Isolation of High Molecular Weight (HMW) BAC DNA: Three BAC clones, in the 2 Mb contig T7D17, T7M7, and T14P1 and one outside this region, T8P21, were chosen from the TAMU Arabidopsis library (Choi et al.,1995) for sequence analysis. F27I1 was from the IGF library (Mozo et al.,1998). HMW BAC DNA was isolated by a modified alkaline lysis method. A single colony of a BAC clone was picked from a Luria-Bertani (LB) agar plate containing 12.5 mg / ml chloramphenicol, inoculated into 5 ml of LB medium with antibiotics and cultured overnight (O/N) at 370 with shaking at 250 rpm. For large scale DNA preparation, 0.5 ml of cell culture was inoculated into 500 ml of LB medium solution with the same antibiotics and cultured as above for 14 hr. The cultured cells were transferred into two centrifuge bottles (250 ml) and centrifuged at 5000 rpm at 40 for 15 min to pellet the cells. The cell pellets were combined, resuspended in 40 ml of cold (40) 10 mM EDTA, pH 8.0, mixed and left at room temperature (RT) for 5 min. Eighty ml of fresh 0.2 M NaOH, 1% SDS was added to the solution, gently mixed 3-4 times by inversion and left at RT for 5-10 min. Sixty ml of cold 1.875 M K-acetate, 11.5% acetic acid was added to the solution, mixed gently by inversion and incubated on ice for 5-10 min. The lysed cell solution was centrifuged at 10,000 rpm at 40 for 15 min. The supernatant was transferred and filtered through cheesecloth into another centrifuge bottle. Ninety ml of isopropanol was added to the solution, mixed and centrifuged at 5000 rpm for 15 min. The pellet was resuspended in 18 ml of 50 mM Tris, 50 mM EDTA, pH 8.0. Nine ml of 7.5 M K-acetate was added, and the solution was transferred into Oak ridge centrifuge tubes and chilled at -800 for 30 min. The tubes were quickly thawed and centrifuged at 5000 rpm at 40 for 10 min. The supernatant was transferred into another tube, 54 ml of 100% ethanol added, and spun at 5000 rpm at 40 for 10 min. The supernatant was removed and the pellet dissolved in 1.4 ml of 50 mM Tris, 50 mM EDTA. Twenty m l of RNase (10 mg /ml) was added and incubated at 370 for 1 hr. The DNA solution was then extracted with phenol, pH 7.4, phenol / chloroform / isoamylalcohol (25:24:1), and chloroform / isoamylalcohol (24:1). DNA was precipitated by additon of an equal volume of isopropanol, and washed with 70% ethanol. The DNA pellet was dried at RT for 15 min, dissolved in 100 m l of 1X TE and stored at 40 for fragmentation and size-selection.
Fragmentation and size-selection of BAC DNA: HMW BAC DNA was fragmented by nebulization. BAC DNA (4-6 m g) was resuspended in 2 ml of 50 mM Tris, pH 8.0, 15 mM MgCl2, 25% glycerol and transferred into a prepared nebulizer (IPI medical product, # 4207). For preparation of the nebulizer, tubing was removed, excessive material from the plastic edge of a soft plastic cone insert was trimmed using a razor blade so that the cone could be inverted inside the nebulizer. The smaller opening faced towards the top and sealed against the lid. The assembled nebulizer was placed on ice and attached to a liquid nitrogen tank (AIRCO, #U.N.1066) by tubing. DNA was nebulized for 150 sec at 30 psi at the tank outlet. The fragmented DNA sample (1800 ml) was collected and concentrated to 400 m l by 5-6 butanol extractions. Concentrated DNA solution was mixed with 2.5 volumes of 100% ethanol and kept at -200 for 2 hr, then pelleted by microcentrifugation at 40 for 15 min. The DNA pellet was washed with 70% cold ethanol, dried at RT for 15 min and then dissolved in an appropriate volume (~100 m l) of 1X TE. Ends of the fragmented DNA (~1.5 m g) were filled by Pfu DNA polymerase (Stratagene, #200409). The reaction solution was loaded onto a 1%, 8 cm long low melt pointing (LMP) agarose (Kodak, # IB70050) gel in 1X TBE and DNA was separated on the gel. Gel slices containing DNA fragments ranging from 500 bp to 3.0 kb were excised on a long UV light (366 nm) box with a razor blade when the loading dye had migrated ~ 2/3 of the way into the gel. The gel slice was recast in another 1% LMP agarose gel and run in the opposite direction to concentrate the DNA into a very thin band. The DNA band was excised and melted at 650 for 10 min. The agarose in the solution was digested with b-agarase I (NEB, # 392S) at 400 for 2 hr. The digested agarose solution was chilled on ice for 15 min and microcentrifuged at 40 for 15 min to pellet the undigested agarose. The supernatant was transferred into another eppendorf tube and extracted with an equal volume of phenol / chloroform, then chloroform. The extracted supernatant was mixed with 2 volumes of isopropanol, chilled on ice for 30 min and pelleted by microcentrifugation at 40 for 15 min. The pellet was washed with 70% cold ethanol, dried at RT for 15 min, dissolved in 20 m l of 1X TE and stored at 40 for later ligation.
