Title Cloning and Nucleotide Sequuence Analysis of xylE Encoding Catechol 2,3-dioxygenase from Pseudomonas putida SU10
Author Kim, Yeon Jung¹ · Lee, Byeong Jae * · Min, Kyung Hee¹
Address ¹Department of biology, Sookmyung Women’s University; ¹Research Center for Molecular Microbiology, Institute for Molecular Biology and Genetics, Seoul National University
Bibliography Korean Journal of Microbiology, 32(6),471-479, 1994
Key Words XylE, catechol 2,3-dioxygenase, Pseudomonas putida
Abstract Pseudomonas putida SU10 isolated from Han River carries a TOL plasmid, which has been confirmed to clone xylE gene encoding catechol 2,3-dioxygenase enzyme. This plasmid was partially digested with Sau3AI, followed by insertion of the resulting DNA fragment into BamHI site of pBluescript SK+vector. The xylE gene was screened from E. coli DH5α transformed with recombinant plasmids. The resulting clones containing xylE gene were able to convert catechol to a yellow hydroxymuconic semialdehyde. In order to isolate the functional area of xylE gene, recombinant plasmid DNAs were mapped with restriction endonuclease, AvaI. Among the 11 recombinant plasmid DNAs, restriction endonuclease mapping of 2kb of insert DNA in the recombinant plasmid, pTY1, was carried out with PvuII, XhoI, AvaI. By means of ExoIII deletion mutagenesis to both orientation, the functional region of xylE was found to be about 1kb in size including two internal PvuII sites. We determined the nucleotide sequence of 2kb fragment DNA containing xylE gene. One open reading frame of 921 bp was found and specially another ORF is located on reverse orientation. Amino acid sequences of this ORF were highly homologous with other genes encoding catechol 2,3-dioxygenase previously published.
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