YAC Protocols

372 Pages·1996·26.552 MB·English

by  David Markie

CHAPTER 1
Generation
of Large Insert YAC Libraries
Zoia Larin, Anthony P. Monaco,
and Hans Lehrach
1. Introduction
The introduction of yeast artificial chromosomes (YACs) as cloning
vectors in 1987 has significantly advanced the analysis of complex
genomes (I). The capability of cloning large DNA (1004000 kb) as
YACs has accelerated the construction of physical maps and contig build-
ing (a contiguous set of overlapping clones). YAC contigs now cover
entire human chromosomes (i.e., Y and 21) (2,3) and small genomes
(i.e., Schizosaccharomyces pombe) (4), and large YAC contigs cover
much of the human genome (5). The main advantages of YACs over
prokaryotic-based cloning systems are their large insert capacity and
ability to maintain sequences that are unstable or not well represented
in bacteriophage or cosmid genomic libraries (6). Therefore, YACs
complement existing cloning vectors (cosmids, bacteriophage) and new
cloning vectors (Pl bacteriophage [Pl], bacterial artificial chromosomes
[BACs], and Pl-derived artificial chromosomes [PACs]; for review, see
ref. 7) in mapping and chromosome walking projects (6,8).
Several laboratories have generated YAC libraries from different
eukaryotic genomes including arabidopsis (9), S. pombe (4), mouse
(IO, II), and human DNA (10,12,13). Libraries usually have been con-
structed in the Saccharomyces cerevisiae strain AB 1380, but other strains
are available with additional genetic markers that may be useful for
selection of products following homologous recombination of YACs
From Methods II) Molecular B/o/ogy, Vol. 54 YAC Protocols
Edlted by D Markle Humana Press Inc , Totowa, NJ
1
2 Larin, Monaco, and Lehrach
(14). In addition, recombination deficient yeast strains (radl or r&52)
have also been used to reduce the problem of chrmerism owing to recom-
bination in YACs (15), and these strains stabilize some sequences cloned
in YACs (16). Analysis of YACs maintained in rud52 and radl yeast
strains compared to standard strains indicate that the frequency of chi-
merism is lower (27). Different YAC vectors with centric and acentric
arms have been constructed that allow rescue of end fragments in yeast
for chromosome walking projects, and a bacteriophage T7 promoter for
generation of riboprobes from the rescued end fragments (14). Other
YAC vectors incorporate a conditional centromere that allows for ampli-
fication of YAC DNA under appropriate conditions (18).
YAC libraries have been constructed by preparing and size fractionat-
mg high molecular weight DNA in solution using sucrose gradients
(1,12), or in agarose by pulsed field gel electrophoresis (PFGE;
IO, 13,I9). When DNA is prepared in agarose, YAC insert sizes are larger
on average because shear forces seen with DNA in solution are mini-
mized. However, partial degradation of DNA occurs when melting aga-
rose containing high molecular weight DNA, perhaps due to metal ion
contamination or denaturation (10). The presence of polyamines (10) or
high concentrations of NaCl (100 mM) (20), protects DNA in agarose
from degradation at the melting step. The authors constructed mouse,
human, and S. pombe YAC hbrarres with average insert sizes of 700,
620, and 500 kb, respectively, by incorporating polyamines in the clon-
ing procedure (10).
This chapter describes in detail the protocols the authors used to con-
struct large insert YAC libraries. This includes preparation of pYAC4
vector partial digestion of genomic DNA in agarose blocks, size frac-
tionation by PFGE both before and after ligation to vector, and transfor-
mation of the yeast host AB 1380.
2. Materials
1. Preparationo f vector: All library construction protocols in this chapter are
basedo n the pYAC4 vector (I), avarlablef rom the American Type Culture
Collection. Vector DNA is prepared by large scale plasmid extractions and
purification by CsCl gradientc entrifugation (22).
2. Restriction enzyme digest buffers: For most restriction digests, buffers rec-
ommendedb y the manufacturera re adequateT. he authorsr ecommend T4
Large Insert YAC Libraries 3
polymerase buffer (21) when digesting vector DNA because it works with
almost all restriction enzymes and calf intestinal alkaline phosphatase (CIP;
Boehringer, Mannheim, Germany, 1 U/pL), thus eliminating precipitation
of DNA and buffer changes between enzyme reactions. 1O X T4 polymerase
buffer: 0.33M Tris-acetate, pH 7.9,0.66M potassium acetate, 0.1 OMmag-
nesium acetate, 0.005M dithiothrettol (DTT), 1 mg/mL bovine serum
albumin (BSA). Store frozen at -20°C m small abquots.
