Liu J, Zhao X, Meng Y, Shen J, Xue Y, Shi S, Cai Y
Department of Genetics, Institute of Laboratory Animal Science, PUMC, CAMS, Beijing, 100021, China.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2000 Apr;22(2):111-4.
To clone complete EcoR II restriction endonuclease gene (ecoR II R) and methyl-transferase (ecoR II M) gene into one vector and to analyzing the expression of the whole system.
Unidirective deletion subclones constructed with Exo III, ecoR II R/M genes were preliminarily located in the cloned fragments according to the enzyme activities of each subclone, exact deletion sites were determined by sequencing, and transcriptional start sites were mapped by S1 mapping.
The DNA fragment which was cloned into pBluescript SK+ contained the complete ecoR II R gene and ecoR II M gene, there are two transcriptional start sites in ecoR II R gene, 132 bp to 458 bp from 3' and of ecoR II R gene are indispensable to enzyme activities and deletion of 202 bp from 3' end of ecoR II M gene made it lose the capability to resist specific cut of EcoR II R enzyme, deletion of coding region and flanking sequence of one gene did not affect the expression of the other gene, the recombinant only containing ecoR II R gene appeared to be lethal to dcm + host.
ecoR II M gene closely linking to ecoR II R gene was very important for the existence of the R-M system in process of evolution, but the key to control EcoR II R enzyme acted later than EcoR II M enzyme did not exist in transcriptional level.
