Expression and deletion analysis of EcoRII endonuclease and methylase gene.
OBJECTIVE
To clone complete EcoRII restriction endonuclease gene (ecoRIIR) and methyltransferase gene (ecoRIIM) in one vector and to analyze the coordinating expression of this whole R-M system.


METHODS
Unidirectional deletion subclones were constructed with ExoIII. ecoRIIR/ M genes were preliminarily located in the cloned fragment according to the enzyme activities of subclones. Exact deletion sites were determined by sequencing, and transcriptional start sites were determined by S1 mapping.


RESULTS
The DNA fragment which was cloned into pBluescript SK + contained intact ecoRIIR gene and ecoRIIM gene, and two transcriptional start sites of ecoRIIR gene were determined. 132bp to 458bp from 3'end of ecoRIIR gene are indispensable to enzyme activities and deletion of 202bp from 3' end of ecoRIIM gene made enzyme lose the capability in DNA protection to resist specific cut with EcoRII endonuclease (EcoRII. R). Deletion of the coding and flanking sequences of one gene did not affect the expression of the other gene, and the recombinants only containing ecoRIIR gene appeared to be lethal to dcm+ host.


CONCLUSIONS
ecoRIIM gene linking closely to ecoRIIR gene is very important for the existence of the R-M system in process of evolution, but the key to control EcoRII R-M order may not exist in transcriptional level .
Liu
J
J
Department of Genetics, Institute of Laboratory Animal Science, CAMS & PUMC, Beijing 100021.
Zhao
X
X
Meng
Y
Y
Shen
J
J
Xue
Y
Y
Shi
S
S
Cai
Y
Y
D001483:Base Sequence; D003001:Cloning, Molecular; D015257:DNA-Cytosine Methylases / Q000235:genetics*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D017421:Sequence Analysis
C029528:DNA modification methylase EcoRII; D015257:DNA-Cytosine Methylases; D015252:Deoxyribonucleases, Type II Site-Specific