3. Preparation and lysis of cells in agarose blocks: High molecular weight
DNA from fibroblast or lymphoblastoid cell lines, whole blood, or fresh
mouse spleen tissue IS prepared in low melting point agarose blocks (‘221,
with 2-5 x lo6 cells/block (approx 1.5-40 pg DNA).
4. EcoRI partial digestion reaction buffer: 1 agarose block with DNA 80-
100 uL, 50 pL (5 mg/mL) BSA, 50 pL 10X EcoRI methylase buffer,
13 pL (O.lM) spermidine, 1 U EcoRI, 50-200 U EcoRI methylase (NEB),
distilled water to 500 pL final volume.
5. 10X EcoRI methlyase buffer: 800 pM S-adenosyl-methionine (SAM,
NEB), 0.02M MgCl,, 1. OM NaCl, OSM Tris-HCl, pH 7.5, O.OlM DTT.
Store frozen at -20°C in small aliquots.
6. 100X Polyamines: 0.075M spermidine-(HCl),, 0.03OM spermine-(HC1)4
Store frozen at -20°C m small aliquots.
7. 10X Ligase buffer: 0.5M Tris-HCl, pH 7.5, O.lM MgC12, 0.03M NaCI,
1O X polyamines.
8. YPD medium: see Chapter 29.
9. Regeneration plates (23): 1. OM sorbitol (Sigma, St. Louis, MO), 2% dex-
trose, 0.67% yeast nitrogen base without amino acids (Difco, Detroit, MI;
add as filter sterile after autoclaving of agar), 1X ammo acid supplements
(without uracil), 2% agar.
10. 1O X Amino acid supplements (23) : 200 pg/mL adenme, 200 pg/mL argi-
nine, 200 ug/mL isoleucine, 200 pg/mL histidine, 600 pg/mL leucine,
200 pg/mL lysine, 200 pg/mL methionine, 500 pg/mL phenylalanine,
200 yg/mL tryptophan (light sensitive, filter sterilize and store at 4OC),
1.5 mg/mL valine, 300 pg/mL tyrosine, 200 yglmL uracil (omit in regen-
eration and selective plates).
11. SCE: I.OM sorbitol, O.lMsodmm citrate, pH 5.8, O.OlMEDTA, pH 7.5,
0.03M 2-mercaptoethanol or O.OlM DTT (add fresh).
12. STC: 1. OM sorbnol, O.OlM Tris-HCl, pH 7.5, O.OlM CaCl,.
13. PEG: 20% Polyethylene glycol6000 (PEG, Serva, Heidelberg, Germany),
O.OlMTris-HCl, pH 7.5, O.OlMCaCl,. Make fresh and filter sterilize.
14. SOS: 1 .OM sorbitol, 25% YPD, O.O065MCaCl,, 10 ug/mL tryptophan,
1 pg/mL uracil. Make fresh and filter sterilize.
4 Larin, Monaco, and Lehrach
15. YAC selective media and plates: 2% dextrose, 0.67% yeast nitrogen base
without amino acids (add filter sterile), 1X amino acid supplements (with-
out uracil and tryptophan), 2% agar for plates.
16. Contour-clamped homogeneous electric field (CHEF) apparatus. The
authors recommend the BioRad (Richmond, CA) system.
17. Small horizontal gel electrophoresis apparatus: Use to check
restriction enzyme digests of vector and test ligations of vector and
genomic DNA.
18. Electrophoresis buffer: For both CHEF and horizontal gels, the authors
recommend TBE. 10X TBE: 0.89M Tris-borate, 0.89M boric acid,
0.016M EDTA.
19. Agarose: The authors recommend regular (SeaKern) and low melting point
(LMP) (Seaplaque GTG) agarose from FMC. Most gels will be 1% (w/v)
(aqueous).
20. Yeast and/or lambda concatamer size markers (BioRad).
21. Agarase (Sigma) dissolved in 50% (v/v) glycerol in water and store at
10 U/uL at -2OOC or P-agarase (NEB, Beverly, MA).
22. T4 DNA ligase (NEB) at 400,000 U/mL.
23. T4 polynucleotide kinase (NEB) at 10 U&L.
24. 1X TE: O.OlMTris-HCl, pH 7.5, O.OOlMEDTA, pH 7.5.
