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KMID : 0364820160520040406
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Korean Journal of Microbiology
2016 Volume.52 No. 4 p.406 ~ p.414
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Construction and characterization of heterozygous diploid Escherichia coli
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Jung Hye-Im
Lim Dong-Bin
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Abstract
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Among 6 leu codons, CUG is the most frequently used codon in E. coli. It is recognized by leu-tRNA(CAG) encoded by four genes scattered on two chromosomal loci (leuT and leuPQV ). In the process of constructing a strain with no functional leu-tRNA (CAG) gene on chromosome, we made two mutant strains separately, one on leuPQV locus (ΔleuPQV ), and the other on leuT locus [leuT *(GAG)], where the anticodon of leuT was changed from CAG to GAG, thereby altering its recognition codon from CUG to CUC. We attempted to combine these two mutations by transduction using leuT *(GAG) strain as a donor and ΔleuPQV strain as a recipient. Large and small colonies appeared from this transduction. From PCR and DNA sequencing, large colony was confirmed to be the reciprocal recombinant as expected, but the small colonies contained both mutant leuT *(GAG) and wild type leuT (CAG) genes in the cell. This heterozygous diploid strain did not show any unusual morphology under microscopic observation, but, interestingly, it showed a linear
growth curve in rich medium with much slower growth rate than wild type cell. It always formed homogenous small colonies in the selection medium, but, when there was no selection, it readily segregated into leuT *(GAG) and leuT (CAG). From th ese observations, we suggested that the strain with both leuT *(GAG) and leuT (CAG) genes was not a partial diploid (merodiploid), but a full diploid cell having two different chromosomes. We proposed a model explaining how such a heterozygous diploid cell was formed and how and why its growth showed a linear growth curve.
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KEYWORD
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Escherichia coli, codon usage, CUG codon, diploid, leuT
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