Materials and Methods

DNA Insert Isolation and Transformation:

A pZL1 plasmid DNA containing an unidentified cDNA insert was obtained from the Arabidopsis Biological Resource Center at Ohio State University. This plasmid DNA, contained in host E.coli cells, was isolated by the alkaline-lysis method. DNA contaminants such as RNA, proteins, and lipids were removed by RNAse treatment and phenol/chloroform extraction [3,6]. The pZL1 plasmid DNA and a given pBluescript vector DNA were digested using three restriction enzymes (EcoRI, BamHI, and Hind III). Using an E.coli host, the insert and vector DNA were ligated together and transformed. The cells were then plated on LB plates containing ampicillin, IPTG, and X-gal. White transformants were subjected to minipreparations to check for actual recombinants [6].

Miniprep of Plasmid DNA and Polymerase Chain Reaction:

The plasmid DNA was again isolated but with a commercial QIAquick Gel Kit [8].This simple and quick procedure allowed for pure elution of the plasmid DNA from RNA, proteins, and other contaminants. For PCR T3 and T7 primers were used to amplify the DNA template along with a thermostable Taq DNA polymerase. Reactions were placed in a thermocycler set for 32 cycles for denaturation, annealing, and extension steps [4].

DNA Sequencing and Computer Analysis:

In sequencing of the DNA, the dideoxy chain termination method of Sanger was used along with Sequenase [9,10]. These samples underwent Polyacrylamide (Long Ranger) Gel Electrophoresis for manual sequencing and data from automated sequencing were entered into Mac Vector software programs for analysis of the best open reading frames and translation into amino acid sequences [5]. ]. Searches for homologous sequences for DNA and protein were utilized in Blast data banks and other Arapidopsis thaliana programs on the Internet [1,7].

Isolation of Genomic DNA and Southern Hybridization:

2 g of frozen Arabidopsis plant tissue was ground in liquid N2. RNA contamination was removed by SDS/NaOH and phenol/chloroform extraction while DNA was separated out by PEG precipitation. This DNA was digested with EcoRI, HindIII, and BamHI restriction enzymes, fractionated on a 0.8% agarose gel, and transferred to nylon membrane by capillary transfer in the Southern technique. Using the random primer method [6],a probe labeled with 32-P dATP was prepared using the EcoRI fragment for hybridization to the Southern blot. A low stringency wash of the blot was performed at 65 degrees C.

Isolation of Total RNA and Northern Hybridization:

Various whole plant, leaf, flower, and stem tissues from Arabidopsis thaliana were subjected to grinding in liquid nitrogen and extraction by phenol/chloroform in SDS/EDTA to purify total RNA. Wounded plant tissues exposed at 0, 1, 5, and 8 hours were also used to isolate the RNA which was fractionated on a 1.2% formaldehyde agarose gel to check quality and then transferred to nylon membranes through Northern transfer. Hybridization of the Northern blot using the EcoRI probe was performed in the same manner as prepared from the Southern experiment [6].