25. Proteinase K (Boehringer-Mannheim): Dissolve in water at 10 mg/mL and
store in small aliquots at -2OOC. Alternatively, use pronase (Boehringer-
Mannhelm). Add directly at 2 mg/mL.
26. Phenylmethylsulfonylflouride (PMSF, Sigma): Prepare at 40 mg/mL m
ethanol or isopropanol and heat several minutes at 68°C to dissolve. Cau-
tion: Use gloves. It is toxic.
27. 0.5M EDTA, pH 8.0.
28. Lyticase (Sigma): Weigh out fresh prior to spheroplast formation (500 U/
20 mL of yeast cells in SCE) and dissolve in SCE or water. Lyticase is
difficult to get in solution and will need extensive vortexing.
29. 2-Mercaptoethanol (BDH, London, UK): Open m hood and use gloves,
30. For the yeast transformation, a spectrophotometer, a student microscope
(lox, 25x, and 40x objectives and phase contrast), and a hemocytometer
cell counter are needed.
3 1. Phenol equilibrated with O.lM Tris-HCl, pH 8.0. Caution: Wear gloves
because phenol burns.
32. Chloroform.
33. 100% Ethanol.
34. Trinitriloacetic acid (BDH): Dissolve in water at O.lSMand store frozen in
small aliquots at -20°C. Used to inactivate CIP.
Large Insert YAC Libraries 5
3. Methods
3.1. Preparation of pYAC 4 Vector
1. Before preparing pYAC4 arms for ligation to genomic DNA, test plasmid
preps for deletions of telomere sequences during propagation in
Escherichia coli. Digest 0.5 pg of the pYAC4 plasmid with Hind111 and
check on a 1% agarose gel. Four bands should be visualized: a 3.5, 3.0,
1.9, and 1.4 kb doublet.
2. If there is an additional smaller fragment below the 1.4 kb doublet, then
telomere sequencesh ave been deleted from the plasmid and another prepa-
ration should be attempted.
3. For preparattve vector arms, digest 100-200 ug of pYAC4 with EcoRI and
BamHI to completion m 500 pL 1X T4 polymerase buffer and check on a
1% agarose gel. Three bands should be visualized: 6.0, 3.7, and 1.7 kb.
4. Heat kill the EcoRI and BamHI at 68OC for 10 min.
5. Add directly 0.03-0.06 U/ug vector of CIP and incubate at 37°C for 30 min.
6. Inactivate the CIP with trinitriloacetic acid to 0.015Mat 68OC for 15 min.
7. Extract twice with phenol, once with chloroform, and precipitate with ethanol.
8. Resuspend the vector arms at a concentration of 1 ug/uL in O.OlM Tris-
HCl, pH 7.5, and O.OOlMEDTA (1X TE).
9. Check the efficiency of dephosphorylation of vector ends and the ability of
these ends to ligate after phosphorylation. Set up two 20-uL ligation reac-
tions (2 pL 10X ligase buffer without polyamines, 0.5 ug of digested and
CIP-treated pYAC4 vector, 1 U T4 DNA ligase), one with and one without
1 U of T4 polynucleotide kinase.
10. Check hgations on a I % agarose gel:
a. Without kinase: 3 bands should be visualized as after digestton; and
b. With kinase.
The 1.7 kb BamHI fragment can ligate to itself and form several super-
coiled forms below 1.7 kb. The upper arms (6.0 and 3.7 kb) should ligate
together by their EcoRI and BarnHI sitesa nd form several larger fragments.
3.2. Partial Digestion of Genomic DNA
1. Partial digestion reactions: Prior to enzyme digestion, wash the blocks
containing genomtc DNA in 1X TE with 40 pg/mL PMSF at 50°C to inac-
tivate the proteinase K and twice in 1X TE to remove the PMSF. Blocks
incubated in pronase instead of proteinase K need only be washed exten-
sively in 1X TE.
2. Perform partial EcoRI digestions by incubating blocks with a combination
of EcoRI and EcoRI methylase. To determine the best mixture of the two
6 Larin, Monaco, and Lehrach
enzymes, set up analytical reactions of 1 U of EcoRI with 0, 20, 40, 80,
160,320, and 640 U of EcoRI methylase.
3. Place mdividual blocks in EcoRI partial digestion buffer (see Section 2.,
item 4) with the various combmations of EcoRI and EcoRI methylase
and incubate on ice for 1 h.
