Development of backbone plasmids
The backbone of the pLL-EHC vector was constructed by linking two DNA fragments. The first fragment (6,212-bp) was isolated from the pBeloBAC11 vector  by digesting with Sal I and Pci I (Figure 1A). The second fragment was produced by multiple rounds of oligonucleotide-extension using six different primers. This fragment contains one I-Sce I site, two I-Ceu I sites, a lox71, an attP1 site, and a multiple cloning site (MCS) consisting of 16 unique restriction sites. The vector pLL-FF was developed by ligating the shared Sal I and Pci I sites between the synthetic fragment and the pBeloBAC11-derived fragment. A Hin dIII fragment containing the Egfp gene isolated from the Pk7GWIWG2D(II) vector (Invitrogen, Carlsbad, California)  was inserted into pLL-FF to create pLL-E. Finally, a Bst XI digested PCR fragment containing the Hpt gene, a plant selectable marker, from pHZWG7 , and a synthetic φC31 attP1 site were all inserted into PI-Psp I digested pLL-E to create pLL-EH (Figure 1). An ~110-kb fragment from the rice BAC OSJNBa0038J12 was then ligated into the Fse I site of pLL-EH to yield pLL-EHC.
To generate a telomeric DNA fragment, a PCR reaction was performed using a synthetic (TTTAGGG)11 DNA fragment as a template and a telomeric 25-mer (TTTAGGG)3TTTA) as a primer. The reaction was driven using Vent polymerase (1U) (New England Biolabs, Ipswich, Massachusetts) in a buffer containing 20 mM Tris-HCl (pH 8.8), 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton X-100, 1 mM dNTPs. The concentrations of both telomeric DNA fragments were 1 μM and the volume of the reaction was 5 μl. The amplified telomeric DNA fragments were cloned into the pGEM-T Easy vector (Promega, Madison, Wisconsin). One recombinant clone containing a 340-bp telomeric DNA fragment, pGEM-TT, was selected and confirmed by direct sequence analysis. The insert of the pGEM-TT plasmid was released using a Nde I and Xba I double digestion, blunt-ended using T4 polymerase (New England Biolabs), and subcloned into a Pst I-digested and blunt-ended pTLT plasmid. The pTLT plasmid is a modified pGEM-T Easy vector containing two additional Bsg I sites. This final clone was named pTLT-R11.
A PCR-based approach was used to insert the I-Sce I and attB1 sites into the pTLT-R11 plasmid. The following primers, which contain the attB1 and I-Sce I sites, were used in the PCR using pTLT-R11 as a template: TBS5' TTAGTCTCGAGACAAGTTTGTACAAAAAAGCAGGCTCTGCATGCCCTAAATCACTAGTGAATTCG; TBS3' TACTTCTCGAGACAAGTTTGTACAAAAAAGCAGGCTTGGTCTAGACCAAGATATCCTTGGC; TSB5' TTAGTCTCGAGTAGGGATAACAGGGTAATCTGCATGCCCTAAATCACTAGTGAATTCG. TSB3' TACTTCTCGAGTAGGGATAACAGGGTAATTGGTCTAGACCAAGATATCCTTGGC. The PCR fragments were digested with Xho I and self-ligated to yield the pLL-TBS and pLL-TSB plasmids.
Synthesis of back-to-back telomeric DNA fragments
To generate long telomeric DNA fragments, the short telomeric DNA inserts were released from pLL-TBS and pLL-TSB plasmids by digesting with Bsg I. Unidirectional telomeric DNA extension was performed using a 5'-(tTTACCC)12-3' oligonucleotide. The oligonucleotides and the released plasmid inserts were mixed at a 1:2 ratio in a 100 μl PCR reaction containing 50 mM Tris-HCl pH 9.1, 16 mM NH4SO4, 3.5 mM MgCl2, 150 μg/ml bovine serum albumin (BSA), 250 μM dNTPs, Klentaq (5 U) (Clontech, Mountain View, California), and Pfu polymerase (0.03 U) (Stratagene, La Jolla, California). The extended DNA fragments were purified and treated with Mung bean nuclease at 30°C for 30 min to remove any single stranded DNA. The DNA fragments were then treated with calf intestinal alkaline phosphatase (New England Biolabs) at 37°C for 60 min to remove a phosphate group, ensuring that one DNA fragment from pLL-TBS and one from pLL-TSB would be ligated in a back-to-back direction.
