| Summary: | 博士 === 國立清華大學 === 生命科學系 === 86 === The dapA gene of Escherichia coli encoding
dihydrodipicolinate synthase (DHDPS), the first enzyme of the
biosynthetic branch leading to diaminopimelate and lysine, was
cloned in this study. We amplified the dapA gene by PCR
(polymerase chain reaction) from a minilibrary of E. coli DH1
chromosome, inserted the PCR product into vector pUC18, and
confirmed the insert first by restriction patterns and finally
by DNA sequence analysis. Plasmid pCR13, the multicopy plasmid
carrying the dapA gene, wasintroduced into both E. coli AT998
and DH1. Expression of dihydrodipicolinate synthase was detected
from SDS-PAGE analysis and theenzyme activity of the crude cell
extract. SDS-PAGE analysis revealed aspecific band of about 35
kDa present on E. coli strains harboring pCR13.SDS-PAGE analysis
and enzyme assay showed that expression of the dapA geneon pCR13
varied with the host strains. The expression level was higher in
DH1 than in AT998. SDS-PAGE analysis and enzyme assay also
indicated thatincrease of the copy number of the dapA gene led
to increase of the DHDPS activity. Moreover, DHDPS activity was
inhibited 80 to 84 % by 20 mM lysinein vitro. A lysC-lac￠Z
fusion plasmid was constructed to study the regulatoryregion of
the lysC gene. Analysis by deletion mutations confirmed
theexistence of an alternative promoter, P2, located upstream of
the previouslyidentified promoter, P1. The transcription start
site of promoter P2 was located 85 base pairs upstream the
transcription start site of promoter P1.Both promoters are
regulated by lysine. The untranslated region of the lysCgene was
analyzed by Bal 31 nested deletion and specific-site insertion
mutations. It seemed that the regulation mechanism might not be
conventionalrepressor-operator binding.
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