Results
A restriction map is constructed of the unknown insert
given in Figure 1. In evaluating the agarose gel of the restriction digestion
process involving the insert and pZL1 plasmid, the recombinant plasmid was
found to be approximately 5.5 kb long while the 92K11T7 inset is about 1.5 kb
long. The map shows an internal restriction site for EcoRI. The Mac Vector
program gave the longest ORF and translated the nucleotide sequences
with corresponding amino acid sequences (Figure 2). Figure 3 shows the
hydropathy analysis and secondarry structures using the Protein
Toolbox Plot in Mac Vector [5]. From the graphs, there appears to be a
smaller percentage of hydrophilicity, low surface probability, and many long
stretches of helices/sheets/turns. All of this indicate that our protein is
perhaps a transmembrane protein with greater hydrophobic nature and tends to
be embedded within the membrane. The longest reading frame from 104 to 378
bases was used to perform homology searches. From BLASTN search in the
Arabidopsis EST analysis database , the highest match to our sequence was
clone 92K11T7, GenBank Accession number T20778 which gave a score of
416 and P value=3.4 x 10-26 but the other homologous sequence has a score of
117 and P=0.60. (Figure 4) The Southern analysis of A. thaliana genomic DNA
showed the hybridization to a 6kb EcoRI fragment, a 1.2kb BamHI fragment, and
a 1.5kb HindIII fragment (Figure 5). One copy of the gene is evident in the
dark bands. The lighter bands consist of homologous genes which appear
because of the low strigency wash. Using a higher stringency would result in
no extra bands. Figure 6 give Northern hybridization results where a 2.4 kb
transcript is detected in the wounded tissue after 5 hours but no gene
expression in samples containing whole plant, leaf, flower, and stem tissues.
This is due to bad transfer of the blot onto the nylon membrane for probing.
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