4. Transfer the reactions to 37°C for 4 h.
5. Add EDTA and protemase K to 0.02M and 0.5 mg/mL, respectively, to
terminate the reactions, and incubate at 37°C for 30 min.
6. Check partial digests on a 1% agarose gel m a CHEF apparatus with yeast
chromosomes as stze markers to see which combination of enzymes gives
most DNA in the range of 200-2000 kb.
7 Then digest many (6-l 2) blocks preparatively for the library construction
usmg several of the best enzyme combmations (usually 1 U EcoRI and 50--
200 U EcoRI methylase).
3.3. First Size Fractionation by PFGE
1. Pool blocks containmg partially digested DNA in a 50-mL Falcon tube
and wash once m 0.0 1M Tris-HCl, pH 7.5, and 0.05M EDTA.
2. Place blocks adjacent to each other in a trough m a 1% LMP agarose gel in
0.5X TBE, and preset for 1 h at 4°C. Place a genomic DNA block in the
adjacent gel slot on either side of the trough and place yeast chromosome
size markers in the outside gel slots.
3. Overlay the gel slots and trough wtth 1% LMP agarose. Subject the gel to
electrophoresis at 160 V (4.7 V/cm), using a switch time of 30 s (which
selects fragments 2400 kb) for 18 h at 15OC in a CHEF apparatus.
4. Remove the gel from the CHEF apparatus. Cut away only the outside lanes,
including one lane each of partially digested genomic DNA and yeast chro-
mosome size markers, and stain with ethidmm bromide (1 ug/mL) for 45
min. Keep the central portion of the preparative gel in 0.5X TBE plus
0.02M EDTA at 4°C.
5. Under UV light, notch the marker lanes at the edges of the limiting mobil-
ity (>400 kb) and take a photograph. Place adjacent to the central portion
of the preparative gel, cut out the limiting mobility using the notches m the
outside lanes as a guide, and place m a 50-mL Falcon tube. Stain all of the
remaining preparative gel with ethidium bromide and take a photograph.
3.4. Ligation to Vector
1. Equilibrate the gel slice (l-2 mL) containing the limiting mobility of
size-selected DNA four times (30 min each) in 1X ligase buffer (see Sec-
tion 2., item 7).
Large Insert YAC Libraries
2. Place the gel slice equilibrated m 1X ligase buffer in an Eppendorf tube
(cl mL agarose/tube) and melt at 68°C for 10 min together with digested
and CIP-treated pYAC4 vector (see Section 3.1.) m a ratio of 1: 1 by weight
of genomic DNA
3. Stir the vector and genomic DNA in molten agarose slowly with a pipet tip
and mcubate at 37°C for l-2 h.
4. Add T4 DNA hgase to 4 U/uL, ATP, pH 7.5 and DTT to O.OOlMeach m
1X hgase buffer by slow stirring at 37°C. Incubate the reaction at 37°C for
an additional 0.5-l h and then overnight at room temperature. For ligation
efficiency controls, see Note 2.
5. Termmate the reaction by adding EDTA pH 8.0 to 0.02A4.
3.5. Second Size Fractionation by PFGE
1. Melt the ligation reaction at 68°C for 10 mm and cool to 37°C.
2. Carefully pipet the molten agarose with a tip of bore diameter >4 mm mto
a trough m a 1% LMP agarose gel m 0.5X TBE, and preset for 1 h at 4°C.
Place some molten agarose ligation mix in the gel slots adjacent to the
trough on each side and place yeast chromosome size markers in the out-
side gel slots. Overlay the gel slots and trough with 1% LMP agarose.
3. Subject the gel to electrophoresis in a CHEF apparatus using the same
conditions as described in Section 3.3. for the first size fractionation.
4. Excise the limiting mobility as described m Section 3.3., step 5. If any
degradation of DNA is seen at this step, see Note 1.
5. Equilibrate the gel slice (approx 2-3 mL), containing the limiting mobility
from the second size fractionation, four times (30 min each) in 0.0 IMTris-
HCI, pH 7.5,0.03M NaCl, O.OOlM EDTA, and 1X polyamines.