Preparation of vector DNA, ligation and transformation: pBluscript II KS+ vector (Stratagene, #212207) DNA was digested with EcoRV at 370 for 3 hr, and dephosphorylated with Calf Intestinal Alkaline Phosphatase, (CIP) (NEB, #290S). The dephosphorylated vector DNA (~100 ng) was mixed with the size-selected BAC DNA (~100 ng) and ligated with T4 DNA ligase (NEB, #202S) at 160 O/N. After ligation, the ligase was heat-inhibited at 650 for 10 min. DNA in the ligation solution was electro-transformed into E. coli DH5a competent cells by using a Bio·RAD gene pulser. Transformants were recovered in 1 ml of SOC (2% Bacto tryptone, 0.5% Bacto yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 10 mM glucose) at 370 with shaking at 300 rpm for 1 hr. Transformants in recovery solution were mixed with an equal volume of 30% glycerol, divided into aliquots (100 ml) and stored at -800 for later use.
Preparation of plasmid DNA from subclones and sequencing: The frozen transformant stock was thawed briefly and diluted 50 times with LB liquid medium and then plated onto selective LB agar plates containing 50 m g / ml ampicillin. Recombinants were identified using a blue-white screening system. White colonies were inoculated into 96-well titer plates (each well containing 1.3 ml of liquid LB medium) and cultured at 370 with shaking at 300 rpm for 22 hr. Plasmid DNA was isolated by using rapid extraction alkaline plasmid kits (QIAGEN #26173), and dissolved in 50 ml of double distilled water for later use as DNA sequencing templates. DNA was sequenced on a 377 DNA sequencer (ABI prismTM). The Phred, Phrap and Consed sequence assembly and viewing programs (Phil Green unpublished; Ewing et al.,1998; Ewing and Green,1998; Gordon et al.,1998) were used to remove BAC vector sequences and to assemble contigs. After large sequence contigs were assembled and only a few gaps remained the gaps were closed by amplifying the intervening DNA by PCR from the original BAC DNA using primers based on adjacent sequences. The PCR products were sequenced on a long-range sequencer ABI 377 using dye terminator chemistry as described by the manufacturer (Perkin Elmer).
Gene prediction from the BAC sequences: Putative genes were identified using a combination of three gene prediction programs. Grail (Xu et al., 1994) was run using client server software, and selecting for Arabidopsis as the DNA source. Gene assembly for the Grail program requires the investigator to define a region of the DNA sequence to assemble. This was done somewhat subjectively based on the clustering of the predicted exons. Genscan (Burge and Karlin,1997) was run with the Arabidopsis organism option and an exon cutoff of 1.0. Genefinder (Phil Green, personal communication) was run with the Arabidopsis tables developed by ESSA (European Scientists Sequencing Arabidopsis). As partial confirmation of the predicted genes, the BAC sequence was compared to sequences deposited in Genbank using blastn and blastx (Gish et al.,1993; Altschul et al.,1990).
This work was supported by a grant from Hoechst to H.M.G.
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