We chose to try direct genetic complementation of the Escherichia coli dapA- auxotroph lacking DHPS enzyme activity. The procedure was similar to that used for direct genetic selection of maize glutamine synthetase cDNA (Snustad et al. Genetics 120:1111, 1988). E. coli dapA- cells require diaminopimelate, a lysine precursor, for growth. These auxotrophic cells were transformed by electroporation with size fractionated Black Mexican Sweet cDNA in pUC13. Selection for transformed, complemented cells on minimal medium containing ampicillin resulted in identification of five colonies. Plasmid DNA isolated from each colony yielded complementation frequencies comparable to the transformation frequency. DHPS activity in the five complemented cell lines (Table 1) was inhibited 86-90% by 100 uM lysine as expected of maize DHPS, but DHPS activity in nontransformed wildtype dapA+ cells was inhibited less than 10%. No DHPS activity was detected in dapA- cells.
Table 1. DHPS activity in crude lysates
of transformed Escherichia coli strains.
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Cell line |
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Wildtype dapA+ |
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Auxotroph dapA- |
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Transformant 1 |
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Transformant 2 |
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Transformant 3 |
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Transformant 4 |
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Transformant 5 |
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The cDNA insert was isolated as an EcoRI
fragment from one of the cell lines and both strands were sequenced. The
start of the coding region for maize DHPS was established by comparing
the amino-terminus amino acid sequence of gel-purified DHPS monomer with
the amino acid sequence derived from the nucleotide sequence. The apoprotein
for DHPS is expected to have an amino-terminus transit sequence for transport
into the plastid, but the purified mature protein is not expected to have
the transit sequence. The amino-terminus sequence of Ala-Ile-Thr-Leu-Asp-Asp-Tyr-Leu
of the purified protein was identified in the cDNA sequence as indicated
in Figure 1. The nearest in-frame 5' ATG was located 162 bp upstream
(designated 1 in Fig. 1) indicating
that the transit peptide consisted of 54 amino acids. The complete DHPS
coding sequence for the apoprotein was 1140 bp. The predicted molecular
weight of the mature protein was 35,854, which was close to the 38,000
Mr estimate obtained from SDS-PAGE.
Figure 1. Representation of maize DHPS cDNA indicating the amino acid sequence of the mature protein and the corresponding cDNA sequence.
The cDNA clone hybridized to a single
transcript of about 1400 nucleotides in northern blots of immature embryo
and endosperm total RNA. A genomic clone containing at least part of the
coding region has been identified in an EMBL-3 library of W22.
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