6. Score the equilibrated gel slice with a sterile scalpel and place lesst han 1 mL
of agarose into individual eppendorf tubes. Melt at 68°C for 10 min, cool to
37°C and add agarase( Sigma 15&200 U/mL of molten agarose or P-agarase
20 U/mL of molten agarose).I ncubate at 37°C for 2-6 h prior to transformation.
3.6. Transformation
Transformation is carried out as described (24) using lyticase (Sigma)
to spheroplast yeast cells. The yeast strain S. cerevisiae AB1380 has
largely been used (I), but libraries have been prepared in recombination
deficient strains (1.5).
1. Streak a fresh YPD plate with the appropriate strain from a frozen glycerol
stock. Grow at 30°C for 2-3 d. Inoculate a single colony into 10 mL of
YPD. Let sit overnight at 30°C.
8 Larin, Monaco, and Lehrach
2. The next evening, inoculate 200 mL of YPD m a 1- L flask with 200 uL of
the 10 -mL overnight culture. Use a larger inoculum (1 /lo0 or l/500) if it is
a recombination deficient strain, because these cells tend to grow more
slowly. Shake at 30°C overnight for 16-l 8 h.
3. When the ODboO, ,,,,o f a l/l 0 dilution of the ABl380 culture IS between
0 12 and 0.15, spht the culture mto 50-mL Falcon tubes. Check some of
the culture under the mtcroscope for bacterial contamination.
4. Spm the tubes at 400-6OOg (3000 rpm on tabletop centrtfuge) for 5-
10 mm at 20°C. Decant media and resuspend pellets m 20 mL of distilled,
sterile water for each tube.
5. Spm 400-6OOg for 5-10 min at 20°C. Decant water and resuspend pellets
m 20 mL of 1 .OM sorbitol.
6. Spm 400-6OOg for 5-10 min at 20°C. Decant sorbrtol and resuspend pel-
lets in 20 mL SCE.
7. Add 46 uL of 2-mercaptoethanol and take 300 j.tL from one tube for a prelyti-
case control. Add 500 U lyticase (Sigma), mix gently, and incubate at 3O’C.
8. At 5, 10, 15, and 20 min, test the extent of spheroplast formation of one
tube by two independent methods:
a. Using a spectrophotometer, measure OD6a0n mo f a l/l0 dilution in dis-
tilled water. When the value is l/10 of the prelyticase value, sphero-
plast formation is 90% complete.
b. Mix 10 yL of cells with 10 uL 2% SDS and check under the micro-
scope using phase contrast.
When cells are dark (“ghosts”) they are spheroplasted.
9. Take the spheroplast formation to 80-90%. This should take 1O -20 min. Then
spin cells at 200-300g (1100 rpm on tabletop centrifuge) for 5 min at 20°C.
10. Decant SCE and resuspend pellets gently in 20 mL of l.OM sorbitol.
Spm 200-300g for 5 min at 20°C. Decant sorbitol and resuspend pel-
lets in 20 mL STC.
11. Take a cell count of one tube by making a l/l 0 to l/50 dilution in STC and
count on a hemocytometer.
12. Spin cells at 200-300g for 5 min at 20°C and then resuspend m a volume
of STC calculated for a final concentration of 4-O-6.0 x 10s cells/ml when
added to genomic DNA.
13. Add approx 0.5-l .O ug of DNA in digested agarose solution (50-75 uL) to
150 uL of spheroplasts in 15-mL conical polystyrene Falcon tubes. For
transformation controls, use:
a. No DNA;
b. 10 ng supercoiled YCp50 (25); and
c. 100 ng restricted and CIP-treated pYAC4.
Let DNA and spheroplasts sit for 10 min at 20°C.
Large Insert YAC Libraries
14. Add 1.5 mL PEG and mix gently by Inverting tubes. Let sit for 10 mm at
20°C. Spin at 200-300g for 8 min at 20°C.
15. Carefully pipet off PEG solution and do not disturb pellets. Gently resus-
pend pellets in 225 pL of SOS. Place at 30°C for 30 mm.
16. Keep molten top regeneration agar at 48°C. If usmg small plates, add 5 mL
of regeneration top agar (without uracil) to each 225 pL of SOS and cells.
If you are using large (22 x 22 cm) plates, pool 10 tubes of 225 uL of SOS
and cells to a 50-mL Falcon tube, and add 50 mL of regeneration top agar
(without uracil). Mix gently by inverting the tube and pour quickly onto
the surface of a prewarmed regeneration plate (without uracil) and let sit.
Incubate plates upside down at 30°C for 34 d.
