase
  • Support Protocol 2: Partial Digestion of Chromosomal DNA Using Restriction Enzyme–Methylase Competition
  • Support Protocol 3: Partial Restriction Enzyme Digestion by Mg2+ Limitation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol: Construction of a Yeast Artificial Chromosome Library

 Materials
For recipes, see Reagents and Solutions in this unit (or cross-referenced unit); for common buffers and stock solutions, see appendix 2D ; for suppliers, see suppliers appendix.
  • E. coli strain carrying a pYAC4 vector (Figs. 5.2.1 and 5.2.2; ATCC #67379)
  • 20 U/µl HindIII, BamHI, and EcoRI restriction endonucleases and appropriate buffers
  • DNA molecular size markers
  • 1 U/µl calf intestine phosphatase (CIP; Boehringer Mannheim)
  • TE buffer
  • Size-selected, partially digested insert DNA in agarose block (second or third support protocol)
  • 1× ligase buffer: 50 mM Tris×Cl (pH 7.6)/10 mM MgCl2 (store at room temperature)
    100 mM ATP (appendix 2)
  • 1 M DTT
  • 400,000 U/ml T4 DNA ligase (in cohesive-end units; New England Biolabs)
  • SeaPlaque GTG agarose (FMC Bioproducts)
  • 0.5× TBE buffer (appendix 2)
  • 0.5 µg/ml ethidium bromide solution (appendix 2)
  • T50E buffer (see recipe)
  • Agarase buffer (see recipe)
  • 100 mM spermidine (store at –20°C)
  • 1000 U/ml -agarase (New England Biolabs)
  • Yeast strain AB1380 (ATCC #20843)
  • YPD medium (unit 5.5)
  • 1 M sorbitol
  • SCE buffer (unit 5.1)
  • 2-mercaptoethanol (2-ME)
  • 10,000 U/ml Lyticase (see first support protocol; Sigma)
  • STC buffer (see recipe)
  • Calf thymus DNA, purified (see recipe)
  • 20% (w/v) polyethylene glycol (PEG) 8000 solution
  • SOS medium (see recipe)
  • –Ura top agar, prewarmed to 50°C (see recipe)
  • 150-mm –Ura SORB dropout plates, 30°C (see recipe)
  • AHC plates (unit 5.5)
  • Glycerol, sterile
  • 50-ml polypropylene centrifuge tubes (Falcon)
  • 4- and 12-ml polypropylene tubes (Falcon), sterile
  • 50° and 68°C water baths
  • Pulsed-field gel electrophoresis (PFGE) apparatus (e.g., Bio-Rad CHEF)
  • 30°C humidified incubator
  • Beckman G6-6R centrifuge with GH-3.8 rotor (or equivalent)
  • 1000-µl wide-bore pipet tips
  • Dissecting microscope
  • Sterile toothpicks or plastic inoculating loops (VWR Scientific)
  • 96-well tissue culture plates (Costar)
  • Transtar device with elevator (Costar)
  • Adhesive sealing membrane (Costar)
  • 96-pin replicator (Dan Kar Scientific)
  • Additional reagents and equipment for CsCl plasmid preps (unit 5.3 and cpmb unit 1.7), agarose gel electrophoresis (unit 2.7), phenol extraction, ethanol precipitation, and quantitation of DNA (appendix 3C & 3D), and PFGE (unit 5.1)
CAUTION: Ethidium bromide and 2-mercaptoethanol are hazardous; see appendix 2A for guidelines on handling, storage, and disposal.

Support Protocol 1: Titration of Lyticase

 Additional Materials
For recipes, see Reagents and Solutions in this unit (or cross-referenced unit); for common buffers and stock solutions, see appendix 2D; for suppliers, see suppliers appendix.
  • Yeast strain containing control YAC
  • 0.03 ng/µl YCp50 plasmid DNA in agarase buffer (see recipe)
    Additional reagents and equipment for preparing YAC DNA in agarose blocks (unit 5.1)

Support Protocol 2: Partial Digestion of Chromosomal DNA Using Restriction Enzyme–Methylase Competition

 Additional Materials
For recipes, see Reagents and Solutions in this unit (or cross-referenced unit); for common buffers and stock solutions, see appendix 2D; for suppliers, see suppliers appendix.
  • Mammalian cells or tissue
  • EcoRI compromise buffer (prepare fresh; see recipe)
  • 40 U/µl EcoRI methylase (New England Biolabs)
  • Yeast chromosomal DNA size standards in agarose blocks (unit 5.1)
  • 100 mM S-adenosylmethionine (SAM; Sigma)
    Additional reagents and equipment for preparation of high-molecular-weight mammalian genomic DNA in agarose blocks(unit 5.1)
    CAUTION: Human cells and tissue are hazardous; see appendix 2A for guidelines on handling, storage, and disposal.