The extended telomeric DNA fragments were separated on 0.7% low-melting agarose gel. Electrophoresis was performed over night at 37 V. DNA fragments of 2 to 10-kb were excised from the gels. The telomeric DNA was purified from the agarose and concentrated using a Microcon YM-50 spin column (Amicon, Houston, Texas) according to the manufacturer's instructions. Equal amounts of the size-fractionated telomeric DNA derived from pLL-TBS and pLL-TSB were mixed and digested with I-Sce I in a total volume of 200 μl for 3 h. The homing endonucleases were heat inactivated, and ATP (Epicentre, Madison, Wisconsin) was added to a final concentration of 1 mM. The telomeric DNA was ligated overnight at room temperature using T4 DNA ligase.
Assembly of AC constructs
For the attB1 × attP1 recombination reaction, 500 ng of the attP1-containg pLL-EHC plasmid DNA and 100 ng of the attB-containing back-to-back telomeric DNA fragments were mixed with 4 μl each of 5 × BP clonase buffer and BP Clonase™ Enzyme Mix (Invitrogen), and adjusted to 20 μl with TE buffer. The mixture was allowed to react at 25°C for 16 h. After the recombinationreaction, the enzymes were inactivated by treatment with Proteinase K for 10 min at 37°C. Similar recombination reactions were also performed using the pLL-EHC vector and expansive telomeric DNA fragments derived from either pLL-TBS or pLL-TSB alone. This resulted in a polarized capping of the in linear centromeric DNA molecules with telomeric DNA at only one of the two ends.
The assembled linear AC constructs were separated by PFGE. The DNA band corresponding to the expected size of the linear AC constructs was excised from the gel and placed into 0.5 × TBE. The electro-eluted DNA was then dialyzed into ddH2O and concentrated using a Microcon YM-100 spin column (Amicon). The purified DNA fragments were used as a template for PCR analysis (see below). We developed a pLL-BKE plasmid as a control for artificial chromosome confirmation analysis. The pLL-BKE plasmid is a modified pLL-E vector with an attB1 site instead of attP1 site. Recombination between pLL-EHC and a linearized pLL-BKE (~10 kb), resulted in a linear molecule that is not capped with telomeric DNA.
Specific primers were designed from the junction regions between the pLL-EHC vector and the two telomeric DNA fragments as follow: L5 5'-TGATTTAGGGCATGCAGAGCCTGC-3', L3 5'-CTGTCAAGGGCAAGTATTGACATGT-3', R3 5'-TGATTTAGGGCATGCAGATTACCC-3', and R5 5'-TCATCTATGTTACTAGAGTACGCGC-3'. The positions of these primers are shown in Figure 6. PCR reaction mixtures contained 1 μM primers, 200 μM dNTPs, 0.01% gelatin, 2.5 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), and 1 U rTaq (Takara Bio, Shiga, Japan) in a final volume of 20 μl. Following an initial denaturing step at 95°C for 5 min, 35 amplification cycles of 30 s at 95°C, 30 s at 60°C and 40 s at 72°C, were followed with a final incubation at 72°C for 7 min.
Southern blot hybridization and fiber-FISH
Southern hybridization of DNA was performed using the digoxigenin (DIG) detection system (Boehringer Mannheim BV, Almere, The Netherlands). The probes used in the Southern hybridization were synthesized and DIG-labeled by random priming. The alkali-labile form of DIG-11-dUTP, the pRCS2 plasmid DNA insert containing the CentO repeats , and the Not I/Nhe I digested 140-bp telomere DNA fragments from pTLT-R11 were used as templates for random labeling with DIG. Hybridization conditions were carried out as recommended by the manufacturer.
Fiber-FISH analysis of assembled AC constructs was performed using published protocols . An appropriate amount of target DNA resulting from ligations between pLL-EHC and 4 to 8-kb of back-to-back oriented telomeric DNA, was directly dropped on a poly-lysine coated glass slide and a 18 × 18 cover glass was carefully placed on the top of the DNA drop. Slides were hybridized with a telomeric DNA probe and the pLL-EHC plasmid. The signals were detected following the procedure of standard fiber-FISH .