Restriction digests of pMMfnuDIM302:20-6 give the following sizes (kb): HindIII/XbaI--4.1, 1.7; EcoRI--6.0, 0.2; BamHI--6.2; HindIII--6.0. Restriction digest of pMMhaeIIIRM127-1 give the following sizes (kb):HindIII/XbaI--3.2, 2.7, 2.3, 2.2, 0.66; SacI--8.4, 2.7; EcoRI--3.8, 3.4, 1.5, 1.15, 0.96, 0.2; BamHI--11.25; HindIII--5.4, 2.7, 2.3, 0.68. This strain contains 2 plasmids. pMMfnuDIM302:20-6 is a 1.4 kb HindIII/XbaI fragment cloned into pACYC184 (cmlR) that expresses the modification methylase from F. nucleatum D. It was derived from pEVCfnuDIRM 102-1 (ATCC 40521) by deletion of internal HincII fragments and cloning into the the vector HindIII and XbaI sites. pMMhaeIIIRM127-1 is a 8.5 kb SacI fragment cloned into pUC19 (ampR) that expresses the restriction endonuclease HaeIII from H. aegyptius (ATCC 11116). The HaeIII methylase is expressed inadequately so the presence of pMMfnuDIM 302:20-6 is necessary to methylate host DNA and permit isolation of the HaeIII endonuclease. pMMhaeIIIRM127-1 is the predominant plasmid because of higher copy number. |
