[Cloning of Erwinia chrysanthemi genes in escherichia coli]

Taichung NCHU Taiwan

1982

41p.

M.Sc

QH442.W3

The chromosomal DNA isolated from Erwinia chrysanthemi SR 120A was divided and digested with Bam HI for 30, 60, 90 and 180 min., so as to obtain some partial and some complete digestions. The digested DNA was ligated with plasmid pBR322, which had been cut with the same enzyme, by using DNA ligase, at 4 C for 30 hr. The ligated mixture were used to transform E. coli CS 412. 2418 transformants were obtained after scoring on L agar containing ampicillin; and 230 of them were scored as clones based on the insertional inactivation response. Chimeric plasmids were detected to exist in the clones. Among them, five were picked and designated pEC1, pEC2, pEC3, pEC4 and pEC5. The chimeric plasmids were isolated by a reapid procedure and digested with Bam HI. When the digestion mixtures were separated electrophoretically on agarose gel, a band corresponding to cut pBR322 was found in each sample, in addition to the inserted fragments. The molecular weight of the inserted DNA fragments from pEC1 was 3.5 Md; from pEC2 5.8 Md; from pEC3 8.1 Md; from pEC4 3.9 and 1.7 Md; and from pEC5 8.0 and 1.6 Md, respectively. Attempts were made to screen the clones expressing insertase or pectinase activities. Two was found to grow on a basal medium containing sucrose as the sole carbon, and one were found to grow on Na-polypectate. However, when the enzyme activities were assayed. none was detected. [AS]

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