17. YAC analysis and replication of transformants. Good transformation effi-
ciencies are between 2-8 x lo5 clones/ug YCp50 and 100-l 000 clones/pg
genomic DNA. For low transformation efficiencies, see Note 3. Ptck YAC
clones individually onto selective plates (without uracil and trypophan,
see Section 2., item 14) to test for both vector arms. When usmg mmlmal
adenine, visualize red color in YAC colonies containmg mserts Grow
YAC clones in selective media and make agarose blocks contammg chro-
mosomes to check the size of YAC clones by PFGE. To replicate clones
for library screening, pick YAC clones individually into mtcrotiter dishes
for screening of pools by polymerase chain reaction (PCR) amplification
(26) or by colony hybridization after spotting onto filters using manual
devices. A multipm transfer device, containmg 40,000 closely spaced pins,
has been used to efficiently replicate YAC clones from the supportive agar
matrix of regeneration plates to the surface of selective plates, for colony
hybridization and picking into microtiter dishes (10).
4. Notes
1. Degradation of DNA: If anywhere m the cloning procedure you encounter
complete or partial degradation of high molecular weight DNA, use yeast
chromosomes in a series of control reactions to pinpoint the problem.
Because yeast chromosomes can be visualized as distinct bands on PFGE,
degradation can be detected much easier than in partial digests of genomic
DNA. Test all buffers and enzymes (EcoRI methylase, T4 DNA hgase,
proteinase K, agarase) for nuclease activity in mock cloning experi-
ments using yeast chromosomes. Also, melt agarose blocks containing
yeast chromosomes in buffers with and without 1X polyamines to test for
partial degradation.
2. Ligation controls for vector and genomic DNA: Test the efficiency of hga-
tion of vector arms to partially digested genomic DNA by incubating a
small sample of the ligation reaction with and without 1 U T4 polynucle-
10 Larin, Monaco, and Lehrach
ottde kmase. Melt the samples and load them on a small 1% agarose gel to
check for no change of vector arms without kmase and disappearance of
vector arms to larger sized fragments when incubated with kmase.
3 Transformation efficiency: If your transformation efficiencies are routinely
lower than expected, check the followmg:
a. Always streak the yeast strain onto a fresh YPD plate before settmg up
cultures. Cultures grown from old plates (>2 wk) seem to transform
less well although they will appear to spheroplast normally.
b. Try different concentrations of lyticase and percent spheroplast forma-
tion for optimum efficiency.
c. Try various batches of sorbitol and PEG to see if there is any difference
in transformatton efficiency.
d. Always use distilled, deionized water to guard against heavy metal
ion contammation that can degrade DNA or decrease transforma-
tion efficiency.
e. Check the temperature of room. Transformation is best at 20-22°C and
decreases dramatically at temperatures around 30°C.
References
1 Burke, D T , Carle, G F , and Olson, M V. (1987) Cloning of large DNA seg-
ments of exogenous DNA mto yeast by means of artificial chromosome vectors
Science 236,806-8 12.
2 Foote, S , Vollrath, D , Hilton, A., and Page, D. C (1992) The human Y chromosome
overlappmg DNA clones spanning the euchromatic region Sczence 258,60-66
3 Chumakov, I , Rigault, P , Gmllou, S , Ougen, P , Billaut, A , Guascom, G , et al
(1992) Continuum of overlappmg clones spanning the entire human chromosome
21q. Nature 359,38&387.
4. Maier, E., Howeisel, J , McCarthy, L., Mott, R , Grigortev, A. P., Monaco, A. P.,
Larm, Z., and Lehrach, H (1992) Complete coverage of the Schzzosaccharomyces
pombe genome m yeast artificial chromosomes. Nature Genet 1,273-297.
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map of the human genome. Nature 366,698-70 1,
6 Coulson, A., Waterston, R , Klff, J., Sulston, J , and Kohara, Y. (1988) Genome
lmkmg with yeast artificial chromosomes Nature 335, 184-I 86
7. Monaco, A. P and Larm, Z. (1994) YACs, BACs, PACs and MACs artificial
chromosomes as research tools Trends BzotechnoZ 12,280-286.
8. Garza, D , Ajioka, J W , Burke, D T., and Hart& D. L (1989) Mapping the Droso-
phtla genome with yeast artitictal chromosomes. Sczence 246,641&646.
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10 Larm, Z., Monaco, A P , and Lehrach, H. (1991) Yeast artificial chromosome
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