A Novel Single-Strand Specific 3′–5′ Exonuclease Found in the Hyperthermophilic Archaeon, Pyrococcus furiosus
Figure 1
Screening and identification of the gene responsible for the DNA cleavage activity.
(A) The E. coli cells, transformed with pUC118 containing each PstI-digested DNA fragment from the positive cosmid clone, were heated. The heat-stable cells extracts from each clone were assayed for DNase activity, using 32P-labeled d27 as the substrate. The reactions were analyzed by PAGE on a 12% gel containing 8 M urea followed by autoradiography. Lane M shows the GA ladder of d49R, generated by the Maxam-Gilbert reaction. The other lanes (1–12) are labeled d27 treated with heat-stable cell extracts from different clones. (B) The ORFs identified on the PstI-DNA fragment obtained from the positive clone #8. (C) The 5 full-length ORFs on the PstI fragment in the positive clone were individually cloned and expressed in E. coli, and the same DNase assay was performed using 32P-labeled d49R as the substrate. PF2046 was identified as the gene responsible for the DNA cleavage activity.