Support Protocol 3: Partial Restriction Enzyme Digestion by Mg2+ Limitation

 Additional Materials
For recipes, see Reagents and Solutions in this unit (or cross-referenced unit); for common buffers and stock solutions, see appendix 2D; for suppliers, see suppliers appendix.
  • Mammalian cells or tissue
  • EcoRI(–Mg2+) buffer (see recipe)
  • 10 and 100 mM MgCl2 (store at –20°C)
  • 100 mM EDTA, ice-cold (store at 4°C)
  • 15-ml conical tubes, sterile
  • 2-ml flat-bottom microcentrifuge tubes, sterile
  • Additional reagents and equipment for preparation of high-molecular-weight DNA in agarose blocks (unit 5.1)

CAUTION: Human cells and tissue can be hazardous; see appendix 2A for guidelines on handling, storage, and disposal.

Looking for materials?  Suppliers Guide
     
 
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Figures

  • Figure 5.2.1
    pYAC4. The pYAC4 vector, propagated as a circular plasmid in E. coli. It contains a unique EcoRI cloning site in the SUP4 gene, as well as ARS1 and CEN4 elements required for stable single-copy propagation of the artifical chromosome and the TRP1, HIS3, and URA3 selectable markers. TEL sequences are derived from Tetrahymena telomeres and function as telomeres in yeast. (See Fig. 5.2.2 for restriction maps of ARS1, CEN4, TRP1, HIS3, and URA3). Selected restriction sites (not necessarily unique) are indicated. Solid bars are chromosomal function elements; stippled bars are yeast genes; open bar is the amp sequence from pBR322. See Burke et al., 1987, for a discussion of the construction of the vector. To clone an insert, the vector is digested with BamHI (which cuts adjacent to TEL sequences) and EcoRI. The resulting vector arms, which contain either TRP1, ARS1, and CEN4 or URA3, are ligated to insert fragments with EcoRI-compatible ends. Ligation products are transformed into ura3– trp– ade2-1 spheroplasts and screened selecting for Ura+ and subsequently Trp+ to ensure the presence of both vector arms. Transformants are screened for the presence of inserts in SUP4 using a color assay: colonies with the ade2-1 ochre mutation suppressed by SUP4 are white, while colonies with inserts in SUP4 are red because SUP4 is inactivated.

    View Image
  • Figure 5.2.2
    Restriction enzyme maps for yeast CEN4, TRP1/ARS1, URA3, and HIS3 in pYAC4. Restriction enzyme–digestion of pYAC4 yeast genes can be used to generate hybridization probes for yeast sequences.

    View Image
  • Figure 5.2.3
    Pulsed-field gel analysis of partial digestion pattern using EcoRI as described in the third support protocol. Increasing Mg2+ concentration results in greater digestion of DNA. The first and last lanes contain intact yeast chromosomes from S. cerevesiae strain YNN 295 as size markers.

    View Image

Literature Cited

Literature Cited
    Albertsen, H.M., Le Paslier, D., Abderrahim, H., Dausset, J., Cann, H., and Cohen, D. 1989. Improved control of DNA restriction digestion in agarose using limited concentrations of Mg++. Nucl. Acids Res. 17:808.
    Albertsen, H.M., Abderrahim, H., Cann, H.M., Dausset, J., Le Paslier, D., and Cohen, D. 1990. Construction and characterization of a yeast artificial chromosome library containing seven haploid equivalents. Proc. Natl. Acad. Sci. U.S.A. 87:4256-4260.
    Burgers, P.M.J. and Percival, K.J. 1987. Transformation of yeast spheroplasts without cell fusion. Anal. Biochem. 163:391-397.
    Burke, D.T., Carle, G.F., and Olson, M.N. 1987. Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science 236:806-812.
    Cooke, H. and Cross, S. 1988. pYAC-4 Neo, a yeast artificial chromosome vector which codes for G418 resistance in mammalian cells. Nucl. Acids Res. 16:11817.
    Shero, J.H., McCormick, M.K., Antonarakis, S.E., and Hieter, P. 1991. Yeast artificial chromosome vectors for efficient clone manipulation and mapping. Genomics 10:505-508.
    Smith, D.R., Smyth, A.P., and Moir, D.T. 1990. Amplification of large artificial chromosomes. Proc. Natl. Acad. Sci. U.S.A. 87:8242-8246.
 Key References
    Albertsen et al., 1989. See above.

Mg2+ limitation for genomic partial digestion.

    Albertsen et al., 1990. See above.

The first description of use of PFGE to size select DNA for ligation.

    Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (eds.). 1993. Current Protocols in Molecular Biology. Greene Publishing Associates and John Wiley & Sons, New York.

Good reference for elementary yeast-associated techniques.

    Burgers and Percival, 1987. See above.

A detailed description of the factors that influence spheroplast transformation.

    Burke et al., 1987. See above.

Original description of YACs and their construction, still of interest even though the method of making YACs has changed.

     
 
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