| Summary: | 碩士 === 國立中興大學 === 分子生物研究所 === 85 === The tdh gene of Xanthomonas campestris pv. campestris 17 (Xc17)
was cloned and sequenced. The gene product has a high degree of
identity to the threonine dehydrogenase (TDH) of E. coli in
amino acid sequence. Primer-extension analysis showed that the
+1 site was located 42 bp upstream from the translational start
codon of the tdh gene in Xc17. The putative -10 (TATAAT) and
-35 (TGGCGC) regions were located bp -11 to -6 and bp -32 to -27
upstream of the transcriptional start site, respectively.
Deletion analysis demonstrated that the -10 sequence, but not
the -35 sequence was critical for the tdh promoter activity.
Two putative Lrp (leucine-responsive regulatory protein)-binding
sites were also located between bp -58 and -46 upstream and bp
+2 and +14 downstream from the transcriptional start site,
respectively. Presence of these sequences suggests that binding
of Lrp may be involved in the regulation of tdh expression in
Xc17. Similar to most of the X. campestris promoters, the tdh
promoter expressed poorly in E. coli. A putative terminator,
locating at 22 bp downstream of the translational stop codon,
was functional in terminator probe assay in E. coli and X.
campestris. These results suggest that transcription from the
tdh promoter may produce a monocistronic mRNA. This prediction
was confirmed by Northern-blot analysis which showed that the
transcript indeed had a size similar to that of the tdh coding
region. The activity of threonine dehydrogenase in the tdh
mutant, constructed by gene replacement, was found to be
regained in the presence of the cloned fragment containing the
complete tdh gene sequence and its promoter.
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